Mechanistic Studies on the Formation and Decomposition
Reactions of Iron(III) Porphyrin Complexes with NO
Mechanistische Untersuchungen der Bildungs- und
Zersetzungsreaktionen
von Eisen(III)-Porphyrin-Komplexen mit NO
Den Naturwissenschaftlichen Fakultäten
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur Erlangung des Doktorgrades
vorgelegt von
Joo-Eun Jee
aus Yeosu (Süd Korea)
Als Dissertation genehmigt von den Naturwissenschaftlichen Fakultäten der
Friedrich-Alexander-Universität Erlangen-Nürnberg
Tag der mündlichen Prüfung:
14. Juni. 2007
Vorsitzender der
Promotionskommission:
Prof. Dr. E. Bänsch
Erstberichterstatter:
Prof. Dr. Dr. h. c. mult. R. van Eldik
Zweitberichterstatter:
Prof. Dr. N. Burzlaff
ACKNOWLEDGEMENTS
This work was carried out from April 2004 to June 2007 at the Institute for
Inorganic Chemistry at the Friedrich-Alexander-University of ErlangenNürnberg under the supervision of Prof. Dr. Dr. h. c. mult. Rudi van Eldik.
I would like to warmly thank Prof. Dr. Dr. h. c. mult. Rudi van Eldik for the
particular opportunity to work in his research group, the proposition of the
interesting and fascinating subject and all his support. His everlasting
enthusiasm and lightened ideas encouraged me in hard times to continue and
develop my own visions and projects.
Also, I gratefully thank Dr. Norbert Jux and his group for providing samples of
the porphyrins complexes investigated in my study.
Furthermore, I want to express my gratitude to Dr. Maria Wolak for her very
helpful support in practice. Her patience and encouragement, as well as her
remarkable ability to impart scientific knowledge were of immense help and
allowed me to rapidly deepen my understanding in the subject.
Special thanks go to Dr. Achim Zahl, Dr. Anton Neubrand, Dr. Carols DückerBenfer, Dr. Gosia Brindell, Dr. Lukasz Orzel, Dr. Erika Ember, David Sarauli,
Geo-feng Liu, Eun-young Ji, Moon-Seok Choi, Jin-Woo Park, Korean
community and all of those who, in one way or another, have contributed to aid
me during my doctor thesis.
The exceptionally pleasant environment and working atmosphere in the ‘Rudi´s
Group’ helped to make my doctor thesis an unforgettable experience for me.
Finally, I wish to give my greatest appreciation to my parents, Tae-Uk Jee and,
Jin-Suk Lee and my family for endless love, support and patience which have
been valuable during my study.
Joo-Eun
To my lovely parents and family
Publications
Jee, J.-E., Eigler, S., Hampel, F., Jux, N., Wolak, M., Zahl, A., Stochel, G., van Eldik, R.
“Kinetic and Mechanistic Studies on the Reaction of Nitric Oxide with a Water-Soluble
Octa-anionic Iron(III) Porphyrin Complex” Inorg. Chem. 2005, 44(22), 7717-7731.
Jee, J.-E., Wolak, M., Balbinot, D., Jux, N., Zahl, A., van Eldik, R. “A Comparative
Mechanistic Study of the Reversible Binding of NO to a Water-Soluble Octa-Cationic
FeIII Prophyrin Complex” Inorg. Chem. 2006, 45(3), 1326-1337.
Jee, J.-E. and van Eldik, R. “Mechanistic Studies on the Nitrite-Catalyzed Reductive
Nitrosylation of Highly Charged Anionic and Cationic FeIII Prophyrin Complexes” Inorg.
Chem. 2006, 45(16), 6523-6534.
Jee, J.-E., Eigler, S., Jux, N., Zahl, A., van Eldik, R. “Influence of an extremely
negatively charged porphyrin on the reversible binding kinetics of NO to Fe(III) and the
subsequent reductive nitrosylation“ Inorg. Chem. 2007, 46(8), 3336-3352
Jee, J.-E., Comas-Vives, A., Dinoi, C., Ujaque, G., van Eldik, R., Lledós, A., Poli, R.
“Nature of Cp*MoO2+ in water and intramolecular proton transfer mechanism by stoppedflow kinetics and DFT calculations” Inorg. Chem. 2007, 46(10), 4103-4113
Conference contributions
Joo-Eun Jee, Maria Wolak, Siegfried Eigler, Nobert Jux, Rudi van Eldik “ Mechanistic
studies on the reaction of nitric oxide with water-soluble octa anionic FeIII-porphyrin”
International Minisymposium on Redox-Active Metal Centers in Homogeneous Electron
Transfer Systems in Erlangen (Germany), Feb 2005.
Joo-Eun Jee, Maria Wolak, Achim Zahl, Rudi van Eldik “Comparative study of the
interaction of NO with water soluble cationic- and anionic FeIII porphyrins” 2th Aqua
Chem Meeting, in Almeria (Spain), Dec 2005.
Joo-Eun Jee, Maria Wolak, Nobert Jux, Rudi van Eldik “Comparative study of the
interaction of NO with highly positively- and negatively-charged iron(III) porphyrins”
Inorganic Reaction Mechanism Meeting (IRMM 35th) in Krakow (Poland), Jan 2006.
Chiara Dinoi, Rinaldo Poli, Joo-Eun Jee, Achim Zahl, Rudi van Eldik. “Acid-base
17
chemistry of Cp*2Mo2O5 and O NMR studies of water exchange in the Cp*Mo(VI) oxo
systems” Les journées de la Division de Chimie de Coordination" in Toulouse (France),
Apr 2006.
Joo-Eun Jee, Rudi van Eldik “Comparative mechanistic studies of the interaction of NO
with
highly
charged
FeIII
porphyrins
and
their
nitrite-catalyzed
reductive
nitrosylation” International Minisymposium on Redox-Active Metal Centers in
Homogeneous Electron Transfer Systems in Erlangen (Germany), Oct 2006.
Joo-Eun Jee, Rudi van Eldik “Comparative study of the interaction of NO with highly
charged cationic- and anionic FeIII porphyrins and their nitrite-catalyzed subsequent
reductive nitrosylation” 3th Aqua Chem Meeting, in Debrechen (Hungary), Jan 2007.
Alexi Comas-Vives, Joo-Eun Jee, Chiara Dinoi, Gregori Ujaque, Agusti Lledós, Rudi van
Eldik, Rinaldo Poli “The Cp*MoO2+ Complex in Water and the Solvent Role in the
Intramolecular Proton Transfer Mechanism:A Theoretical Study” 3th Aqua Chem
Meeting, in Debrechen (Hungary), Jan 2007.
Joo-Eun Jee, Norbert Jux, Achim Zahl, and Rudi van Eldik “Influence of an extremely
negatively charged porphyrin on the kinetics of NO binding to Fe(III) and subsequent
reductive nitrosylation” Inorganic Reaction Mechanism Meeting (IRMM 36th) in York,
(England), Mar 2007.
All my life through, the new sights of nature made me rejoice like a child
(Marie Curie, 1867-1934)
Abbreviations and Acronyms
Chemicals
Ag/AgCl
silver/silver chloride
Me
methyl
Ph
phenyl
MeOH
methanol
CH2Cl2
dichloromethane
HCl
hydrochloric acid
NaOH
sodium hydroxide
Et2O
diethylether
NO2-
nitrite
MgSO4
magnesium sulfate
CH3COOC2H5
ethyl acetate
TMS
tetramethylsilane
Abbreviations
K
equilibrium constant
k
rate constant
kobs
observed rate constant
A0
initial absorbance
A∞
final absorbance
Ax
the absorbance at any given ligand concentration
I
ionic strength
ε
molar extinction coefficient
T
temperature
S
spin state
ΔH≠
activation enthalpy
ΔS≠
activation entropy
ΔV≠
activation volume
R
gas constant, 8.3145 J K-1 mol-1
kb
Boltzmann constant, 1.3807·10-23 J K-1
h
Planck constant, 6.6261·10-34 J s
DFT
density functional theory
NMR
nuclear magnetic resonance
NMR spectra
s
singlet
d
doublet
t
triplet
q
quartet
m
multiplet
br
broad
δ
chemical shift
n
coupling constant
J
Table of Contents
1. General Introduction………………………………………………………….. 1
1.1. References……………………………………………………….……………….. 4
2. Kinetic and Mechanistic Studies on the Reaction of Nitric Oxide with a
Water-Soluble Octa-Anionic FeIII Porphyrin Complex……………........……6
2.1. Abstract……………….……………………………………..……………………6
2.2. Introduction……………….…………………..…………………….…………….7
2.3. Experimental Section…………......…………………………………….…….…10
2.4. Results and Discussion...……..…………………………………………...….….17
2.4.1. Synthesis of (P8-)FeIII(OH)……………………………………………...….17
2.4.2. Speciation of (P8-)FeIII(OH) as a Function of pH………………….……….19
2.4.3. Reactivity of (P8-)FeIII(H2O)2 toward NO…………..…..…..……..….…….26
2.4.4. Reactivity of Monohydroxo-Ligated (P8-)FeIII(OH) toward NO………..….34
2.5. References and Notes……………………………………………………………44
3. A Comparative Mechanistic Study of the Reversible Binding of NO to a
Water-Soluble Octa-Cationic FeIII Porphyrin Complex…………………….48
3.1. Abstract……………………….……………………………………...……….….48
3.2. Introduction……………………………………………………………………....49
3.3. Experimental Section……………….……………………..….…...……………..50
3.4. Results and Discussion……..........................................................................……53
3.4.1. Spectroscopic Data for the Speciation of (P8+)FeIII…………………..…….53
3.4.2. Reversible Binding of NO to (P8+)FeIII(H2O)2………………….……...…..61
3.4.3. Reversible Binding of NO to (P8+)FeIII(OH)(H2O)………………..……….63
3.4.4. Mechanism of Reversible NO Binding to (P8+)FeIII
I
Comparison with Other Water-Soluble Iron(III) Porphyrins………….…...66
A. Reactivity of (P8+)FeIII(H2O)2 toward NO……………...………..…….…..66
B. Reactivity of (P8+)FeIII(OH)(H2O) toward NO…………..….……………..69
3.5. References and Notes……………………………………………………………75
4. Mechanistic Studies on the Nitrite-Catalyzed Reductive Nitrosylation of
Highly-Charged Anionic and Cationic FeIII-Porphyrin Complexes…….....78
4.1. Abstract……………………………………………………………………..……78
4.2. Introduction………………………………………………………………………79
4.3. Experimental Section……………………………………………………….……81
4.4. Results and Discussion....………………...………………………………………83
4.4.1. Reaction of (P8+)FeIII(H2O)2 with Nitrite……………………………...…...83
4.4.2. Reaction of (P8+)FeIII(OH)(H2O) with Nitrite…………………………..….87
4.4.3. Spontaneous Reductive Nitrosylation of (P8-)FeIII and (P8+)FeIII………..…90
4.4.4. Nitrite-Catalyzed Reductive Nitrosylation of (P8-)FeIII and (P8+)FeIII…...…98
4.4.5. Suggested Mechanism and Comparison of Iron(III) Porphyrins………....101
4.5. References and Notes………………………………………………………..…107
5. Influence of an Extremely Negatively Charged Porphyrin on the
Reversible Binding Kinetics of NO to Fe(III) and the Subsequent Reductive
Nitrosylation.……………………………………………….….….…….……110
5.1. Abstract…………………………………………………………………………110
5.2. Introduction…………………………………………………………………..…111
5.3. Experimental Section………………………………………………...…………113
5.4. Results and Discussion.………..………………………………………..………119
5.4.1. Synthesis of (P16-)FeIII…………………………………….……...……….119
5.4.2. Studies on (P16-)FeIII………………………………………………...…….120
II
5.4.3. Reaction of (P16-)FeIII(H2O)2 with Nitric Oxide……………………….….127
5.4.4. Reaction of (P16-)FeIII(OH) with Nitric Oxide………………………...…..131
5.4.5. Spectroscopic and Kinetic Studies on the Subsequent Reactions……...…139
5.4.6. Suggested Mechanisms and Comparison with Other Iron(III) Porphyrins…...
………………………………………………………………………….....146
A. Reactivity of (P16-)FeIII(H2O)2 and (P16-)FeIII(OH) toward NO…………..…146
B. Subsequent Reductive Nitrosylation of (P16-)FeIII(H2O)(NO+)…….…..…...149
5.5. References ……………….…………….……………………………………….151
6. Mechanistic Studies on the Reaction of Nitric Oxide with a Synthetic HemeThiolate Complex Relevant to Cytochrome P450……….….….…….……155
6.1. Introduction…………………………………………………………………..…155
6.2. Experimental Section………………………………………………...…………157
6.3. Results and Discussion.………..………………………………………..………158
6.3.1. Spectrophotometric Studies……………………………….……...……….158
6.3.2. Reactivity of (RSR4+)FeIII toward NO at pH 8.8……….…………...…….159
6.3.3. Reactivity of (RSR4+)FeIII toward NO at pH 3.5………...…………….….168
6.3.4. Discussion of Kinetic Data …………………………………………...…..173
6.4. References ...............................................................................................………175
Summary……………………………......……………………………………….176
Zusammenfassung………………….....………………………………………...180
Appendix…………………………...….....……………………………………...185
Curriculum Vitae…………………….…………………………………………186
III
1. General Introduction
The reaction between NO and iron(III) porphyrin complexes in biology
Nitric oxide – small, reactive, potentially toxic, diatomic free radical – was regarded
as noxious, polluting gas until the mid-1980s.1,2 It has become one of the most fascinated
and vital entities in biological chemistry as a signaling molecule. Endogeneous formation
of NO has been implicated in an astonishing range of physiological processes in human
and other animals, including smooth muscle relaxation, inhibition of platelet aggregation,
neurotransmission, penile erection, and in the ability of immune system to kill tumor cell
and intracellular parasites.3 Based on its diverse role in physiological processes, Science
proclaimed NO “molecule of the year” in 1992.4 This growth was spurred by the
identification by Ignarro et al., who won the Nobel Prize in Physiology or Medicine, that
nitric oxide plays a role in the endothelium derived relaxation factor (EDRF).5-7
The reactive nitric oxide species (RNOS) which are generated, transferred and
consumed within biological and medical processes, are viewed as NO•, NO– and NO+
species.8 They show a wide range of behavior from oxidizing to reducing, from
nucleophilic to electrophilic NO• as a stable-free radical participating very readily in oneelectron events such as coupling to other free radicals, for example, OH•, O2•–, lipid
peroxyl radicals and thiol radicals. Its reaction with OH• to give nitrite and O2•– to yield
peroxynitrite, ONOO–, proceeds at rates near to the diffusion limit (k ∼ 3.7×107 M-1s-1 ).
The reduced form of nitric oxide is nitroxyl or hyponitrous acid (HNO/NO–, pKa= 11.4) in
which HNO is predominant under physiological conditions and isoelectronic with
dioxygen. Nitroxyl is short-lived, very reactive towards nucleophiles (especially thiols)
and dimerizes to form H2N2O2 which then dehydrates to nitrous oxide, N2O. Nitrosonium,
NO+ is a strong oxidant. It undergoes readily hydrolysis to nitrite NO2–. The NO+ species
is mainly characterized by addition and substitution reactions with nucleophiles (Nu:) as
an acid generating the nitroso compounds Nu–NO. The self exchange rates for NO/NO+
General Introduction
and NO/NO– are quite slow, thus the kinetics of nitric oxide electron transfer reactions are
strongly influenced by redox active transition metal complexes.
The interactions of NO with metal centers are of particular interest since transition
metal centers such as hemes are well established as targets for NO activation in
mammalian biology. In general, NO mediates its effect by stimulating or inhibiting
transition metal-containing proteins and by post-translational modification of proteins
such as formation of nitrosothiol adducts.9,10 Much of their significance is related to the
interaction of NO with iron porphyrins and non-heme iron complexes. How rapidly these
complexes are formed and their relative stability dictates their biological relevance. The
most relevant reactions of NO with metals in biological systems include heme proteins
such as guanylate cyclase. The reaction between NO and guanylate cyclase produces an
Fe-nitrosyl complex that becomes activated to form cGMP, a key secondary messenger
that mediates numerous regulatory functions.3,11,12
Other important heme-proteins which are targets of NO are cytochrome P450
representing a super family of heme-type monooxygenase that catalyze the conversion of
diverse substrate, P450nor that catalyzes reductive dimerization of NO to N2O, and NO
synthase (NOS) responsible for catalyzing the oxidation of L-arginine to produce NO. NO
synthesis is quite different in mammals, where NOS catalyzes the stepwise oxidation of
the amino acid L-arginine to NO and L-citrulline with the aid of oxygen.13 All forms of
the enzyme studied contain four prosthetic groups: flavin-adenine dinucleotide (FAD);
flavin mononucleotide (FMN), tetrahydrobiopterin (H4biopterin) and a heme complex,
iron protoporphyrin IX (heme), NOS is the unique enzyme known to use all four. Three
distinct isoforms of NOS have been identified, where three different but very similar
enzymes are involved in the diverse tissues. These are named after the tissue in which
they were first purified and cloned, iNOS in macrophase, eNOS in endothelial cell, and
nNOS in brain neuron, respectively.
On the other hand, NO inhibits cytochrome P450 activity by binding to the heme,
generating a Fe–NO complex and preventing oxygen binding. It has been proposed that
NO can regulate the synthesis of certain hormones like testosterone in this way.
-2-
General Introduction
Analogously, binding of NO to the heme-site of NO synthase prevents the binding of
oxygen and the oxidation of arginine. NO serves as a negative feed back factor, regulating
the NO level in this way. Red blood cells, for example, are actively involved in the
regulation of vascular tone, largely through their capacity to stimulate production of, and
transport the vasodilator substance nitric oxide. In particular, the irreversible oxidation of
NO to form metHb and nitrate, and the reversible reactions of NO with hemoglobin (Hb)
to form S-nitrosoHb (SNO-Hb) and nitrosyl(heme)Hb are of potential major
importance.14-16 S-nitroso-hemoglobin (SNO-Hb) arises from S-nitrosylation of cystein
β93 of oxygenated hemoglobin(Hb) in erythrocytes. It has been thought that SNO-Hb
behaves as a nitric oxide (NO) donor at low oxygen concentrations to regulate the blood
pressure control, in homogeneous reactions of NO with met-hemoglobin (met-Hb) and in
heterogeneous reactions with red blood cells.17 In this process, reductive nitrosylation has
considerably received attention as a possible pathway for the formation of S-nitrosohemoglobin (SNO-Hb).
The variation of spin and ligation states of the central iron atom is reflected in
distinct structural features observed for different types of model and naturally occurring
iron(III) porphyrins. Heme proteins that can react with NO under biological conditions, an
improved characterization of typical reactivity patterns observed in the reactions of NO
with model five- and six-coordinate iron(III) porpyhrins with high-spin, low-spin, and
admixed intermediate-spin states of the iron center, respectively, will help to establish the
relationship between the structure of the heme prosthetic group and its reactivity toward
NO in various heme proteins.18 In addition, the subsequent reductive nitrosylation
reaction of iron(III) porphyrin followed by the binding of NO is also influenced by
features of the heme group, which is catalyzed by nitrite as well as general base such as
OH-.19 Therefore, systematic studies into the binding of NO by synthetic iron porphyrins
should aid our understanding of how NO interacts with heme-containing biomolecules.
This dissertation will provide a detailed study of the mechanism of reaction of NO with
iron(III) porphyrins of different nature and number of substituents and their subsequent
reactions (such as reductive nitrosylation) in aqueous medium.
-3-
General Introduction
1.1.
References
1. Lammel, G; Cape, J. N. Chem. Soc. Rev., 1996, 25, 361
2. Lancaster, J. Jr. (ed) Nitric Oxide Principles and Actions, Academia press, San Diego,
1996
3. Culotta, E.; Koshland, D. E, Jr. Science, 1992, 258, 1862
4. Ignarro, L. J.; Buga, G. M.; Word, k. S., Byrns, R. E. Chaudhuri, G. Proc. Natl. Acad.
Sci. U.S.A. 1984, 84, 9265
5. (a) Moncada, S.; Higgs, E. A. Br. J. Pharmacol. 2006, 147, S104. (b) Feelisch, M.;
Stamler, J. S. Methods in Nitric Oxide Research; John Wiley and Sons: Chichester,
England, 1996. (c) Verma, A.; Hirsch, D. J.; Glatt, C. E.; Romnett, G. V.; Snyder, S.
H. Science (Washington, D.C.) 1993, 259, 381.
6. (a) Moncada, S.; Palmer, R. M. J.; Higgs, E. A. Pharmacol. Rev. 1991, 43, 109. (b)
Ignarro, L. J. A. Rev. Pharmacol. Toxicol. 1990, 30, 535.
7. (a) Moncada, S.; Radomski L. W.; Palmer, R. M. Biochem. Pharmacol. 1988, 37,
2495. (b) Palmer, R. M. J.; Ferrige, A. G.; Moncada, S. Nature. 1987, 327, 524.
8. (a) Stamler, S. S.; Single, D. J.; Loscalzo, J. Science, 1992, 258, 1898 (b) Macyk, W.;
Franke, A.; Stochel G. Coord. Chem. Rev. 2005, 249, 2437
9. (a) Butler, A. R.; Lyn, D.; Williams, H. Chem. Soc. Rev. 1993, 22, 233 (c) Williams,
R. J. P. Chem. Soc. Rev. 1996, 25, 77 (d) Stochel, G.; Ilkowska, E.; Pawelec, M.;
Wanat, A.; Wolak, M. ACH-Models Chem. 1998, 135, 847
10. (a) Ford, P. C.; Laverman, L. E.; Lorkovic, I. M. Adv. Inorg. Chem. 2003, 54, 2003.
(b) Ford, P. C.; Lorkovic, I. M. Chem. Rev. 2002, 102, 993 and references therein.
11. (a) Hoshino, M.; Laverman, L.; Ford, P. C. Coord. Chem. Rev. 1999, 187, 75. (b)
Laverman, L. E.; Ford, P. C. J. Am. Chem. Soc. 2001, 123, 11614. (c) Ford, P. C.,
Fernandez, B. O.; Lim, M. D. Chem. Rev. 2005, 105, 2439
12. (a) Bredt, D. S.; Hwang, P. M.; Glatt, C. E.; Lowenstein, C.; Reed, R. R.; Snyder, S. H.
Nature (London) 1991, 351, 714. (b) Moncada, S.; Palmer, R. M.; Higgs, E. A.
-4-
General Introduction
13. Biochem. Pharmacol. 1989, 38, 1709. (c) Hibbs, J. B., Jr.; Tainter, R. R.; Vabrin, Z.
Science. 1987, 235, 473.
14. (a) Stamler, J. S.; Lamas, S.; Fang, F. C. Cell. 2001, 106, 675 (b) Foster, M. W.;
McMahon, T. J.; Stamler, J. S. Trends. Mol. Med. 2003, 9, 160
15. Liu, L.; Yan, Y.; Zeng, M.; Zhang, J.; Hanes, M. A.; Ahearn G.; McMahon, T. J.;
Dickfeld, T.; Marshall H. E.; Que, L. G.; Stamler, J. S. Cell. 2004, 116, 617
16. Jia, L.; Bonaventura, J.; Stamler, J. S. Nature(London), 1996, 380, 221
17. (a) Allen, B. W.; Piantadosi, C. A. Am. J. Physiol. 2006, 291, H1507 (b) Deem, S.;
Kim, S. S.; Min, J.-H.; Eveland, R.; Moulding, J.; Martyr, S.; Wang, X.; Swenson, E.
R.; Gladwin, M. T. Am. J. Physiol. 2004, 287, H2561
18. (a) Wyllie, G. R. A.; Scheidt, W. R. Chem. Rev. 2002, 102, 1067 (b) Ellison, M. K. ;
Schulz, Ch. E.; Scheidt, R. W. J. Am. Chem. Soc. 2002, 124, 13833 (c) Scheidt, W.R.;
Ellison, M. K. Acc. Chem. Res. 1999, 32, 350
19. (a) Ford, P. C.; Fernandez, B. O.; Lim, M. D. Chem. Rev. 2005, 105, 2439. (b)
Stamler, J. S.; Jia, L.; Eu, J. P.; Mcmahon, T. J.; Demchenko, I. T.; Bonaventura, J.;
Gernert, K.;
Piantadosi, C. A. Science. 1997, 276, 2034. (c) Fernandez, B. O.;
Lorkobic, I. M.; Ford, P. C. Inorg. Chem. 2004, 43, 5393. (d) Jee, J.-E.; van Eldik,
Rudi. Inorg. Chem. 2006, 45, 6523
-5-
2. Kinetic and Mechanistic Studies on the Reaction of Nitric Oxide
with a Water-Soluble Octa-Anionic FeIII-Porphyrin Complex*
2.1 Abstract
The polyanionic water-soluble and non-μ-oxo dimer forming iron porphyrin,
iron(III)-54,104,154,204-tetra-t-butyl-52,56,152,156-tetrakis-(2,2-bis-carboxylato-ethyl)5,10,15,20-tetraphenylporphyrin, (P8–)FeIII (1), was synthesized as the octasodium salt by
applying
well-established
porphyrin
and
organic
chemistry
procedures
to
bromomethylated precursor porphyrins, and fully characterized by standard techniques
such as UV-vis and 1H NMR spectroscopy. A single pKa1 value of 9.26 was determined
for the deprotonation of coordinated water in (P8–)FeIII(H2O)2 (1-H2O) present in aqueous
solution at pH < 9. The porphyrin complex reversibly binds NO in aqueous solution to
give the mononitrosyl adduct, (P8–)FeII(NO+)(L), where L = H2O or OH–. The kinetics of
the binding and release of NO was studied as a function of pH, temperature and pressure
by stopped-flow and laser flash photolysis techniques. The diaqua-ligated form of the
porphyrin complex binds and releases NO according to a dissociative interchange
mechanism based on the positive values of the activation parameters ΔS‡ and ΔV‡ for the
“on” and “off” reactions. The rate constant, kon = 6.2 x 104 M-1 s-1 (24 °C) determined for
NO binding to the monohydroxo-ligated (P8–)FeIII(OH) (1-OH) present in solution at pH
> 9, is markedly lower than the corresponding value measured for 1-H2O at lower pH (kon
= 8.2 x 105 M-1 s-1, 24 °C, pH 7). The observed decrease in reactivity is contradictory to
that expected for the diaqua- and monohydroxo-ligated forms of the iron(III) complex,
and is accounted for in terms of a mechanistic changeover observed for 1-H2O and 1-OH
in their reactions with NO. The mechanistic interpretation offered is further substantiated
by the results of water exchange studies performed on the polyanionic porphyrin complex
as a function of pH, temperature and pressure.
Octa-Anionic FeIII-Porphyrin Complex
* Joo-Eun Jee, Siegfried Eigler, Frank Hampel, Norbert Jux, Maria Wolak, Achim Zahl, Grazyna
Stochel, and Rudi van Eldik, Inorg. Chem. 2005, 44(22), 7717-7731
2.2 Introduction
Iron porphyrins, an important class of transition-metal complexes, continue to
attract considerable attention because of their importance in biology and catalysis. Roles
played by these complexes in electron-transfer processes, metabolic control, transport and
activation of oxygen under biological conditions, and versatile catalytic processes in vitro
are surprisingly diverse. This rich chemistry stems partially from the fact that the
reactivity of iron porphyrins is finely tuned by the surroundings of the central iron atom,
such as the identity of axial ligands, the nature of the substituents on the tetrapyrrole ring,
the polarity of the reaction medium (solvent or amino acid residues around the active site
in heme proteins), and several other factors. To understand these structure-function
relationships, numerous mechanistic and structural studies on heme proteins and their
biomimetic models have been performed. In particular, synthetic iron(III) porphyrins, the
structural analogues of prosthetic groups in ferriheme proteins, have been extensively
studied in relation to their catalytic and biomimetic properties.
An important aspect of studies on the reactivity of model iron(III) porphyrins and
ferriheme proteins concerns their interaction with nitric oxide.1-10 These studies were
directed, on the other, to explain the nature of interactions underlying the diverse
functions of NO in vivo and, on the other hand, to probe the reactivity of various iron
porphyrins in ligand substitution reactions. In the latter context, studies on the mechanism
of formation and decay of iron porphyrin nitrosyls provided information on the influence
of the porphyrin ligand (modified in size and overall charge by the substituents on the
tetrapyrrole ring)1,2,4-7 and protein environment (in studies concerning heme proteins)1,2,5,810
on the reactivity of the central FeIII ion in ligand substitution reactions. Because the
nature and lability of axial ligands coordinated to the iron center are of crucial importance
for its overall reactivity, information gained from such mechanistic studies contributed to
-7-
Octa-Anionic FeIII-Porphyrin Complex
the understanding of various mechanistic pathways exhibited by iron porphyrins in their
biological and catalytic functions.
It is now firmly established that binding of NO to an iron-(III) porphyrin leads to the
formation of a low-spin diamagnetic (S = 0) mononitrosyl complex, in which the Fe–NO
unit adopts a linear (or close to linear) geometry.1,3 Because NO binding is accompanied
by charge transfer from the NO ligand to the metal center, the resulting product
(representing a {Fe-NO}6 nitrosyl3) can be formally described as (P)FeII–NO+. Detailed
mechanistic investigations performed in our5,6,9 and other1,2,4,8,10 laboratories provided
evidence that the dynamics of NO binding in heme proteins and synthetic iron porphyrins
is to a large extent controlled by the ligand substitution step. Considerable differences in
the rates of binding and release of NO,1,2,4,6,8-10 and varying tendencies of the resulting
nitrosyls to undergo subsequent reactions (such as reductive nitrosylation)8,11,12 reported
in these studies, apparently reflect the influence of variation in the immediate
surroundings of the iron(III) center (mainly the types of axial ligands and porphyrin ring
substituents) on the observed reactivity patterns. Despite these studies, a coherent pattern
of a structure-reactivity relationship in the studied reactions is far from complete. In this
context, systematic studies on the reactivity of water-soluble iron porphyrins (considered
as model compounds for aqueous porphyrin chemistry) with different types of positively
and negatively charged meso substituents toward NO have been initiated in order to better
assess the correlation between the porphyrin structure and its reactivity.4,6,7,11-14 The
results of high-pressure NMR, stopped-flow, and laser flash photolysis investigations
performed on these complexes provided evidence that the negatively charged peripheral
substituents tend to labilize the axial metal-ligand bond by increasing the electron density
on the metal center. A survey of literature data, however, shows that porphinatoiron(III)
complexes may exist as five- or six-coordinate species in which the d electrons of the
central FeIII ion can be arranged into three possible spin states, viz., the low spin state (S =
1
/2), intermediate spin state (S = 3/2) and the high spin state (S = 5/2).15 In addition, a range
of (P)FeIII(L)n complexes (with n = 1 or 2) with quantum mechanically admixed
intermediate spin states (S = 5/2 and 3/2) have been reported, in which varying ratios of S =
-8-
Octa-Anionic FeIII-Porphyrin Complex
5
/2 and 3/2 admixing were observed, depending on the nature of the axial ligands and
porphyrin ring substituents.15-23 This variety of spin and ligation states is reflected in
distinct structural features observed for different types of model and naturally occurring
iron(III) porphyrins. While the low-spin (P)FeIII(L)n complexes are typically sixcoordinate and exhibit short axial and equatorial bonds (due to depopulation of eg*
orbitals), purely high-spin analogues are often five-coordinate, with elongated Fe-Np
bonds and considerable displacement (0.4-0.6 Å) of the iron(III) center out of the
porphyrin plane.15,22 The structural manifestations of the intermediate spin state (admixed
or pure) include short Fe–Np equatorial bonds (resulting from partial or complete
depopulation of the dx2–y2 orbital) and strongly elongated axial bonds.15,17,21,23-25 Although
these various features are critical in controlling the reactivity of the iron(III) porphyrins,
their influence on the kinetic and mechanistic features of NO binding and release in
different classes of (P)FeIII(L)n complexes remains obscure. Taking into account a variety
of spin and ligation states in heme proteins that can interact with NO under biological
conditions, an improved characterization of typical reactivity patterns observed in the
reactions of NO with model five and six-coordinate iron(III) porphyrins with high-spin,
low-spin, and admixed intermediate-spin states of the central iron atom, respectively,
would help to establish the relationship between the structure of the heme prosthetic
group and its reactivity toward NO in various heme proteins.
In a continuation of our earlier mechanistic work on the reactivity of iron porphyrins
toward NO,5,6,9,14,26,27 we now report on the synthesis and spectroscopic characterization
of a highly negatively charged water-soluble iron(III) porphyrin, (P8–)FeIII (1; compare
Figure 1) and present the results of detailed mechanistic studies on its interaction with
nitric oxide. Within this context, in addition to kinetic studies on the binding and release
of NO from the diaqua (P8–)FeIII(H2O)2 species present in aqueous solution at pH <9, the
reactivity of a high-spin monohydroxo form of the complex formed at higher pH is
reported. Temperature and pressure effects on the rates of NO binding and release
obtained from stopped-flow and laser flash photolysis experiments at ambient and
elevated pressure at low and high pH enabled the determination of activation parameters
-9-
Octa-Anionic FeIII-Porphyrin Complex
(ΔH‡, ΔS‡, and ΔV‡) for the studied reactions. Activation volumes determined in these
studies enabled the construction of volume profiles for the binding of NO to the diaqua
and monohydroxo forms of the complex, respectively. On the basis of these data, a
feasible explanation for the significant kinetic and mechanistic differences observed in the
reactivity of these two species is offered. The results are compared with those reported for
other water-soluble porphyrins and discussed in reference to the relevant literature data on
the structure and reactivity of iron(III) porphyrins toward NO.
2.3. Experimental Section
O
O
O
O
OEt
EtO
EtO
N Zn
O
OEt
EtO
N
N
N Fe
EtO
O
N
R
EtO
O
OEt
O
O
OEt
N
N
MeOH, lutidine
OEt
O
Cl R
FeCl2,
R
EtO
O
OEt
EtO
OEt
R
N
O
O
O
OEt
4
EtO
O
O
2: M = Zn
HCl
NaOH,
EtOH
3: M = 2H
O
O
O
O
O
O
O
OH R
N Fe
N
O
N
N
R
O
O
O
O
O
O
1-OH
O
O
8 Na
Figure 1. Synthesis of (P8–)FeIII; R = para-tert-butyl-C6H4.
Synthesis and Characterization of 1. Chemicals and solvents employed for the synthesis
of 1 were used as received unless otherwise noted. Solvents were dried using standard
- 10 -
Octa-Anionic FeIII-Porphyrin Complex
procedures. Column chromatography was performed on silica gel 60, 32-63 μm, 60 Å
(MP Biomedicals). Standard 1H and 13C NMR spectra were recorded on a Bruker Avance
300 spectrometer (Bruker Analytische Messtechnik GmbH). Fast atom bombardment
(FAB) mass spectrometry was performed with Micromass Zabspec and Varian MAT
311A machines. Electrospray ionization mass spectra (ESIMS) were measured in the
negative ion mode on an ESI-FT-ICR-MS mass spectrometer (Fa. Agilent, ICR: APEX II,
Fa. Bruker Daltonics, 7 T magnet). Standard UV-vis spectra were recorded on a
Shimadzu UV-3102 PC UV-vis-NIR scanning spectrophotometer. IR spectra (KBr
pellets) were recorded with a FT-IR IFS 88 infrared spectrometer (Bruker Analytische
Messtechnik GmbH). Elemental analyses were carried out on a CHNMikroautomat
(Heraeus). Thin-layer chromatography was carried out on E. Merck silica gel 60 F254
plates. Zinc(II) 54,104,154,204-tetra-tert-butyl-52,56,152,156-tetrakis[2,2-bis(ethoxycarbonyl)ethyl]-5,10,15,20-tetraphenylporphyrin (2) was synthesized as described previously.28
54,104,154,204-Tetra-tert-butyl-52,56,152,156-tetrakis[2,2-bis-(ethoxycarbonyl)ethyl]5,10,15,20-tetraphenylporphyrin (3). HCl (6 M, 50 mL) was added to a solution of 2
(174 mg, 0.109 mmol) in CH2Cl2 (50 mL), and the two layers were shaken vigorously.
The green (organic) layer was shaken once again with 2 M HCl (50 mL) and twice with
water (50 mL each). After neutralization with a saturated NaHCO3 solution (50 mL) and a
final washing with brine (50 mL), the organic layer was dried with MgSO4 and the
solvent was removed under reduced pressure. The compound was further cleaned by
column chromatography (silica gel, CH2Cl2/ethyl acetate 19:1) and obtained as a purple
powder. Yield: 160 mg (96%, 0.105 mmol). 1H NMR (300 MHz, CDCl3, rt): δ 8.88 (d,
4H, 3J = 4.78 Hz, β-pyrr-H), 8.69 (d, 4H, 3J = 4.8 Hz, β-pyrr-H), 8.15 (d, 4H, 3J = 8.1 Hz,
o-Ar*-H; an asterisk indicates proton resonances of the charge-carrying aryl ring), 7.76 (d,
4H, 3J = 8.1Hz, m-Ar*-H), 7.46 (s, 4H, m-Ar-H), 3.62 (m, 16H, OCH2CH3), 3.04 (t, 4H,
3
J = 7.8 Hz, CH), 2.80 (d, 8H, 3J = 7.8 Hz, Ar-CH2), 1.60 (s, 36H, CH3), 1.51 (s, 36H,
CH3), 0.69 (t, 24H, 3J =7.2 Hz, OCH2CH3).
13
C NMR (75 MHz, CDCl3, rt): δ 168.4,
151.4, 150.5, 139.3, 138.7, 138.4, 134.5, 132.0, 130.1, 124.4, 123.6, 120.2, 115.2, 60.7,
52.2, 34.8, 33.6, 31.6, 31.5, 29.6, 13.4. MS (FAB, NBA): m/z 1528 (M+.). IR (KBr): ν
- 11 -
Octa-Anionic FeIII-Porphyrin Complex
[cm-1] 3318, 2961, 2933, 2906, 2867, 1755, 1735, 1632, 1468, 1367, 1221, 1146, 1034,
807. UV-vis (CH2Cl2): λ [nm] (ε [L mol-1 cm-1]) 421 (4.58 × 105), 517 (1.98 × 104), 551
(8.1 × 103), 591 (6.4 × 103), 648 (4.3 × 103). Anal. Calcd for C92H110N4O16: C, 72.32; H,
7.26; N, 3.67. Found: C, 71.97; H, 7.31; N, 3.66.
Chloroiron(III) 54,104,154,204-Tetra-tert-butyl-52,56,152,156-tetrakis[2,2-bis(ethoxycarbonyl)ethyl]-5,10,15,20-tetraphenylporphyrin (4). A solution of FeCl2 (400 mg,
3.15 mmol) in ethanol (30 mL) was added to a solution of 3 (343 mg, 0.225 mmol) in
CHCl3 and the mixture heated under reflux for 18 h. The solvent was evaporated and the
residue dissolved in CH2Cl2 and washed with 6 M HCl. The organic layer was separated
and washed twice with water. After drying over MgSO4, the compound was cleaned by
column chromatography (silica gel, 19:1 CH2Cl2/ethyl acetate) to give a dark green
powder. Yield: 318 mg (89 %, 0.201 mmol). 1H NMR (300 MHz, CDCl3, rt): δ 82.9 (br s,
β-pyrr-H), 80.8 (br s, β-pyrr-H), 15.8, 14.1, 13.3, 12.2 (br s, aryl-H). MS (FAB, NBA):
m/z 1582 (M+). IR (KBr): ν [cm-1] 2963, 2939, 2907, 2869, 1751, 1734, 1626, 1464, 1445,
1395, 1367, 1332, 1149, 1031, 997, 859, 803, 722. UV-vis (CH2Cl2): λ [nm] (ε [L mol-1
cm-1]) 422 (1.18 × 105), 509 (1.4 × 104), 583 (7.3 × 103). Anal. Calcd for
C92H108ClFeN4O16·CH2Cl2: C, 65.62; H, 6.51; N, 3.29. Found: C, 65.58; H, 6.68; N, 3.33.
Octasodium Hydroxoiron(III) 54,104,154,204-Tetra-tert-butyl- 52,56,152,156-tetrakis[2,2-bis(carboxylato)ethyl]-5,10,15,20-tetraphenylporphyrin (1-OH). NaOH (1.50 g,
37.5 mmol) was added to a solution of 4 (200 mg, 0.126 mmol) in ethanol (20 mL), and
the reaction mixture was heated under reflux for 1 h. After cooling to room temperature,
the precipitate was filtered, washed with ethanol (200 mL), and dried under reduced
pressure. Gel permeation chromatography (Sephadex LH20) in methanol und subsequent
precipitation with diethyl ether gave a dark brown powder, which, according to the
microanalysis, contains sodium hydroxide in the lattice. Yield: 230 mg (83%, 0.105
mmol; based on the formula obtained by microanalysis). 1H NMR (300 MHz, unbuffered
D2O, pD ) 13.4, rt): δ 82.7 (br s, β-pyrr-H), 13.2, 12.3 (br s, aryl-H). ESIMS
(MeOH/H2O):
1376.45
{[(P)FeIII(COOH)5(COO)2(COONa)]–},
1354.46
{[(P)FeIII(COOH)6(COO)2]–}. IR (KBr): ν [cm-1] 3429, 2963, 2924, 2854, 1594, 1445,
- 12 -
Octa-Anionic FeIII-Porphyrin Complex
884, 805. UV-vis (H2O, pH 11): λ [nm] (ε [L mol-1 cm-1]) 417 (1.1 × 105), 532 (1.2 × 104).
Anal. Calcd for C76H69FeN4Na8O17·16NaOH: C, 41.68; H, 3.91; N, 2.56. Found: C, 41.81;
H, 4.04; N, 1.56.
Materials. NO gas (Messer Griesheim or Riessner Gase, ≥ 99.5 vol %) was cleaned from
trace amounts of higher nitrogen oxides by passing it through a concentrated KOH
solution and an Ascarite II column (NaOH on silica gel, Sigma-Aldrich). CAPS, MES,
Tris, and Bis-Tris buffers were purchased from Sigma-Aldrich. All other chemicals used
in this study were of analytical reagent grade.
Solution Preparation. All solutions were prepared from deionized water. Buffered
solutions of the appropriate pH for laser flash photolysis and stopped-flow measurements
were prepared with the use of Tris (0.05 M), Bis-Tris (0.05 M), CAPS (0.05 M), and
TAPS (0.05 M) buffers. The desired pH was adjusted by the addition of HClO4 or NaOH.
The ionic strength (0.1 M) was adjusted with NaClO4. Argon or nitrogen and gastight
glassware were used for the preparation and handling of deoxygenated solutions.
Measurements. pH measurements were performed on a Methrom 623 pH meter. An NO
electrode (World Precision Instruments isolated nitric oxide meter, model ISO-NO) was
used to determine the concentration of NO gas in aqueous solution. The NO electrode was
calibrated with a freshly prepared KNO2 solution according to the method suggested by
the manufacturer. UV-vis spectra were recorded in gastight cuvettes on a Shimadzu UV2100 spectrophotometer equipped with a thermostated (±0.1°C) cell compartment.
Kinetic Studies. (a) Laser Flash Photolysis. Laser flash photolysis was carried out with
the use of the LKS-60 spectrometer from Applied Photophysics for detection and a
Nd:YAG laser (SURLITE I-10, Continuum) pump source operating in the third harmonic
(λexc = 355 nm) (100-mJ pulses with ∼7 ns pulse widths). Spectral changes at 427 and 432
nm (at pH 7 and 11, respectively) were monitored using a 100-W xenon lamp,
monochromator, and photomultiplier PMT-IP22. The absorbance reading was balanced to
zero before the flash. Data were recorded on a digital storage oscilloscope DSO HP
54522A and transferred to a computer unit for subsequent analysis. Gastight quartz flow
cuvettes and a pillbox cell combined with a high-pressure unit were used at ambient and
- 13 -
Octa-Anionic FeIII-Porphyrin Complex
elevated pressures (up to 170 MPa), respectively. In ambient pressure experiments, the
deoxygenated solution of the iron porphyrin was mixed in an appropriate volume ratio
with the NO saturated solution, transferred to a gastight flow cuvette, and equilibrated in a
thermostated cell holder for 10 min. In the high pressure studies, deoxygenated solutions
of iron porphyrin and NO were mixed in an appropriate ratio with the use of gastight
syringes, transferred under an inert atmosphere to the pill-box cell, and equilibrated for 10
min at the appropriate temperature and pressure in the high-pressure cell compartment.
All kinetic experiments were performed under pseudo-first-order conditions, i.e., with at
least 10-fold excess of NO over the iron porphyrin. Rate constants reported are the mean
values of at least five kinetics runs, and the quoted uncertainties are based on the standard
deviation.
(b) Stopped-Flow Measurements. Stopped-flow kinetic measurements were performed
on an SX 18.MV (Applied Photophysics) stopped-flow apparatus. In a typical experiment,
a deoxygenated buffer solution was mixed in varying volume ratios with a NO saturated
solution in a gastight syringe to obtain the appropriate NO concentration (0.2−1.8 mM).
The NO solution was then rapidly mixed with deoxygenated iron(III) porphyrin in a 1:1
volume ratio in a stopped-flow apparatus. The kinetics of the reaction was monitored at
427 and 432 nm at pH 7 and 11, respectively. The rates of NO binding and release (kon
and koff) were determined from slopes and intercepts of linear plots of kobs versus [NO],
respectively, as described in more detail in the Results and Discussion section. The NO
dissociation rates at different temperatures and pressures were also measured directly by
the NO-trapping method. This involved rapid mixing of a (P8–)FeII(NO+)(L) solution (2 ×
10-5 M; L = H2O and OH– at pH 7 and 11, respectively) containing a small excess of NO
with aqueous solutions of [RuIII(edta)(H2O)]– (1−2 mM) to give [RuIII(edta)NO]– and (P8–
)FeIII(L), as evidenced by the observed spectral change. The kinetics of NO release was
followed in a stopped-flow spectrophotometer at 427 nm (pH 7) or 432 nm (pH 11). The
first-order rate constants determined from the kinetic traces were in acceptable agreement
with those determined from the intercepts of the plots of kobs versus [NO].
- 14 -
Octa-Anionic FeIII-Porphyrin Complex
High-pressure stopped-flow experiments were performed at pressures up to 130 MPa on a
custom-built instrument described previously.29 The kinetic traces were analyzed with the
use of the OLIS KINFIT (Bogart, GA, 1989) set of programs.
(c) 17O NMR Water-Exchange Measurements. Rate and activation parameters for water
exchange on the paramagnetic (P8-)FeIII(H2O)2 complex and the corresponding activation
parameters ΔH‡ex, ΔS‡ex and ΔV‡ex were measured by use of the 17O NMR line-broadening
technique. Aqueous solutions of 1 (20 mM) were prepared at pH 7 (0.05 M Bis-Tris
buffer) and 11 (0.05 M CAPS buffer), and 10% of the total sample volume of enriched
17
O-labeled water (normalized 19.2% 17O H2O, D-Chem Ltd.) was added to the solution.
A sample containing the same components except for 1 was used as the reference.
Variable-temperature and -pressure Fourier transform 17O NMR spectra were recorded at
a frequency of 54.24 MHz on a Bruker Advance DRX 400WB spectrometer. The
temperature dependence of the
17
O line broadening was determined in the range of
278−353 K. A homemade high-pressure probe30 was used for the variable-pressure
experiments performed at pH 7 at the selected temperature (283 K) and in the pressure
range of 1-150 MPa. The sample was placed in a standard 5-mm NMR tube cut to a
length of 45 mm. The pressure was transmitted to the sample by a movable macor piston,
and the temperature was controlled as described elsewhere.30 The reduced transverse
relaxation times (1/T2r) were calculated for each temperature and pressure from the
difference in the line widths observed in the presence and absence of the metal complex
(Δνobs - Δνsolvent). The reduced transverse relaxation time is related to the exchange rate
constant kex = 1/τm (where τm is the mean-coordinated solvent lifetime) and to the NMR
parameters by the Swift and Connick equation (1),31a,b
1
T2r
= π
1
Pm
(Δνobs − Δνsolvent) =
1
T2m-2 + (T2mτm)-1 + Δω2m
τm
(T2m-1 + τm-1)2 + Δω2m
+
1
T2os
(1)
where Pm is the mole fraction of water coordinated to the FeIII ion, T2os represents the
outer-sphere contribution to T2r arising from long range interactions of unpaired electrons
of FeIII with the water outside the coordination sphere, T2m is the transverse relaxation
- 15 -
Octa-Anionic FeIII-Porphyrin Complex
time of water in the inner coordination sphere in the absence of chemical exchange, and
Δω2m is the difference in the resonance frequency of 17O nuclei in the first coordination
sphere of the metal and in the bulk solvent. In the present system, the contributions of
1/T2m and 1/T2os to 1/T2r are negligible, so that eq 1 can be reduced to eq 2.
1
T2r
= π
1
Pm
(Δνobs – Δνsolvent) =
Δω2m
1
τm
(2)
τm-2 + Δω2m
Taking into account that the temperature dependence of kex is given by eq 3 (taken from
transition-state theory), the NMR and kinetic parameters were calculated by the use of a
nonlinear least-squares method applied to eq 2, in which 1/τm was replaced by eq 3.
kex = τ1m
= (kBT/h)exp{(ΔS‡ex/R) – (ΔH‡ex/RT)}
(3)
The temperature dependence of Δωm was assumed to be a simple reciprocal function
A/T,31a-c where A was determined as a parameter in the treatment of the line-broadening
data. The exchange rate constant is assumed to have a simple pressure dependence given
by eq 4, where kex0 is the rate constant for solvent exchange at atmospheric pressure.
kex =
1
τm
= (kex0exp{(−ΔV‡ex/RT )P}
(4)
The pressure-dependent measurements were performed at a temperature close to the
optimal exchange region [i.e., around the maximum of the plot of ln(1/T2r) versus 1/T].
The reduced relaxation time T2r and the value of Δωm (calculated using the value of A
determined from the temperature dependence and assumed to be pressure-independent31d)
were substituted into eq 2 to determine kex at each pressure. The resulting plot of ln(kex)
versus pressure was linear, and the volume of activation was calculated directly from the
slope (−ΔV‡ex/RT). The value of kex0 obtained from the plot of ln(kex) versus P by
extrapolation to atmospheric pressure was in good agreement with the corresponding
value of kex0 from the temperature-dependent measurements at ambient pressure.
In analogous temperature-dependent
17
O NMR measurements performed for the studied
iron(III) porphyrin at pH 11 (0.05 M CAPS buffer), the line widths observed in the
- 16 -
Octa-Anionic FeIII-Porphyrin Complex
presence and absence of the metal complex changed little (6-13 Hz within the temperature
range of 278−353 K), indicating the absence of a significant water-exchange process for
the 1 form present at higher pH. Because of the small observed effects (close to the
experimental error limits), the data obtained in the variable-temperature study did not
allow a reliable fit to the Swift and Connick equation.
2.4. Result and Discussion
2.4.1. Synthesis of (P8-)FeIII(OH).
Some of us recently reported the synthesis of a water-soluble anionic zinc
porphyrin (2 in Figure 1) that carries eight carboxylates in four malonate units.28 The
precursor porphyrin 2 carries the malonates in the form of ethyl esters, which renders the
compound soluble in organic but not in aqueous solvents. We choose 2 as a starting
material because it can be easily made from a bromomethylated zinc porphyrin
precursor32 by a typical malonate ester alkylation protocol. By virtue of its easy
demetalation with concentrated hydrochloric acid, 2 is transformed into the freebase
porphyrin 3, as shown in Figure 1. Subsequent reaction with ferrous chloride in methanol
using lutidine as a proton scavenger gives the neutral iron(III) porphyrin tetramalonic
ester system, 4. 1H NMR and UV-vis spectroscopic data show that 4 is a paramagnetic
iron(III) porphyrin with a single chloro axial ligand. The two β-pyrrole 1H resonances at
82.9 and 80.8 ppm (half-width ∼ 300 Hz) indicate the overall C2V symmetry of the
porphyrin and prove the S = 5/2 spin state.33,34 Saponification of the malonic esters of 4
with sodium hydroxide in ethanol leads to precipitation of a brownish material that is
soluble in water but totally insoluble in apolar solvents. Gel permeation chromatography
(Sephadex LH20) in methanol and subsequent precipitation with diethyl ether gave a solid
material containing 75% of 1 (with the impurities being NaOH and H2O). Standard NMR
spectra measured in unbuffered D2O ([1] = 1.5 × 10-2 M, pD = 13.4) have shown that
- 17 -
Octa-Anionic FeIII-Porphyrin Complex
under these conditions 1 exists as a high-spin monohydroxo complex (P8-)FeIIIOH (βpyrrole 1H at 82.7 ppm; see also further text).
To visualize the structure of the porphyrin ligand in 1, the crystal structure of its zinc
precursor 2 crystallized from THF is shown in Figure 2. As can be seen from the structure,
the zinc atom lies in the N4 porphyrin plane (average Zn-N distance, 2.053 Å) and
interacts weakly with oxygen atoms of the two axially bound THF molecules (Zn-O
distance, 2.419 Å).
Figure 2. ORTEP diagram of [(P)Zn(THF)2] (2-THF) visualizing structural features of
the porphyrin ligand in 2. General crystallographic data for 2-THF are reported in Table
A1 of Appendix.
Two structural features important in terms of the investigations presented in this report
can be extracted from this structure. First, the malonate groups are located above and
below the porphyrin plane, thus decreasing the possibility of μ-oxo-dimer formation for
steric and electrostatic reasons (as confirmed by in-depth spectroscopic studies described
below). Second, the malonate groups cannot coordinate to the metal center in an
intramolecular fashion without extreme distortion of the molecule (the average distance of
the malonate oxygen atoms from the zinc atom, ∼7 Å; the closest distance, ∼4 Å).
- 18 -
Octa-Anionic FeIII-Porphyrin Complex
Nevertheless, ester or carboxylate groups of malonate substituents may interact with axial
ligands coordinated to the metal center, as visualized in Figure 3 for the diaqua-ligated
porphyrin (P8–)FeIII(H2O)2.
Figure 3. Visualization of possible through-space interactions (hydrogen bonds, d(OH2 −
-
OOC) = 2.33 Å) between coordinated water and carboxylic groups of flexible malonato
substituents in (P8–)FeIII(H2O)2. Models shown are based on a PM3 minimization of the
zinc porphyrin 2 with Spartan’02, Wavefunction Inc., 18401 Von Karman Avenue, Suite
370, Irvine, CA 92612. The two t-butylphenyl meso substituents of the porphyrin ligand
are omitted for clarity.
2.4.2. Speciation of (P8-)FeIII(OH) as a Function of pH.
To establish the nature of the iron porphyrin species present in aqueous solution as
a function of pH, a spectrophotometric titration in the pH 1 – 12 range and 1H NMR
measurements at selected pD values (7 and 11) were performed for 1. Figure 4 shows the
UV-vis spectral changes observed during the spectrophotometric titration of 1 at 0.1 M
ionic strength (adjusted with NaClO4). The corresponding plot of absorbance at 418 nm
versus pH is shown in the inset of Figure 4b. As can be seen from these data, the observed
spectral changes can be separated into two regions. In the pH 1 – 6 range, a continuous
increase in absorbance over the whole spectral range is observed. This results mainly
from an increase in the solubility of the porphyrin complex due to deprotonation of the
carboxylic acid groups upon increasing pH. A concomitant gradual shift of the broad band
- 19 -
Octa-Anionic FeIII-Porphyrin Complex
centered at ca. 420 nm to shorter wavelengths leads to the formation of a Soret band at
400 nm (ε = 9.8 × 104 M-1 cm-1) at pH > 5. Further spectral changes, accompanied by
clean isosbestic points at 360, 438, 514, and 555 nm, are observed in the pH 8 – 11 range.
1,0
1,2
(b)
(a)
1.2
1,0
1.0
Abs (418nm)
0,8
0,6
Absorbance
Absorbance
0,8
0,4
0.8
0.6
0.4
0,6
0.2
0
2
4
6
8
10
12
14
pH
0,4
0,2
0,2
0,0
300
400
500
600
700
0,0
300
400
500
600
700
800
Wavelength, nm
Wavelength, nm
Figure 4. UV-vis spectral changes observed for aqueous solutions of (P8–)FeIII (1) in the
pH range 1 – 6 (a) and 6 – 12 (b). Inset: plot of absorbance at 418 nm versus pH.
Experimental conditions: [1] = 1 × 10-5 M, 25 oC, I = 0.1 M (adjusted with NaClO4)
The nature of these spectral changes, i.e., shift of the Soret band to 417 nm (ε = 1.1 × 105
M-1 cm-1) and formation of peaks at 334 and 532 nm (ε = 1.2 × 104 M-1 cm-1), is
analogous to that observed for other water-soluble iron(III) porphyrins on deprotonation
of coordinated water at the Fe(III) center.35-39 The spectral features observed at high pH
are typical for that reported for monomeric monohydroxo-coordinated iron(III)
porphyrins in organic33,34 and aqueous35-39 solvents. No further spectral changes occurred
in solution at high pH on extended standing, and those observed in the pH 8 – 11 range
were fully reversible and indicated rapid interconversion of species present at high and
low pH upon changing of the pH. These observations strongly suggest that the formation
and hydrolysis of μ-oxo dimers do not occur in the studied system, as was further
confirmed by NMR data reported below.
- 20 -
Octa-Anionic FeIII-Porphyrin Complex
The pKa1 values characterizing the acid-base equilibria in the studied system were
determined by Specfit40 analysis of the UV-vis spectra recorded in the pH 1 – 12 range.
To minimize experimental errors resulting from changes in absorbance due to the
observed precipitation of protonated (H8P)FeIII in the pH 1 – 5 range, the results of four
independent titrations were subjected to the Specfit analysis. The pKa values of 2.9 ± 0.8
and 4.4 ± 0.6 estimated for the low pH range41a are ascribed to deprotonation of the
carboxylic acid groups on the porphyrin (compare simplified eq 5) on the basis of their
similarity with the pKa1 of carboxylic groups in benzylmalonic acid (2.56 and 5.22),41b
and the observed changes in the solubility of 1 in this pH range.
(H8P)FeIII
(P8–)FeIII + 8 H+
2.5 < pKa(RCOOH) < 5.2
(5)
The pKa1 value of 9.26 ± 0.01 determined at higher pH is ascribed to deprotonation of a
water molecule in the (P8–)FeIII(H2O)2 (1-H2O) species present in solution at pH < 9,
according to eq 6. This conclusion is further supported by 1H NMR data and 17O NMR
(P8–)FeIII(H2O)2
(P8–)FeIII(OH) + H+
pKa1
(6)
water-exchange measurements performed for 1 at pD 7 and 11. According to numerous
literature reports, 1H NMR spectroscopy is a widely recognized method to characterize
(P)Fe(L)n (n = 1 and 2) complexes in solution.16-19,24,42-45 The hyperfine shift patterns
observed for paramagnetic iron porphyrins were shown to strongly depend on the spin
and ligation states of the central iron atom. In particular, the chemical shift of the βpyrrole protons in iron(III) porphyrins has proven to be an excellent probe to determine
the spin state of the iron(III) center.16-19,24,42-45 In the case of pure high-spin (S = 5/2)
iron(III) porphyrins, the β-pyrrole proton signals are shifted downfield to δ ≥ 80 ppm at
25°C.18,19,33,34 In contrast, the pure intermediate spin state (S = 3/2) complexes exhibit βpyrrole proton signals at extremely upfield positions, i.e., at ca. –60 ppm.18 In the case of
admixed intermediate spin state complexes (S = 5/2, 3/2), β-pyrrole resonances appear
between these two extremes, viz., +80 and −60 ppm.
- 21 -
Octa-Anionic FeIII-Porphyrin Complex
Table 1. β-pyrrole 1H NMR Chemical Shiftsa and pKa1 Values of Synthetic WaterSoluble Iron(III) Porphyrins
Iron(III)
porphyrin
meso phenyl
substituent
O
-O
8-
(P )Fe
β-pyrrole 1H (ppm)a
pKa1b
(P)Fe(H2O)2
(P)Fe(OH)
45.6;
46.7d
82.7
9.3c
NA
NA
8.0
52.4
NAg
7.0
45
80
7.0
43
82
6.9
70.5
NA
5.5
73.5
84
5.1
O
O-
c
O-
-O
O
O
(TanP4-)Fe e
O
SO3HN
(TPPS4-)Fe f
SO3-
Me
SO3-
(DMPS4-)Fe h
4-
(TMPS )Fe
Me
Me
i
SO3Me
Me
(4-TMPyP4+)Fe f
+
N Me
Me
4+
(2-TMPyP )Fe
j
+
N
a
Referenced to TMPS, NA – not assigned. b Values from ref. 53 unless
otherwise stated. cThis work. d Two separate resonances result from chemical
inequivalency of β-pyrrole protons in P8-. e Ref. 7a f Ref. 44. g The small peak
at 33.4 ppm assigned to hydroxo form of (TPPS)FeIII in ref. 44 is probably
shifted upfield from the typical value of ca. 80 ppm due to the exchange of
diaqua, monohydroxo and μ-oxo dimer present in solution at pD 5-6. h
Reference 35. i Reference 27. j Reference 43 .
- 22 -
Octa-Anionic FeIII-Porphyrin Complex
As can be seen from the data summarized in Table 1, the β-pyrrole shifts observed
for selected water-soluble iron(III) porphyrins fall in the range of 43 – 85 ppm. Chemical
shifts of δ ≥ 80 ppm, diagnostic for monomeric monohydroxo-ligated iron porphyrins in
aqueous35,42,43 and nonaqueous18,33,34 solvents, clearly indicate that these complexes exist
in solution as purely high-spin species. The more upfield signals (43 – 73 ppm) observed
for diaqua-ligated forms35,43,44 reflect a varying contribution of S = 3/2 in the admixed S =
3
/2, 5/2 spin system featuring (P)FeIII(H2O)2, in which the axial sites are occupied by two
weak field H2O ligands (see also the text below). The position of the β-pyrrole proton
resonances at ca. 47 ppm for (P8–)Fe at pH 7 (compare Table 1 and Figure 5) is similar to
that observed for the diaqua forms of other negatively charged water-soluble iron(III)
porphyrins and differs remarkably from that observed for the monohydroxo species. A
peak at ca. 83 ppm (Table 1 and Figure 5b) appears, however, in the 1H NMR spectrum of
(P8-)Fe at pD 11, indicating the presence of the monohydroxo-coordinated form. In
addition, the lack of resonances in the spectral region of 13–14 ppm (characteristic for βpyrrole protons in μ-oxo-bridged dimers18,34,44), shows unequivocally that the studied
porphyrin complex does not undergo dimerization to a μ-oxo dimer at high pH.
a)
m
β−Py
85
b)
80
75
70
65
60
55
50
45
40
35
30
25
20
15
(ppm)
10
m
β−Py
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
(ppm)
Figure 5. (a) The β-pyrrole (β-Py, 44.7, 46.3 ppm) and aryl meta 1H (m, 9.5, 10,2 ppm)
resonances observed in the 1H NMR spectrum of (P8–)FeIII(H2O)2 (10 mM, pD 7) (b) the
corresponding spectrum recorded for (P8–)FeIII(OH) at pD 11 (β-Py: 82 ppm, m: 11.5,
11.8 ppm).
- 23 -
Octa-Anionic FeIII-Porphyrin Complex
Temperature- and pressure-dependent 17O NMR studies performed at pH 7 (compare
the Experimental Section) indicated that the (P8–)Fe(H2O)2 form (1-H2O) present at this
pH undergoes a rapid water-exchange reaction. As can be seen from data presented in
Table 2, the rate constant measured for this process (kex = 7.7 × 106 s-1 at 25 °C) falls
within the range of kex values reported for diaqua-ligated forms of other water soluble
iron(III) porphyrins. Significantly positive ΔS‡ex and ΔV‡ex observed for 1-H2O as well as
for other diaqua-ligated porphyrins indicate the operation of a dissociative (Id or D)
pathway for water exchange on (P)FeIII(H2O)2 complexes studied to date.
Table 2. Rate Constants (at 25 °C) and Activation Parameters for Water-Exchange
Reactions on a Series of Water-Soluble Iron(III) Porphyrins
a
δβ-pyrrole Int%
Iron(III) porphyrin
(ppm)
1-H2O b
(TMPS4-)Fe(H2O)2c
(TPPS4-)Fe(H2O)2 c
4+
c
(TMPyP )Fe(H2O)
a
kex ×10-6
(s-1)
ΔH
‡
ex
‡
ΔS
ex
‡
ΔV
ex
(kJ mol-1) (J mol-1 K-1) (cm3 mol-1)
ca. 46
43
24
26
7.7 ± 0.1
21 ± 1
61 ± 6
61 ± 1
+91 ± 23
+100 ± 5
+7.4 ± 0.4
+11.9±0.3
52
20
2.0 ± 0.1
67 ± 2
+99 ± 10
+7.9 ± 0.2
70.5
6.8
0.45 ± 0.10
71 ± 2
+100 ± 6
+7.4 ± 0.4
Calculated according to eq 7. b This work. c Reference 14.
In contrast to this observation, variable temperature 17O NMR measurements performed
for 1 at pH 11 indicated very small variation of the reduced relaxation time for the bulk
water signal. This strongly suggests that the monohydroxo-ligated form of 1 exists in the
five-coordinate form (P8–)FeIII(OH) [possibly in equilibrium with a minor fraction of the
six-coordinate (P8–)FeIII(OH)(H2O) species]. It is concluded on the basis of the UV-vis,
1
H NMR, and 17O NMR data reported above that the studied porphyrin complex exists as
1-H2O in the pH 5 – 8 range and forms the monomeric monohydroxo species (P8–
)FeIII(OH) as a predominant porphyrin form at pH > 9. No evidence for the formation of a
- 24 -
Octa-Anionic FeIII-Porphyrin Complex
dihydroxo species, (P8-)Fe(OH)2, or μ-oxo bridged dimers was obtained in the studied pH
range, i.e., pH < 13.
As can be seen from data summarized in Table 1, the pKa1 determined for 1-H2O is
higher than the reported pKa1 values characterizing deprotonation of a water molecule in
other water-soluble (P)FeIII(H2O)2 porphyrins. Such a high pKa1 apparently reflects the
electronic effects of the negatively charged meso substituents that increase the electron
density on the metal center (and thus disfavor the release of a proton from coordinated
water), as well as through-space interactions of RCOO– groups with the axially
coordinated water molecules. In the latter case, the formation of hydrogen bonds with
deprotonated carboxylate groups of flexible malonate substituents (compare Figure 3) is
expected to stabilize coordinated water and the local 1+ charge of the [FeIII(Np)4]+ unit,
thus increasing the pKa1 value. A similar influence of through-space interactions with
negatively charged SO3– groups increasing the pKa1 value of coordinated water has been
reported for another negatively charged water-soluble ferric porphyrin, viz., (TanP4–)FeIII(H2O)2.7a
An interesting observation that can be added on the basis of the data reported in
Tables 1 and 2 is that the lability of water coordinated to the iron(III) center apparently
correlates with the contribution of the intermediate S = 3/2 spin state in the admixed S = 5/2,
3
/2 spin system of (P)FeIII(H2O)2 complexes. This contribution can be estimated for
iron(III) complexes of meso-tetraarylporphyrins on the basis of the β-pyrrole proton
chemical shift (δ) according to eq 7.19
Int% = [(80 - δ)/140] x 100 (%)
(7)
The Int% values calculated from eq 7 for water-soluble porphyrins reported in Table 2
clearly show that an increasing contribution of the S = 3/2 state correlates with an
increasing labilization of the axial water molecules, as indicated by higher rates of water
exchange observed for these complexes. Taking into account that the strong tetragonal
distortion resulting in long axial bonds is the representative feature of the S = 3/2 spin state
in iron(III) porphyrins, the increasing lability of axial H2O apparently reflects the
- 25 -
Octa-Anionic FeIII-Porphyrin Complex
lengthening of the Fe–OH2 bond in spin-admixed S = 5/2, 3/2 porphyrins with increasing
contribution of the S = 3/2 spin state. It has been shown that population of the S = 3/2 spin
state results from destabilization of the dx2–y2 orbital; i.e., as the energy of the dx2–y2 orbital
increases, the ground state in iron(III) porphyrins with weak field ligands (such as H2O)
changes from mainly S = 5/2 to mainly S = 3/2. Hence, the larger contribution of the S = 3/2
state in negatively charged porphyrins, in comparison to that observed for (Pn+)Fe(H2O)2,
apparently reflects destabilization of the dx2–y2 orbital by repulsive interactions with the
increased electron density on the pyrrole nitrogens. A similar effect has been reported for
water-insoluble ferric porphyrins with meso substituents of varying electron-releasing
ability.16 It can be expected that the trend of increasing lability of axial positions observed
for water-soluble porphyrins included in Table 2 will also be reflected in their reactivity
toward NO. In fact, a correlation of the S = 3/2 spin admixture and the rates of NO binding
exists for diaqua-ligated (P)FeIII(H2O)2, as discussed in detail in the following section.
2.4.3. Reactivity of (P8-)Fe(H2O)2 (1-H2O) toward NO.
The addition of NO gas to a deoxygenated solution of 1-H2O at pH < 9 resulted
in the spectral changes presented in Figure 6. The decrease in the absorbance at 400 nm
accompanied by the appearance of new bands at 427 (Soret, ε = 1.5 × 105 M-1 cm-1) and
540 nm indicates the formation of a typical low-spin iron(III) porphinatonitrosyl
complex4,6 in which the formal charge distribution can be described as (P8–
)FeII(NO+)(H2O) (eq 8).
8–
III
(P )Fe (H2O)2 + NO
kon
koff
(P8–)FeII(NO+)(H2O) + H2O
KNO = kon/koff
(8)
Bubbling of an inert gas through the resulting solution leads to reversed spectral changes
indicating reversibility of the reaction, in agreement with eq 8.
- 26 -
Octa-Anionic FeIII-Porphyrin Complex
1,0
45
0,9
40
35
0,8
30
Absorbance
0,7
(Α−Α ο)
-1
25
20
15
0,6
10
0,5
5
0
0
0,4
20
40
60
80
100
120
140
-1
[NO] , 1/mM
160
180
200
0,3
0,2
0,1
0,0
300
400
500
600
700
800
Wavelength, nm
Figure 6. Spectral changes resulting from NO binding to (P8–)FeIII(H2O)2. Inset: plot of
(A-A0)-1 versus [NO]-1 (where A0 – absorbance at 400 nm at [NO] = 0, A – absorbance at
400 nm at a given NO concentration10). Experimental conditions: pH = 7.0 (0.05 M BisTris), I = 0.1 M (NaClO4), 22 oC
The combination of UV-vis spectroscopy and amperometric detection of free NO in
solution allowed the determination of the thermodynamic equilibrium constant KNO = (1.5
± 0.2) × 104 M-1 (compare the inset in Figure 6).
The kinetics of the reversible binding of NO to 1-H2O at pH 7 was studied by laser
flash photolysis and stopped-flow techniques for the “on” and “off” reactions,
respectively. Laser flash photolysis of (P8–)FeII(NO+)(H2O) solutions resulted in transient
spectral changes consistent with the spectral difference between (P8–)FeII(NO+)(H2O) and
(P8–)FeIII(H2O)2. In the presence of excess NO, the transient spectrum decayed
exponentially to the initial one. Thus, the processes occurring in the laser flash
experiment can be summarized as follows.
hv
H2O + (P8–)FeII(NO+)(H2O)
kon
koff
- 27 -
(P8–)FeIII(H2O)2 + NO
(9)
Octa-Anionic FeIII-Porphyrin Complex
The kinetics of the reaction was followed under pseudo first-order conditions with at
least a 10-fold excess of NO. As can be expected for the reversible process (eq 9), the kobs
values determined under such conditions by fitting the kinetic traces to a singleexponential function depend linearly on [NO] according to eq 10 (see Figure 7a).
kobs = kon[NO] + koff
(10)
A linear fit of kobs versus [NO] obtained at 24 °C allowed the determination of kon = (8.2 ±
0.1) × 105 M-1 s-1 and koff = 217 ± 16 s-1 from the slope and intercept, respectively. The
overall equilibrium constant calculated from the kinetic data, KNO = kon/koff = (3.8 ± 0.2) ×
103 M-1 (24 oC), is in reasonable agreement with the corresponding thermodynamic value
of Keq determined from a combination of UV-vis and electrochemical measurements. To
determine the activation parameters ΔS‡, ΔH‡, and ΔV‡ for the binding and release of NO,
the kinetics was studied at different temperatures (6 – 30 °C) and hydrostatic pressures
(0.1 – 170 MPa). The kon and koff values determined from linear dependences of kobs
versus [NO] at each temperature (Figure 7a and b) and pressure (Figure 8a and b) allowed
the construction of Eyring plots for the “on” and “off” reactions and a linear plot of ln(kon)
versus pressure. Activation parameters calculated from the plots are summarized in Table
3.
3200
3000
2800
9
o
30 C
(a)
(b)
8
2600
2400
kon
2200
7
o
2000
24 C
ln(kon/T)
kobs, s
-1
1800
1600
1400
o
18 C
1200
1000
1,5
0,0
o
12 C
800
600
-1,5
o
6C
400
koff
-3,0
200
0
0,0000
0,0004
0,0008
0,0012
0,0016
0,0020
-4,5
0,00320
[NO], M
0,00328
0,00336
0,00344
1/T
- 28 -
0,00352
0,00360
0,00368
Octa-Anionic FeIII-Porphyrin Complex
Figure 7. (a) Plots of kobs versus [NO] for the reaction of 1-H2O with nitric oxide in the
temperature range 6 – 30 °C measured by laser flash photolysis. (b) The corresponding
Eyring plots for the “on” and “off” reactions. Experimental conditions: [1-H2O] = 2.0 ×
10-5 M, pH 7.0 (0.05 M Bis-Tris), λirr = 355 nm, λdet = 417 nm, I = 0.1 M (NaClO4)
12,7
600
(a)
500
400
10 MPa
50 MPa
90 MPa
130 MPa
170 MPa
(b)
12,6
12,5
ln kon
kobs, s
-1
12,4
300
12,3
200
12,2
100
12,1
0
0,0000 0,0002 0,0004 0,0006 0,0008 0,0010 0,0012 0,0014 0,0016 0,0018 0,0020
12,0
0
20
40
60
80
100
120
140
160
180
200
Pressure, MPa
[NO], M
Figure 8. (a) Plots of kobs versus [NO] for the reaction of 1-H2O with NO in the pressure
range 10 – 170 MPa. (b) Plot of ln(kon) versus pressure. Experimental conditions: [1-H2O]
= 2.0 ×10-5 M, pH 7.0 (Bis-Tris, 0.05 M), λirr = 355 nm, λdet = 417 nm, I = 0.1 M
(NaClO4)
Because of the small intercepts in the plots of kobs vs [NO] in the pressure-dependent
study, the activation volume for the “off” reaction could not be determined accurately in
this way. This value, however, could be measured in a stopped-flow experiment using the
NO-trapping method. Rapid mixing of a (P8–)FeII(NO+)(H2O) solution with a large excess
of [Ru(edta)(H2O)]– (an efficient scavenger for NO)46 led to re-formation of the (P8–
)FeIII(H2O)2 complex, as indicated by the observed spectral change. The kinetics of the
reaction was followed at 427 nm. The kinetic traces gave good mathematical fits to a
single-exponential function, and the observed reaction rates did not depend on the
- 29 -
Octa-Anionic FeIII-Porphyrin Complex
[Ru(edta)(H2O)]– in the concentration range used in the NO-trapping experiments (1–2
mM). These observations indicate that the release of NO from (P8–)FeII(NO+)(H2O)
follows the reaction sequence in Scheme 1, in which koff is the rate-determining step, such
that the kobs values determined from the kinetic traces equal koff.
Scheme 1
(P8-)FeII(NO+)(H2O) + H2O
koff
kon
(P8-)FeIII(H2O)2 + NO
fast [RuIII(edta)(H2O)][RuIII(edta)(NO)]- + H2O
As can be seen from the data in Table 3, the rate constants obtained from variabletemperature NO-trapping measurements are in reasonable agreement with those resulting
from the laser flash photolysis experiments. Small deviations of the koff values obtained
by these two techniques were, however, observed, particularly at higher temperatures (a
similar tendency was also observed in measurements at pH 11; see further text). This
presumably reflects a diminished efficiency of [RuIII(edta)(H2O)]– as the NO trap at pH >
6 because of its deprotonation to a less reactive [RuIII(edta)(OH)]2- form.46 Nevertheless,
the similarity of the activation parameters ΔH‡off and ΔS‡off determined from laser flash
photolysis and NO-trapping studies (Table 3) indicates that the NO-trapping method
provides a reliable mechanistic description of the “off” reaction at pH 7. This technique
was therefore used to determine ΔV‡ for the “off” reaction. The stopped-flow NOtrapping measurements performed in the pressure range of 0.1–130 MPa allowed the
determination of ΔV‡off = +16.8 ± 0.4 cm3 mol-1.
- 30 -
Octa-Anionic FeIII-Porphyrin Complex
Table 3. Rate Constants and Activation Parameters Determined by Laser Fash Photolysis
and Stopped-Flow (NO-Trapping Method) Techniques for the Reversible Binding of NO
to (P8-)FeIII(H2O)2 at pH 7.0
temp
pressure
(o C)
(MPa)
6
10
12
18
24
30
10
10
50
90
130
170
ΔH‡, (kJ mol-1)
ΔS‡, (J mol-1 K1
)
‡
ΔV , (cm3 mol-1)
koff
(M-1 s-1)
(s-1)
koffa
2.0 ± 0.1
15.0 ± 1.5
3.4 ± 0.1
5.3 ± 0.2
8.2 ± 0.1
12.7 ± 0.1
34 ± 2
96 ± 28
217 ± 16
494 ± 24
10.5 ± 0.5
23.0 ± 0.5
31.0 ± 0.4
81 ± 2
220 ± 2
432 ± 12
2.7 ± 0.1
2.4 ± 0.1
2.2 ± 0.1
1.9 ± 0.1
1.7 ± 0.1
8.4 ± 0.2b
6.7 ± 0.1b
4.8 ± 0.3b
3.5 ± 0.2b
kon × 10-5
51.1 ± 0.5
+40 ± 2
+6.1 ± 0.1
101 ± 2
+140 ± 7
−
(s-1)
107 ± 3
+160 ± 10
+16.8 ± 0.4
a
Data obtained by NO-trapping method using [RuIII(edta)(H2O)]–
(unbuffered solution, pH 7) b Data obtained by NO-trapping
method at 3 °C
A comparison of the rate and activation parameters for the binding and release of
NO obtained for 1-H2O with those reported in the literature for other water-soluble ferric
porphyrins (compare Table 4) shows that the reactivity of the diaqa-ligated 1-H2O toward
NO is similar to that observed for other (Pn-)Fe(H2O)2 complexes. Although the “on” and
“off” rate constants reported for (TMPyP4+)FeIII(H2O)2 with positively charged meso
substituents are markedly smaller in comparison to those observed for (Pn-)FeIII(H2O)2,
the similarity of the activation parameters (in particular, ΔS‡ and ΔV‡) shows that the
- 31 -
Octa-Anionic FeIII-Porphyrin Complex
mechanistic features of the binding and release of NO are analogous for both positively
and negatively charged (P)FeIII(H2O)2 species. Thus, the volume profile constructed from
the activation volumes, ΔV‡on and ΔV‡off, obtained for 1-H2O (Figure 9) reflects a
common reaction mechanism for the diaqua forms of water-soluble porphyrins, in which
the “on” reaction is controlled by substitution of a water molecule according to a
dissociative (Id or D)47 mechanism, as evidenced by the positive values of ΔV‡on.4,6
Table 4. Comparison of Rate Constants and Activation Parameters for Binding and
Release of NO for Water-Soluble Diaqua-Ligated Iron(III) Porphyrins
Iron(III) porphyrin
Int %
k at 25 °C
ΔH‡
ΔS‡
ΔV‡
(kJ mol-1)
(J mol-1 K-1)
(cm3 mol-1)
kon, M-1 s-1
(P8-)Fe(H2O)2
24 %
8.2 × 105
51 ± 1
+40 ± 2
+6.1 ± 0.1
(TMPS4-)Fe(H2O)2 a
26 %
3.8 × 106
57 ± 3
+69 ± 11
+13 ± 1
(TPPS4-)Fe(H2O)2 a
20 %
5 × 105
69 ± 3
+95 ± 10
+9 ± 1
(TMPyP4+)Fe(H2O)2 b
7%
2.9 × 104
67 ± 3
+67 ± 11
+4 ± 1
koff, s-1
(P8-)Fe(H2O)2
24 %
217
101 ± 2
+140 ± 7
+17 ± 2
(TMPS4-)Fe(H2O)2 a
26 %
900
84 ± 3
+94 ± 10
+17 ± 3
(TPPS4-)Fe(H2O)2 a
20 %
500
76 ± 6
+60 ± 11
+18 ± 2
(TMPyP4+)Fe(H2O)2 b
7%
66
113 ± 5
+169 ± 18
+16.6 ± 0.2
a
Data from ref. 4 b Data from ref. 6
The rate-determining substitution step is followed by a large volume collapse due to a
spin state change and solvent reorganization accompanying the formation of the low-spin
(P)FeII(NO+)(H2O) product. The large positive volumes of activation typical for the “off”
- 32 -
Octa-Anionic FeIII-Porphyrin Complex
reaction clearly indicate that the release of NO also follows a dissociative mechanism.
The rate-determining breakage of the FeII–NO+ bond (resulting in a positive contribution
to ΔV‡off) is in this case accompanied by a further volume increase due to formal oxidation
(FeII → FeIII) and spin-state change (S = 0 → S = 5/2, 3/2) on the iron(III) center, as well as
solvent reorganization due to neutralization of the partial charge on the FeII–NO+ unit.
Partial molar volume, cm3 mol-1
OH2
NO ‡
FeII
OH2
OH2
FeIII
OH2
+ NO + 6.1 ± 0.1
+ 16.8 ± 0.4
- 10.7 ± 0.5
Reactants
Transition state
NO+
FeII
+ H2O
OH2
Products
Reaction coordinate
Figure 9. Volume profile for the reversible binding of nitric oxide to (P8–)FeIII(H2O)2
It can also be seen from the data in Table 4 that the increase in the rate of NO
binding correlates directly with the rising contribution of the intermediate S = 3/2 spin
state (Int %) in the admixed S = 5/2, 3/2 spin system of the (P)-FeIII(H2O)2 complexes, as
previously observed for the water exchange rates. This provides additional evidence that
the lability of the axial water controls to a large extent the kinetics of NO binding. It can
be further concluded from data in Table 4 that the rate of NO release from a (P)FeII(H2O)(NO+) complex tends to increase with increasing electron-donating influence of
meso-porphyrin substituents [reflected by the rising contribution of S = 3/2 in the spin
admixed state of (P)FeIII(H2O)2]. Although the correlation of koff and Int % is not as clear
- 33 -
Octa-Anionic FeIII-Porphyrin Complex
as in the case of the NO binding rates, it suggests that the FeII-NO+ bond in (P)FeII(NO+)(H2O) is better stabilized by the positively charged porphyrins than by the
negatively charged ones. This is in line with recent literature reports that, based on
resonance Raman and density functional theory (DFT) results, indicate that a gradual
increase of the electron density within the Fe-NO unit induced by meso substituents
destabilizes the Fe−NO bond in the porphyrin {FeNO}6 nitrosyls.48
2.4.4. Reactivity of Monohydroxo-Ligated (P8–)Fe(OH) (1–OH) toward NO.
Spectroscopic and kinetic studies on the reaction of 1 with NO performed at pH >
9 indicated differences in the kinetics of NO binding and release, as well as in the nature
of the nitrosyl species formed at high pH in comparison to that observed at pH < 9. The
spectral changes that accompany the reaction of 1-OH with NO at pH 11 (Figure 10a)
lead to a final spectrum in which the characteristic peaks at 317, 432 (Soret, ε = 1.9 × 105
M-1 cm-1), and 544 nm occur at longer wavelengths as compared to those observed at
lower pH (viz., 310, 427, and 540 nm). The spectral changes reported in Figure 10b show
a gradual change in the spectrum of the nitrosyl product obtained by the reaction of the
ferric porphyrin with NO in buffered aqueous solutions as a function of pH in the range
7–11. A fit of the plot of ΔAbs434 vs pH (inset in Figure 10b) allowed the determination of
pKa(NO) = 9.1 for the pH-dependent equilibrium associated with the observed spectral
change.
1,2
1,8
1,6
1,0
(b)
(a)
0,9
Abs(434 nm)
1,0
1,4
1,2
0,8
pKa = 9.1
0,8
0,7
Absorbance
Absorbance
0,6
1,0
0,8
0,6
0,5
7
0,6
8
9
10
11
pH
0,4
0,4
0,2
0,2
0,0
250
0,0
300
350
400
450
500
550
600
650
700
300
350
400
450
500
550
Wavelength, nm
Wavelength, nm
- 34 -
600
650
700
Octa-Anionic FeIII-Porphyrin Complex
Figure 10. (a) Spectral change accompanying the reaction of 1-OH with NO at pH 11; (b)
Spectra of the product obtained in the reaction of 1 with NO in buffered aqueous solutions
in the pH range 7 – 11. Plot of ΔAbs434 versus pH is shown in the inset. Experimental
conditions: [1] ≈ 7 × 10-6 M, [NO] = 1 mM, I = 0.1 M (NaClO4); buffers used: pH 7.0 –
Bis-Tris (0.05 M), pH 7.5 - 8.8 TAPS (0.05 M), pH 9.1 borate (0.05 M), pH 9.5 – 11
CAPS (0.05 M)
This equilibrium is ascribed to deprotonation of a coordinated water molecule in (P8–
)FeII(NO+)(H2O) according to eq 11.
(P8–)FeII(NO+)(H2O)
(P8–)FeII(NO+)(OH) + H+
pKa(NO) = 9.1
(11)
Combined UV-vis and NO electrode measurements allowed the determination of
equilibrium constant KNO = (4.8 ± 0.5) × 103 M-1 for the reversible binding of NO to 1OH at pH 11. This value is similar to that derived kinetically from the rate constants kon =
5.1 × 104 M-1 s-1 and koff = 14.9 s-1 (24 °C) determined from the dependence of kobs on
[NO] at pH 11, viz., KNO = kon/koff = (3.4 ± 0.1) × 103 M-1. A comparison of kon and koff
measured at pH 11 with those obtained at pH 7 (kon = 8.2 × 105 M-1 s-1 and koff = 217 s-1)
shows that both coordination and release of NO decreases ca. 15 times at high pH. Figure
11 presents the pH dependence of kon and koff values determined from kobs versus [NO]
plots in the pH 6.5–11.5 range. It can be seen from these data that the rates of NO binding
and dissociation gradually decrease between pH 7.5 and 10.5 and become constant at pH
>11. The pKa value determined by fitting the data in Figure 11a to a sigmoidal function,
viz., 9.04 ± 0.01, is close to the pKa1 = 9.26 determined from a spectrophotometric
titration of 1, which characterizes deprotonation of a water molecule in 1-H2O (eq 6).
- 35 -
Octa-Anionic FeIII-Porphyrin Complex
300000
40
(a)
35
(b)
250000
30
-1
25
150000
koff, s
kon, M s
-1 -1
200000
20
15
100000
10
50000
5
0
0
6
7
8
9
10
11
12
7
pH
8
9
10
11
12
pH
Figure 11. pH dependence of the rate constants for binding (a) and release of NO (b)
from 1 determined from the slopes and intercepts of kobs versus [NO] plots measured in
buffered aqueous solutions in the pH range 6.5 – 11.5. Experimental conditions [1] = 2.0
×10-5 M, Temp = 10 oC, I = 0.1 M (NaClO4)
This indicates that the change in reactivity observed on increasing pH reflects differences
in the kinetics of NO binding to 1-H2O and 1-OH present in solution at low and high pH,
respectively. An analogous sigmoidal fit of the less precise data in Figure 11b
(constructed on the basis of koff values obtained by extrapolation of kobs versus [NO] plots
to [NO] = 0) resulted in pKa = 8.9 ± 0.1, which is also close to that characteristic for the
formation of the hydroxo-ligated porphyrin (eq 6), as well as that estimated for the
process depicted in eq 11 (pKa(NO) = 9.1). It is apparent from these data that the presence
of the hydroxo group decreases the rate of NO binding to (P8–)FeIII, as well as the rate of
NO release from (P8–)FeII(NO+)(OH). The observed reactivity pattern is summarized in
Scheme 2, in which the rate constants for 1-H2O and 1-OH species are denoted by kH2O
and kOH, respectively.
- 36 -
Octa-Anionic FeIII-Porphyrin Complex
Scheme 2
H O
2
8–
III
(P )Fe (H2O)2 + NO
H3O+
kon
H O
2
koff
(P8–)FeII(NO+)(H2O) + H2O
H+
pKa1 = 9.26
pKa(NO) = 9.1
OH
(P8–)FeIII(OH) + NO
kon
OH
koff
(P8–)FeII(NO+)(OH) + H2O
The observation that the rate of NO binding to (P8–)FeIII decreases on going from a
trans-diaqua to an hydroxo complex is surprising in view of kinetic data on the
substitution behavior of such metal complexes in solution, where in the majority of cases
an increase in the reactivity of the hydroxo form is observed as compared to the diaqua
species. Such a trend reflects the significant labilizing influence of the hydroxo group,
which facilitates substitution of the ligand coordinated in the trans position to OH–.49 The
reversed reactivity pattern observed for 1-OH indicates that binding of NO to 1-OH is no
longer controlled by the lability of the metal center (where, in such a case, a very fast
diffusion-controlled rate for NO coordination to this fivecoordinate iron(III) complex
would be expected). Apparently, other factors determine the rate of formation of the
nitrosylated species at high pH. This conclusion is further substantiated by a detailed
study of the reversible binding of NO to 1-OH at pH 11. Figures 12, Figure 13 and Table
5 report the results of stopped-flow experiments performed at different temperatures and
pressures for the reaction of 1-OH with NO at pH 11. As can be seen from the data
summarized in Table 5, the activation parameters determined for NO binding to 1-OH,
viz., ΔH‡on = 34.6 kJ mol-1, ΔS‡on = – 39 J mol-1 K-1, and ΔV‡on = – 6.1 cm3 mol-1, are very
different from that characterizing NO binding to the diaqua forms of water-soluble
porphyrins given in Table 4.
- 37 -
Octa-Anionic FeIII-Porphyrin Complex
26
100
(a)
o
30 C
(b)
24
130 MPa
90 MPa
50 MPa
10 MPa
22
80
20
o
24 C
18
16
kobs, s
kobs, s-1
-1
60
o
18 C
40
14
12
10
o
12 C
8
o
6
6C
20
4
2
0
0
0,0
-4
2,0x10
-4
-4
4,0x10
6,0x10
-4
-3
8,0x10
1,0x10
-4
0,0
2,0x10
-4
4,0x10
-4
-4
6,0x10
8,0x10
-3
1,0x10
-3
1,2x10
[NO], M
[NO], M
Figure 12. (a) Plots of kobs versus [NO] measured by stopped-flow method for the
reaction of 1-OH with NO in the temperature range 6 – 30 oC. (b) Plots of kobs versus
[NO] for the reversible binding of NO to (P8-)FeIII(OH) by high-pressure stopped flow
measurements in the pressure range 10 – 130 MPa. temp. 1.5 °C Experimental conditions:
[1-OH] = 2.0 × 10-5 M, pH 11 (0.05 M CAPS buffer), I = 0.1 M (NaClO4), λdet = 432 nm
12
8
(a)
6
(b)
11
kon
kon
10
4
9
ln(k)
ln(k/T)
2
0
8
-2
koff
-4
koff
-6
0
0,00330
0,00340
0,00350
0,00360
0
-1
20
40
60
80
100
120
140
Pressure, MPa
1/T, K
Figure 13. (a) Eyring plots of ln(k/T) versus 1/T obtained for the “on” and “off” reaction
on reversible binding of NO to 1-OH. (b) Plots of ln(k) versus pressure determined for the
same reaction. Experimental conditions: see Figure 12.
- 38 -
Octa-Anionic FeIII-Porphyrin Complex
Table 5. Rate Constants and Activation Parameters for Binding and Release of NO from
(P8-)FeIII(OH) at pH 11
kon × 10-4
koffa
koffb,c
6
1.9 ± 0.1
0.70 ± 0.02
0.86 ± 0.01
12
2.6 ± 0.1
2.2 ± 0.3
1.97 ± 0.02
18
3.7 ± 0.1
6.0 ± 0.5
5.1 ± 0.1
24
5.1 ± 0.2
14.9 ± 0.3
11.4 ± 0.3
30
6.7 ± 0.1
33.9 ± 0.7
23.7 ± 0.3
10
1.67 ± 0.06
2.7 ± 0.3
0.16 ± 0.01d
50
1.83 ± 0.03
2.4 ± 0.2
0.12 ± 0.01 d
90
2.06 ± 0.02
1.8 ± 0.1
0.09 ± 0.01 d
130
2.29 ± 0.05
1.1 ± 0.3
0.06 ± 0.01 d
34.6 ± 0.4
–39 ± 1
– 6.1 ± 0.2
107 ± 2
+136 ± 7
+17 ± 3
96 ± 1
+97 ± 4
+21.3 ± 0.4
temp
(oC)
1.5
pressure
(MPa)
ΔH‡, kJ mol-1
ΔS‡, J mol-1 K-1
ΔV‡, cm3 mol-1
(M-1 s-1)
(s-1)
(s-1)
a
Data obtained from intercepts of plots of kobs versus [NO]; b
Data obtained with the use of [RuIII(edta)(H2O)]- as the NO
scavenger. c Systematic deviations of the koff values from those
determined as intercepts of kobs versus [NO] plots point to a low
efficiency of NO trapping by [RuIII(edta)] at pH 11. Therefore,
the activation parameters obtained in alternative measurements
(Figure 13) are preferentially used for mechanistic
characterization of NO release at high pH. d Measured at 3oC.
In particular, negative values of ΔS‡on and ΔV‡on contrast the positive ones observed at low
pH and indicate a changeover in the mechanism of NO binding from dissociatively
activated (Id) in 1-H2O to associatively activated in 1-OH. This difference is particularly
evident on comparing the volume profile in Figure 9 with that constructed for the
reversible binding of NO to (P8–)FeIII(OH), as shown in Figure 14.
- 39 -
Partial molar volume, cm3 mol-1
Octa-Anionic FeIII-Porphyrin Complex
FeIII
OH
+ NO
- 6.1 ± 0.2
NO
‡
FeIII
OH
+ 17 ± 3
- 23 ± 3
NO+
FeII
Reactants
Transition
OH
Products
Reaction coordinate
Figure 14. Volume profile for the reversible binding of nitric oxide to (P8–)FeIII(OH)
Notably, the latter profile is very similar to that reported in the literature for reversible
binding of NO to the high-spin five-coordinate (P)FeIII(Cys) center in substrate-bound
Cytochrome P450cam.9a The activation volumes ΔV‡on = –7.3 cm3 mol-1 and ΔV‡off = +24
cm3 mol-1 determined for this ferric protein were interpreted in terms of the mechanistic
scheme outlined in eq 12, in which rapid formation of an encounter complex, {(P)FeIII ||
NO}, is followed by the activation step involving the formation of the Fe-NO bond.9a
(P)FeIII(Cys) + NO
kD
k-D
{(P)FeIII(Cys)||NO}
ka
(P)FeII(Cys)(NO+)
(12)
In the above scheme, kD and k-D represent the rate constants for the diffusion-limited
formation and dissociation of the encounter complex and ka is the rate constant for Fe-NO
bond formation. The relatively small negative value of ΔV≠on = –7.3 cm3 mol-1 observed
- 40 -
Octa-Anionic FeIII-Porphyrin Complex
for Cyt P450cam was ascribed to an activation-controlled mechanism with an “early”
transition state, in which only partial (P)FeIII-NO bond formation and a change in the spin
state of the FeIII center (S = 5/2 → S = 0) occurs on going from the encounter complex to
the transition state {(P)FeIII- - -NO}≠. A large positive ΔV≠off value of 24 cm3 mol-1
observed for the backward reaction evidenced a subsequent large volume collapse on
going from the transition state to the low-spin (P)FeII(NO+) product. The data obtained in
the present study for (P8-)FeIII(OH) strongly suggest an analogous reaction mechanism, as
summarized in Scheme 3. According to this scheme, initial partial formation of the FeNO bond in the transition state (accounting for the negative ΔV≠on = –6.1 cm3 mol-1) is
followed by volume collapse (ca. 17 cm3 mol-1) due to complete formation of the FeIINO+ bond, S = 5/2 → S = 0 spin change, and solvent contraction accompanying partial
charge transfer from NO to FeIII.
Scheme 3
kD
FeIII
OH
+ NO
k-D
NO
FeIII
|| NO
OH
ka
III
Fe
OH
‡
NO+
FeII
OH
The mechanistic difference observed in the binding of NO to 1-H2O and 1-OH is
further reflected in the kon values observed for the diaqua- and hydroxo-ligated species,
respectively. The ca. 15-fold decrease in the rate of NO binding to the five-coordinate (or
weakly six-coordinate) hydroxo form implies that the formation of (P8–)FeII(NO+)-(OH) is
not controlled by the lability of the FeIII center but rather by the enthalpy and entropy
changes associated with spin reorganization and structural rearrangements upon the
formation of the FeII-NO+ bond. This situation parallels that previously observed in the
reactions of five-coordinate Fe-(II) hemes with CO, for which an analogous mechanistic
scheme was suggested. In these earlier studies,4 considerably slower rates of CO
coordination to (P)FeII as compared to NO binding rates determined for the same
- 41 -
Octa-Anionic FeIII-Porphyrin Complex
complexes (which approached the diffusion limit in water) and an associative activated
mechanism were interpreted in terms of a significant activation barrier associated with
spin state/structural changes upon coordination of CO (which, however, was absent in the
reactions with NO). This conclusion was further substantiated by recent DFT
computations performed for the (P)Fe(II) + CO system.50 The decrease in kon observed in
the present case for NO coordination to the Fe(III) porphyrin complex 1 at high pH is
ascribed to a larger intrinsic activation barrier for the S = 5/2 → S = 0 spin state change,
which occurs upon the formation of (P8-)FeII(NO+)(OH) from the purely high-spin
hydroxo species 1-OH, as compared to that associated with the (S = 5/2, 3/2 → S = 0) spin
change occurring for the diaqua-ligated spin-admixed 1-H2O complex. As pointed out in
recent mechanistic studies on a spin-forbidden proton-transfer reaction in solution,51 such
an increased “intersystem barrier” can be related to a spin-forbidden transition itself
and/or to a considerable structural reorganization required to enable the spin change.
Consideration of characteristic structural features reported for five-coordinate high-spin,
six-coordinate spin-admixed, and low-spin (P)FeIII(L)n porphyrins, respectively, suggests
that rather minor structural changes are required for the formation of low-spin
(P)FeII(NO+)(H2O) from spin-admixed 1-H2O species, where in both complexes the iron
atom lies in the porphyrin plane, is six-coordinate, and exhibits short equatorial Fe-Np
bonds. In contrast, the binding of NO to 1-OH presumably involves a change in the
coordination number of the Fe(III) atom, its movement into the porphyrin plane, and
contraction of the Fe-Np equatorial bond; i.e., a considerable structural change (and thus
higher energy barrier) is expected to occur upon going from the substrate to product.
An additional contribution to the observed decrease in kon at high pH may arise from
a difference in the reducibility of the FeIII center in (P)FeIII(H2O)2 and (P)FeIII(OH). As
indicated by literature data, deprotonation of a water molecule in (P)FeIII(H2O)2 species
shifts the potentials of FeIII reduction to more negative values in comparison to that
observed for diaqua-ligated forms.52,53 Because the formation of the FeIII-NO bond is
accompanied by charge transfer from NO and formal reduction of the FeIII center, a
- 42 -
Octa-Anionic FeIII-Porphyrin Complex
decrease in the rate of this process can be expected for less easily reducible hydroxoligated species in comparison to the (P)-FeIII(H2O)2 counterparts.
In view of the fact that the spin changes (and the accompanying structural changes)
are clearly involved in the rate-determining step for the “off” reaction (i.e., breakage of
the FeII-NO+ bond) in both (P8–)FeII(NO+)(H2O) and (P8–)FeII(NO+)(OH), it is suggested
that the decreased reaction rate observed for the release of NO at high pH reflects a larger
demand for reorganization of d electrons upon re-formation of 1-OH from the
corresponding nitrosyl complex (i.e., S = 0 → S = 5/2) in comparison to that occurring for
(P8–)FeII(NO+)(H2O) (S = 0 → S = 5/2, 3/2).
Taken together, the kinetic and mechanistic data described above provide strong
evidence that the rate of NO binding and release observed for hydroxo-ligated species is
to a large extent controlled by the degree of spin reorganization occurring at the FeIII
center on going from reactants to products. Notably, the kon and koff rates determined in
the present study for 1-OH (viz., kon = 5.1 × 104 M-1 s-1 and koff = 14.9 s-1 at 24 °C) are
quite similar to those reported for the (TMPyP4+)Fe(H2O)2, which is almost purely high
spin (Int %) 7%, kon = 2.9 × 104 M-1 s-1, koff = 66 s-1 at 25 °C; compare Table 4), but
considerably smaller than those observed for (Pn-)Fe(H2O)2 species exhibiting a relatively
large contribution of the S = 3/2 spin state (ca. 26%), further supporting the above
conclusion. The observations on the influence of the spin and ligation states of the FeIII
center on the dynamics of the reaction with NO are important to understand kinetic and
mechanistic factors governing the interactions of NO with naturally occurring heme
proteins, in which a variety of spin and ligation states are observed. This stimulated
further in-depth mechanistic studies on the reversible binding of NO to model (P)FeIII(L)n
complexes with different types of porphyrin and axial ligands, which will be described in
a subsequent report.
- 43 -
Octa-Anionic FeIII-Porphyrin Complex
2.5. References and Notes
1. (a) Ford, P. C.; Lorkovic, I. M. Chem. Rev. 2002, 102, 993 and references cited t
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2. Hoshino, M.; Laverman, L.; Ford, P. C. Coord. Chem. Rev. 1999, 187, 75 and
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3. (a) Wyllie, G. R. A.; Scheidt, W. R. Chem. Rev. 2002, 102, 1067 and references cited
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124, 13833 and references cited therein.
4. Laverman, L. E.; Ford, P. C. J. Am. Chem. Soc. 2001, 123, 11614 and references cited
therein.
5. Wolak, M.; van Eldik, R. Coord. Chem. Rev. 2003, 230, 263 and references cited
therein.
6. Theodoridis, A.; van Eldik, R. J. Mol. Catal. A: Chem. 2004, 224, 197.
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Acta 2004, 357, 2503. (b) Nakagawa, S.; Yashiro, T.; Munakata, H.; Imai, H.; Uemori,
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8. Hoshino, M.; Maeda, M.; Konishi, R.; Seki, H.; Ford, P. C. J. Am. Chem. Soc. 1996,
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Am. Chem. Soc. 2001, 123, 285.
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Octa-Anionic FeIII-Porphyrin Complex
16. Toney, G. E.; Gold, A.; Savrin, J.; Haar, L. W.; Sangaiah, R.; Hatfield, W. E. Inorg.
Chem. 1984, 23, 4350.
17. Nesset, M. J. M.; Cai, S.; Shokhireva, T. K.; Shokhirev, N. V.; Jacobson, S. E.;
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Inorg. Chem. 1989, 28, 1591.
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Chem. Soc. 2005, 127, 5360.
27. Wolak, M.; van Eldik, R. J. Am. Chem. Soc. 2005, 127, 13312.
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- 45 -
Octa-Anionic FeIII-Porphyrin Complex
31. (a) Swift, T. J.; Connick, R. E. J. Chem. Phys. 1962, 37, 307. (b) Swift, T. J.; Connick,
R. E. J. Chem. Phys. 1964, 41, 2553. (c) Bloembergen, N. J. J. Chem. Phys. 1957, 27,
595. (d) Newman, K. E.; Meyer, F. K.; Merbach, A. E. J. Am. Chem. Soc. 1979, 101,
1470.
32. Jux, N. Org. Lett. 2000, 2, 2129.
33. Woon, T. C.; Shirazi, A.; Bruice, T. Inorg. Chem. 1986, 25, 3845.
34. Cheng, R.-J.; Latos-Grazynski, L.; Balch, A. Inorg. Chem. 1982, 21, 2412.
35. Zipplies, M. F.; Lee, W. A.; Bruice, T. C. J. Am. Chem. Soc. 1986, 108, 4433.
36. Tondreau, G. A.; Wilkins, R. G. Inorg. Chem. 1986, 25, 2745.
37. Kobayashi, N. Inorg. Chem. 1985, 24, 3324.
38. Miskelly, G. M.; Webley, W. S.; Clark, Ch. R.; Buckingham, D. A. Inorg. Chem.
1988, 27, 3773.
39. El-Awady, A. A.; Wilkins, P. C.; Wilkins, R. G. Inorg. Chem. 1985, 24, 2053.
40. Binstead, R. A.; Jung, B.; Zuberbu¨hler, A. D. Specfit. 32., Spectrum Software
Associates, 2000.
41. (a) Mean pKa values determined in four independent measurements. (b) Kawassiades,
C. Th.; Kouimtzis, Th. A.; Tossidis, J. A. Chem Chron. A 1968, 33, 1. The Handbook
of Chemistry and Physics (76th ed.; CRC Press: New York, 1995) reports the values
pKa(1) = 2.83 and pKa(2) = 5.69.
42. (a) La, T.; Miskelly, G. M. J. Am. Chem. Soc. 1995, 117, 3613. (b) La, T.; Miskelly, G.
M.; Bau, R. Inorg. Chem. 1997, 36, 5321.
43. Reed, R. A.; Rodgers, K. R.; Kushmeider, K.; Spiro, T. Inorg. Chem. 1990, 29, 2883.
44. Ivanca, M. A.; Lappin, A. G.; Scheidt, W. R. Inorg. Chem. 1991, 30, 711.
45. Walker, F. A. Proton and NMR spectroscopy of paramagnetic metalloporphyrins. In
The Porphyrin Handbook; Kadish, K. M., Smith, K. M., Guilard, R., Eds.; Academic
Press: New York, 1999; Vol. 5.
46. Wanat, A.; Schneppensieper, T.; Karocki, A.; Stochel, G.; van Eldik, R. J. Chem. Soc.,
Dalton Trans. 2002, 941.
- 46 -
Octa-Anionic FeIII-Porphyrin Complex
47. (a) Helm, L.; Merbach, A. E. Chem. Rev. 2005, 105, 1923. (b) Richens, D. T. Chem.
Rev. 2005, 105, 1961.
48. (a) Linder, D. P.; Rodgers, K. R.; Banister, J.; Wyllie, G. R. A.; Ellison, M. K.;
Scheidt, W. R. J. Am. Chem. Soc. 2004, 126, 14136. (b) Linder, D. P.; Rodgers, K. R.
J. Am. Chem. Soc. 2005, 44, 1367.
49. Cusanelli, A.; Frey, U.; Ritchens, D. T.; Merbach, A. E. J. Am. Chem. Soc. 1996, 118,
5265 and references cited therein.
50. Harvey, J. N. J. Am. Chem. Soc. 2000, 122, 12401.
51. Shafirovich, V.; Lymar, S. V. J. Am. Chem. Soc. 2003, 125, 6547.
52. Batinic-Haberle, I.; Spasojevic, I.; Hambright, P.; Benov, L.; Crumbliss, A. L.;
Fridovich, I. Inorg. Chem. 1999, 38, 4011.
53. Liu, M.; Su, Y. O. J. Electroanal. Chem. 1998, 452, 113.
- 47 -
3. A Comparative Mechanistic Study of the Reversible Binding of NO
to a Water-Soluble Octa-Cationic FeIII-Porphyrin Complex*
3.1. Abstract
The water soluble, non-μ-oxo dimer forming porphyrin, [5,10,15,20-tetrakis-(4’tert-butyl-2’,6’-bis(4-tert-butylpyridinium)-phenyl)porphinato]iron(III)
octabromide,
(P8+)FeIII, with eight positively charged substituents in the ortho positions of the phenyl
rings, was characterized by UV-vis and 1H NMR spectroscopy and
17
O NMR water-
exchange studies in aqueous solution. Spectrophotometric titrations of (P8+)FeIII indicated
a pKa1 value of 5.0 for coordinated water in (P8+)FeIII(H2O)2. The monohydroxo-ligated
(P8+)FeIII(OH)(H2O) formed at 5 < pH < 12 has a weakly bound water molecule that
undergoes an exchange reaction, kex = 2.4 × 106 s-1, significantly faster than water
exchange on (P8+)FeIII(H2O)2, viz. kex = 5.5 × 104 s-1 at 25 °C. The porphyrin complex
reacts with nitric oxide to yield the nitrosyl adduct, (P8+)FeII(NO+)(L) (L = H2O or OH-).
The diaqua-ligated (P8+)FeIII(H2O)2 binds and releases NO according to a dissociatively
activated mechanism, analogous to that reported earlier for other (P)FeIII(H2O)2
complexes. Coordination of NO to (P8+)FeIII(OH)(H2O) at high pH follows an associative
mode, as evidenced by negative ΔS‡on and ΔV‡on values measured for this reaction. The
observed ca. 10-fold decrease in the NO binding rate on going from six-coordinate
(P8+)FeIII(H2O)2 (kon = 15.1 × 103 M−1 s−1) to (P8+)FeIII(OH)(H2O) (kon = 1.56 × 103 M-1 s-1 )
is ascribed to the different nature of the rate-limiting step for NO binding at low and high
pH, respectively. The results are compared with data reported for other water-soluble
iron(III) porphyrins with positively and negatively charged meso substituents. Influence
of the porphyrin periphery on the dynamics of reversible NO binding to these (P)FeIII
complexes as a function of pH is discussed on the basis of available experimental data.
* Joo-Eun Jee, Maria Wolak, Domenico Balbinot, Norbert Jux, Achim Zahl, and Rudi van Eldik,
Inorg. Chem. 2006, 45(3), 1326-1337
Octa-Cationic FeIII-Porphpyrin Complex
3.2. Introduction
Interactions of nitric oxide, an important biological messenger synthesized in vivo
by NO synthase, with heme-containing proteins form the basis of many physiological and
pathophysiological processes mediated by NO. Due to the fact that NO is reactive toward
both iron(III) and iron(II) hemes, nature used a variety of structural and electronic
features to tune the affinity of (P)Fe porphyrin centers for NO, such that its physiological
roles can be affected while at the same time its potential pathophysiological actions are
prevented. In addition to the oxidation state of iron, a number of other factors associated
with the immediate heme surrounding (such as number and identity of axial iron ligands,
type of heme substituents, etc.) influence the rate of NO binding and release, as well as
the stability and chemical properties of the resulting heme nitrosyls.1 To elucidate the role
of these different factors, numerous spectroscopic, structural, and mechanistic studies
focus on the interactions of NO with heme centers in hemoproteins and its synthetic
models.1
Our own contribution in this area involves in-depth mechanistic studies on
interactions of nitric oxide with ferriheme proteins2 and synthetic iron(III) porphyrin
models.3,4 These investigations are aimed at understanding the influence of the porphyrin
microenvironment in a given (P)FeIII system on the rate and mechanism of NO binding
and release, and on the stability of the resulting (P)FeII(NO+) species (i.e., {FeNO}6
nitrosyls according to the Enemark and Feltham notation1) toward subsequent reactions in
solution. In this context, we have recently undertaken systematic studies on the influence
of the porphyrin ligand (modified in size, overall charge and electron donating ability by
introducing various meso phenyl substituents) on the properties and reactivity of the iron
center for water-soluble ferric porphyrins.3-5 As a continuation of our earlier work in this
field, we report here on the speciation of a highly positively charged water-soluble
iron(III) porphyrin (P8+)FeIII (Figure 1) in aqueous medium, and present the results of
mechanistic studies on its reactivity toward NO. In the latter context, variable pH,
temperature and pressure dependent stopped-flow measurements provided a detailed
- 49 -
Octa-Cationic FeIII-Porphpyrin Complex
kinetic and mechanistic description of the reversible binding of NO to (P8+)Fe(H2O)2 and
(P8+)Fe(OH)(H2O) present in aqueous solution at low and high pH, respectively. The
results are compared with water exchange data for these complexes and with kinetic data
reported for other water-soluble porphyrins. A feasible interpretation of the reactivity
patterns observed within the series of complexes studied is presented on the basis of these
data. The biological significance of our results is highlighted.
N
L
N
N Fe N
N
R
L
N
N
N
R
R=
N
N
8 Br
Figure 1. Structure of (P8+)FeIII(L)2, L = H2O, OH–
3.3. Experimental Section
Materials. The water-soluble (P8+)FeIII (1) was synthesized and characterized as
described before.6 [5,10,15,20-Tetrakis(4-N-methylpyridyl)porphinato]iron(III) (2), [(4TMPyP)FeIII(H2O)(OH)](Tos)4, was purchased from Frontier Scientific Ltd. Fine
Chemicals, Utah, USA. NO gas (Messer Griesheim or Riessner Gase, ≥ 99.5 vol %) was
cleaned from trace amounts of higher nitrogen oxides by passing it through a concentrated
KOH solution and an Ascarite II column (NaOH on silica gel, Sigma-Aldrich). All other
chemicals used in this study were of analytical reagent grade. Tris, Bis-Tris, and
ClCH2COOH, used for the buffer solutions, were purchased from Sigma-Aldrich.
- 50 -
Octa-Cationic FeIII-Porphpyrin Complex
Solution Preparation. Solutions were prepared from deionized water and handled in
gastight glassware under oxygen-free conditions due to the oxygen sensitivity of NO and
(P)Fe(NO) nitrosyls. Oxygen-free nitrogen was used to deoxygenate the solutions. Tris,
Bis-Tris, and ClCH2COOH/ClCH2COO− buffers (0.05 M) were used to prepare solutions
of desired pH, which was adjusted by the addition of HNO3 or KOH. Due to the relative
pressure sensitivity of the pH of the ClCH2COOH/ClCH2COO− buffer system, the highpressure stopped-flow measurements at pH 2 were performed in the absence of a buffer,
and the solution acidity was adjusted with HNO3. The ionic strength of the solutions (0.1
M) was kept constant by the addition of KNO3.
Measurements. pH measurements were performed on a Methrom 623 pH meter. UV−vis
spectra were recorded in gastight cuvettes on a Shimadzu UV-2100 spectrophotometer
equipped with a thermostated (±0.1 oC) cell compartment. 1H NMR spectra of iron
porphyrin solutions in D2O (10 mM) were measured on a Bruker Avance DPX300NM
spectrometer. The desired pD was adjusted with DCl and NaOD, and TMSP
(trimethylsilyl propionate) was used as a reference.
Kinetics Measurements. Stopped-Flow Studies. Stopped-flow kinetic measurements at
ambient pressure were performed on an SX 18.MV (Applied Photophysics) stopped-flow
apparatus. In a typical experiment, a deoxygenated buffer solution was mixed in varying
volume ratios with a saturated NO solution in a gastight syringe to obtain the appropriate
NO concentration (0.2−1.8 mM). The NO solution was then rapidly mixed with
deoxygenated iron(III) porphyrin in a 1:1 volume ratio in a stopped-flow apparatus.
Kinetics of the reaction of 1 with NO were monitored at 395 and 430 nm at pH 2 and 8,
respectively. All kinetic experiments were performed under pseudo-first-order conditions,
i.e., with at least 10-fold excess of nitric oxide over (P8+)FeIII. The rates of NO binding
and release (kon and koff) were determined from the slopes and intercepts of linear plots of
kobs versus [NO], respectively, as described in Results and Discussion. Rate constants
reported are mean values of at least five kinetic runs, and the quoted uncertainties are
based on standard deviation. To confirm the kinetic data for the release of NO obtained
from the intercepts of plots of kobs vs [NO], an alternative NO-trapping method was used
- 51 -
Octa-Cationic FeIII-Porphpyrin Complex
to measure the rate of NO dissociation from (P8+)FeII(NO+) directly at pH 8. This was
done by rapid mixing of a (P8+)FeII(NO+) solution containing a small excess of NO with
aqueous solutions of [RuIII(edta)(H2O)]− (1 mM) (an efficient NO scavenger7) to give
[RuIII(edta)NO]− and free (P8+)FeIII, as evidenced by the observed UV−vis spectral
changes. The kinetics of NO release were followed in a stopped-flow spectrophotometer
at 432 nm. The first-order rate constants determined in this way were in good agreement
with those determined from the intercepts of the plots of kobs versus [NO] for the
formation of (P8+)FeII(NO+). High-pressure stopped-flow experiments were performed at
pressures up to 130 MPa on a custom-built instrument, described previously.8 Kinetic
traces were analyzed with the use of the OLIS KINET (Bogart, GA, 1989) set of
programs.
17
O NMR Water-Exchange Measurements. Rate constants for water exchange on the
paramagnetic (P8+)FeIII complex, and the corresponding activation parameters ΔH‡ex,
ΔS‡ex, and ΔV‡ex, were measured at pH = 2.0 and 8.0 by
17
O NMR line-broadening
technique. Aqueous solutions of (P8+)FeIII (20 mM) were prepared at pH 2.0 (adjusted
with HNO3) and pH 8 (0.05 M Tris buffer). Both solutions were 0.1 M in KNO3 for an
approximate adjustment of the ionic strength. To each solution was added 10 % of the
total sample volume of enriched 17O-labeled water (normalized 19.2% 17O H2O, D-Chem
Ltd). A sample containing the same components, except for (P8+)FeIII, was used as a
reference. Variable-temperature and pressure FT
17
O NMR spectra were recorded at a
frequency of 54.24 MHz on a Bruker Advance DRX 400WB spectrometer. The
temperature dependence of 17O line broadening was studied in the range 278 – 353 K. A
homemade high-pressure probe10 was used for the variable-pressure experiments
performed at the selected temperature (313 K at pH 2 and 298 K at pH 8) and in the
pressure range 1–150 MPa. The sample was placed in a standard 5 mm NMR tube cut to a
length of 45 mm. Hydrostatic pressure was transmitted to the sample by a movable macor
piston, and the temperature was controlled as described elsewhere.10 The reduced
transverse relaxation times (1/T2r) were calculated for each temperature and pressure from
the difference in the line widths observed in the presence and absence of the metal
- 52 -
Octa-Cationic FeIII-Porphpyrin Complex
complex, (Δνobs – Δνsolvent). The reduced transverse relaxation time is related to the
exchange rate constant kex = 1/τm (where τm is the mean coordinated solvent lifetime) and
to the NMR parameters by the Swift and Connick equation (1),11,12 where Pm is the mole
fraction of water coordinated to the FeIII ion, T2os represents the outer-sphere contribution
to T2r arising from long-range interactions of unpaired electrons of FeIII with the water
outside of the coordination sphere, T2m is the transverse relaxation time of water in the
inner coordination sphere in the absence of chemical exchange, and Δωm is the difference
in the resonance frequency of 17O nuclei in the first coordination sphere of the metal and
in the bulk solvent. The exchange rate constants and the corresponding activation
parameters were obtained by fitting the experimental data to Swift and Connick equation
(eq 1) as described previous section 2.4b
1
T2r
= π
1
Pm
(Δνobs - Δνsolvent) =
1
T2m-2 + (T2mτm)-1 + Δω2m
τm
(T2m-1 + τm-1)2 + Δω2m
+
1
T2os
(1)
3.4. Results and Discussion
3.4.1. Spectroscopic Data for the Speciation of (P8+)FeIII
The speciation of 1 in an aqueous medium of different pHs was studied by
UV−vis, 1H NMR, and 17O NMR techniques. Spectrophotometric titration of a (P8+)FeIII
solution in the pH range 1–10 resulted in the spectral changes presented in Figure 2a. As
can be seen from these data, an increase in pH from 3 to 7 leads to a gradual shift of the
peaks at 402 nm (ε = 8.6 × 104 M−1cm−1) and 529 nm (ε = 7.4 × 103 M-1 cm-1) to 416 nm (ε
= 9.2 × 104 M-1 cm-1) and 595 nm (ε = 7.2 × 103 M-1 cm-1), respectively. This is
accompanied by the formation of a band at 340 nm. The observed pattern of spectral
changes is analogous to that reported for other water-soluble iron(III) porphyrins for the
formation of monohydroxo-ligated species from the corresponding diaqua-ligated
(P)Fe(H2O)2. 9,13−16
- 53 -
Octa-Cationic FeIII-Porphpyrin Complex
0,6
0,6
(a) pH 1 –10
0,15
0,45
460 nm
(b) pH 10 -13
0,32
0,40
Absorbance
0,09
0,25
390 nm
0,20
0,06
0,3
0,15
2
4
6
8
10
pH
0,24
0,20
0,4
Absorbance
Absorbance
pKa1 = 5.0 ± 0.1
0,30
Absorbance
0,4
0,5
0,12
0,35
Abs (437 nm)
0,28
0,5
0,16
0,12
0,3
0,2
0,2
0,1
0,1
2
4
6
pH 8
10
12
14
0,0
0,0
300
350
400
450
500
550
600
650
700
750
300
350
400
450
500
550
600
650
700
Wavelength, nm
W avelength , nm
Figure 2. (a) Changes in the UV-vis spectrum of (P8+)FeIII (1) in the pH range 1- 10.
Inset: fit of the plots of Abs(390) and Abs(430) versus pH to a sigmoidal function. (b) UVVis spectral changes for 1 in the pH range 10 – 13. Inset shows the evolution of
absorbance at 437 nm in the whole studied pH range. Experimental conditions: [1] = 1.2 ×
10-5 M, I = 0.1 M (KNO3), temp 25 oC.
The pKa1 = 5.05 ± 0.07, determined from a plot of absorbance at 390 and 460 nm versus
pH (inset in Figure 2a), is thus ascribed to equilibrium 2 (the presence of water in the
deprotonated form of 1-H2O is inferred from NMR data reported below).
(P8+)FeIII(H2O)2
(P8+)FeIII(OH)(H2O) + H+
pKa1 = 5.0
(2)
As can be seen from the data in Table 1, the pKa1 determined for 1-H2O lies in the lower
range of pKa1 values reported for water-soluble iron(III) porphyrins, thus reflecting the
increased positive charge on the iron(III) center in (P8+)FeIII(H2O)2.
On further increasing the pH, an onset of spectral changes featuring a second pHdependent equilibrium is observed, as shown in Figure 2b.
- 54 -
Octa-Cationic FeIII-Porphpyrin Complex
Table 1. The pKa1 Values and β-pyrrole 1H NMR Chemical Shifts of Synthetic Water-Soluble
Iron(III) Porphyrins
Iron(III)
porphyrin a
meso phenyl
substituent
8+
pKa1 b
(P8+)
(P )Fe d
(P)Fe(H2O)2 (P)Fe(OH)
5.0 d
66
83
5.5
70 e
NA e
5.9
NA
NA
5.1
73 f
84 f
+
NMe3
6.0
72 g
85 g
SO3-
7.0
52 e
NA e
7.2
45
80
6.9
43
82
9.3
45.6; 46.7
82
+
N Me
(4-TMPyP4+)Fe
β-pyrrole 1H (ppm) c
Me
N+
(3-TMPyP4+)Fe
Me
+
N
4+
(2-TMPyP )Fe
F
F
(TF4TMAP4+)Fe
F
(TPPS4-)Fe
Me
4-
(TMPS )Fe
Me
Me
i
O
8-
SO3Me
-O
(P )Fe
SO3-
h
(DMPS )Fe
4-
F
Me
O
O-
j
O-
-O
O
O
a
The quoted charge represents the overall charge of the meso substituents
in a given porphyrin ligand; b Values taken from: Gruhn, N. E.;
Lichtenberger, D. L.; Ogura, H.; Walker, F. A. Inorg. Chem. 1999, 38,
4023, unless otherwise stated; c Referenced to TMPS; d This work; e Ref.
22; f Ref. 21; g Ref. 24b; h Ref.13; i Ref. 4a; j Ref. 4b
- 55 -
Octa-Cationic FeIII-Porphpyrin Complex
A pattern of UV-vis spectral changes similar to these in Figure 2b was previously
reported for the formation of (TMPyP)FeIII(OH)2 complexes from the corresponding
monohydroxo-ligated species, for which pKa2 values of 10.9, 12.2, and 12.3 (for 2TMPyP, 3-TMPyP, and 4-TMPyP porphyrins, respectively) were measured.14 It is
therefore assumed that (P8+)FeIII(OH)(H2O) (denoted as 1-OH in the remainder of text)
deprotonates to (P8+)FeIII(OH)2 at pH > 12. However, because this process is of minor
importance for the topic of this report, further speciation studies concentrated on the
nature of porphyrin forms occurring in the pH range 1 – 11.
In this respect, 1-H2O and 1-OH were studied by 1H and
17
O NMR spectroscopy.
The 1H NMR spectra of (P8+)FeIII recorded at pD 2 and 8 indicated that the β-pyrrole 1H
signal (which serves as a sensitive probe for the spin and ligation state of iron(III)
porphyrins)17,18 appears at 66 ppm at pD 2, and moves to ca. 83 ppm at high pH. The
chemical shift of the β-pyrrole resonance (δβ-py) observed at a low pH is close to that
reported for other positively charged (P)Fe(H2O)2 complexes (Table 1), and evidences the
presence of six-coordinate (P8+)Fe(H2O)2 species in which the Fe(III) center is weakly
spin-admixed (ca. 10%
contribution of the intermediate S = 3/2 spin state to the
predominant S = 5/2 spin state can be estimated for this complex on the basis of δβ−py18).
The broad β-pyrrole resonance at 83 ppm observed at a high pH is diagnostic for a
monomeric high-spin monohydroxo-ligated iron(III) porphyrin.19-22 The lack of
resonances at ca. 13 ppm (i.e. in the spectral region where β-pyrrole resonances of
(FeIII(P))2O dimers are typically observed19,22), excludes the formation of dimeric μ-oxobridged species at high pH.
Figure 3 shows the results of relaxation-time measurements for 17O NMR nuclei of
bulk water in solutions of 1 at pH 2 and 8. It is evident from these data that water
exchange at the Fe(III) center occurs in both diaqua- and monohydroxo-ligated forms of
1. Detailed variable temperature and pressure measurements allowed for the
determination of rate constants (kex) and activation parameters (ΔH‡ex, ΔS‡ex, and ΔV‡ex)
for this process, as summarized in Table 2. For comparison reasons, Table 2 also reviews
- 56 -
Octa-Cationic FeIII-Porphpyrin Complex
the corresponding values reported for other water-soluble iron(III) porphyrins studied to
date.4,5
13,2
12,9
(a)
(b)
13,0
12,8
12,8
ln(1/T2r)
ln(1/T2r)
12,7
12,6
12,6
12,4
12,5
12,2
12,0
0,0027
0,0028
0,0029
0,0030
0,0031
0,0032
0,0033
0,0034
12,4
0,0035
0
20
40
60
1/T
14,0
100
120
140
160
(d)
(c)
13,5
80
Pressure, MPa
13,2
13,1
12,5
ln(1/T2r)
ln( 1/T2r )
13,0
12,0
11,5
13,0
12,9
11,0
12,8
10,5
0,0028
0,0030
0,0032
0,0034
0,0036
0,0038
0
20
40
60
80
100
120
Pressure, MPa
1/T
Figure 3. (a) Plot of ln(1/T2r) versus 1/T for water exchange on 1-H2O at ambient
pressure. Fit of experimental data to eq 1 allows determination of A = (2.1 ± 0.1) × 108
and kex, ΔH‡ex and ΔS‡ex as reported in Table 2. (b) plot of ln(1/T2r) versus pressure
measured at 313 K. (c) Plot of ln(1/T2r) versus 1/T for water exchange on 1-OH at
ambient pressure. Fit of experimental data to eq 1 yields A = (3.67 ± 0.04) × 108 and kex,
ΔH‡ex and ΔS‡ex as reported in Table 2. (d) plot of ln(1/T2r) versus pressure measured at
298 K.
As can be seen from these data, the kex determined for 1-H2O (viz. 5.5 × 104 s−1) is
the smallest of the values reported for (P)Fe(H2O)2 porphyrins. Thus, the lability of the
- 57 -
Octa-Cationic FeIII-Porphpyrin Complex
metal center in 1-H2O is decreased by the influence of the positively charged meso
substituents on the porphyrin periphery, which apparently stabilize the FeIII−H2O bond
(presumably mainly through inductive electronic effects). The positive signs of ΔS‡ex and
ΔV‡ex suggest a dissociatively activated mode for the water exchange process in 1-H2O, as
also found for the other six-coordinate (P)FeIII(H2O)2 species. However, the absolute
values of these activation parameters are much smaller than those observed for the other
diaqua-ligated ferric porphyrins. Clearly, the degree of bond breaking in the transition
state changes from very substantial in negatively charged 5-H2O to almost negligible in
the highly positively charged 1-H2O, for which the water exchange mechanism can be
described as pure interchange (I). The data presented in Figure 3b indicate a rapid water
exchange reaction at the Fe(III) center of the monohydroxo-ligated form 1-OH. The kex
value determined for this process (2.4 × 106 s−1 at 298 K) is ca. 50-fold larger as
compared to that measured for 1-H2O at a low pH. The slightly negative activation
entropy and small positive activation volume (viz. ΔS‡ex = −14 ± 1 J mol−1 K-1, ΔV‡ex = +
1.1 ± 0.2 cm3 mol-1) are indicative of an interchange mechanism in which the degree of
bond breakage and bond formation in the transition state are comparable. The observed
acceleration of water exchange on 1-OH as compared to 1-H2O is in agreement with the
expected trans-labilizing effect of the OH- ligand on the dynamics of simple substitution
processes such as water exchange reactions.23
As indicated by earlier literature reports, the high-spin monohydroxo-ligated
iron(III) porphyrins may exist in solution as five-coordinate (P)Fe(OH)19,20,24 or sixcoordinate (P)Fe(OH)(Solv) species.24 Although the formation of (P)Fe(OH) is promoted
in noncoordinating solvents,19,20 dynamic equilibria between the five- and six-coordinate
(P)Fe(OH) and (P)Fe(OH)(H2O) forms, respectively, were observed in water-containing
solutions (eq 3), with the latter form being the main porphyrin species at high water
contents.24
H 2O
FeIII
FeIII
+ H2O
OH
OH
- 58 -
(3)
Table 2. Rate Constants (at 298 K) and Activation Parameters for Wwater-Exchange Reactions on Water-Soluble Iron(III)
Porphyrins
Iron(III)
porphyrin
(P)Fe(H2O)2
(P)Fe(OH)
‡
‡
‡
‡
‡
‡
kex/105
kex/105
ΔH ex
ΔH ex
ΔS ex
ΔV ex
ΔS ex
ΔV ex
-1
-1
3
(kJ/mol) (J/mol·K)
(kJ/mol) (J/mol·K) (cm3/mol)
(s )
(s )
(cm /mol)
1 (P8+)Fe a
0.55 ± 0.01 53 ± 3
+28 ± 9
+1.5 ± 0.2
24 ± 6 32.5 ± 0.4 -14 ± 1 +1.1 ± 0.2
4+
b
2 (TMPyP )Fe
4.5 ± 0.1
71 ± 2 +100 ± 6 +7.4 ± 0.4
c
3 (TPPS4-)Fe b
20 ± 1
67 ± 2 +99 ± 10 +7.9 ± 0.2
c
8d
77 ± 1
61 ± 6 +91 ± 23 +7.4 ± 04
4 (P )Fe
e
4f
5 (TMPS )Fe
210 ± 10
61 ± 1 +100 ± 5 +11.9 ± 0.3
g
a
b
c
This work; Ref. 5; Formation of μ-oxo dimers at porphyrin concentrations required for NMR measurements
precludes reliable studies on water exchange; d Ref. 4b; e No water exchange process was detected for nondimerising 4-OH, compare Ref. 4b; f Ref. 4a; g The effect of 5-OH on the bulk water line width observed in variable
temperature 17O NMR studies, although detectable, was too small to allow its quantitative analysis and determination
of kex and the corresponding activation parameters for the water exchange reaction, compare Ref. 4a
- 59 -
Octa-Cationic FeIII-Porphpyrin Complex
Relevant literature data24 and the results of our
17
O NMR water-exchange studies
performed for the monohydroxo-ligated forms of 1, 4 and 5 (Table 2) suggest that the
tendency of (P)FeIII(OH) to bind the sixth (H2O) ligand, although evidently smaller than
that exhibited by (P)Fe(H2O),24 can be modulated by the nature of the porphyrin ring
substituents. Thus, although no water exchange could be observed by
17
O NMR
measurements for highly negatively charged (P8-)Fe(OH) (4) (suggesting that 4-OH exists
predominantly in the five-coordinate form), small (but clearly detectable) line broadening
effects observed for 5-OH point to the presence of a fraction of (TMPS)Fe(OH)(H2O) in
equilibrium with the prevailing five-coordinate (TMPS)Fe(OH) form. In the case of 1OH, the exchange of bulk and coordinated water at the iron(III) center is clearly evident
from the line broadening data. This shows that the affinity of the iron(III) center in
(P8+)Fe(OH) for the axial H2O ligand is increased by the highly positively charged
porphyrin periphery, such that (P8+)Fe(OH)(H2O) is the main (or exclusive) porphyrin
form present in solution at pH > pKa1. Such a conclusion is in line with UV-vis data
indicating deprotonation of the aqua ligand in (P8+)Fe(OH)(H2O) at pH > 12.
In principle, two possible structures can be envisaged for 1-OH in aqueous solution,
as depicted below.19,20,24,25
(a)
H2O
FeIII
OH
H2O
OH
fast
FeIII
H2O
≡
FeIII
OH
(b)
H2O
FeIII
OH
Although the exact coordination geometry cannot be unequivocally assigned on the basis
of our experimental results, structure (a) is assumed to be more probable on the basis of
structural data reported for other high-spin six-coordinate iron(III) porphyrins26 and, thus,
will be used throughout this work to depict the mono-hydroxo species 1-OH.
All in all, the spectroscopic studies described above indicate the presence of sixcoordinate (P8+)Fe(H2O)2 at pH < 5, in which the Fe-H2O bond is strengthened by the
electron-withdrawing influence of the porphyrin meso substituents. The six-coordinate
- 60 -
Octa-Cationic FeIII-Porphpyrin Complex
(P8+)Fe(OH)(H2O) (1-OH) is assumed to be the predominant (or sole) form of 1 in the pH
range 6 – 12. The additional acid–base equilibrium observed for 1 at pH > 12 is ascribed
to the formation of a dihydroxo-ligated complex, (P8+)Fe(OH)2.
3.4.2. Reversible Binding of NO to (P8+)FeIII(H2O)2
The addition of NO gas to a deoxygenated solution of 1-H2O at pH 2 leads to a
rapid spectral change that involves a shift of the Soret and Q-bands from 402 and 526 nm
to 430 and 542 nm, respectively, as reported in Figure 4a. The initially formed
(P8+)FeII(NO+) product (representing an {FeNO}6 nitrosyl) is not stable but undergoes
further reductive nitrosylation27 to form (P8+)FeII(NO) (an {FeNO}7 nitrosyl) on a time
scale of several minutes. The final spectrum with Soret and Q-bands at 420 and 552 nm
(Figure 4b), respectively, is identical with that of (P8+)FeII(NO), formed by the addition of
NO to the reduced iron porphyrin, (P8+)FeII. The observed reaction pattern is outlined in
eqs 4 – 5.
0,90
(a)
0.75
0.70
Absorbance
0,75
0.80
0.60
0.55
0.50
0.45
0,45
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Time, sec
0,30
0,15
0,00
300
350
400
450
500
550
600
Wavelength, nm
0,5
(b)
0,4
Absorbance
Absorbance
0,60
0.65
0,3
0,2
0,1
0,0
350
400
450
500
Wavelength, nm
- 61 -
550
600
Octa-Cationic FeIII-Porphpyrin Complex
Figure 4. (a) Spectral changes accompanying rapid binding of NO (1 x 10-3 M) to 1-H2O
(2 × 10-5 M) at pH 2 (temp. 5 °C). Inset shows a fit of a kinetic trace recorded at 432 nm
to a single-exponential function. (b) Subsequent slow (minute time scale) spectral
changes featuring conversion of the (P8+)FeII(NO+) nitrosyl complex into the final
product (P8+)FeII(NO) via reductive nitrosylation.
Because reaction 5 is considerably slower than 4 over the whole pH range studied, it did
not interfere with the stopped-flow kinetic investigations on the reversible NO binding to
(P8+)FeIII. For clarity reasons, only kinetic data and mechanistic information for reaction 4
will be reported here. Studies on the subsequent reaction 5 for (P8+)FeIII (and other
differently charged water-soluble iron(III) porphyrins) will be addressed in detail in a
subsequent report.
(P8+)FeIII(H2O)2 + NO
(P8+)FeII(NO+)(H2O)
kon
koff
+ NO
(P8+)FeII(NO+)(H2O) + H2O
(4)
(P8+)FeII(NO) + NO2- + 2 H+
(5)
Stopped-flow kinetic studies performed under pseudo-first-order conditions with NO
in excess indicated that reaction 4 follows first-order kinetics, and the observed rate
constants depend linearly on [NO], according to eq 6.
kobs = kon[NO] + koff
(6)
Detailed kinetic measurements in the temperature range 5 – 25 oC allowed for the
determination of kon and koff from the slopes and intercepts of the linear plots of kobs versus
[NO] at different temperatures. Linear Eyring plots allowed for the calculation of the
activation parameters ΔH‡ and ΔS‡ for the “on” and “off” reactions. The corresponding
activation volumes were obtained from stopped-flow kinetic measurements on the
pressure dependence of kon and koff performed in the pressure range 10 – 130 MPa. The
- 62 -
Octa-Cationic FeIII-Porphpyrin Complex
rate constants and activation parameters determined in these studies are summarized in
Table 3.
3.4.3. Reversible Binding of NO to (P8+)FeIII(OH)(H2O)
Figure 5 reports the initial rapid spectral change resulting from mixing
(P8+)FeIII(OH)(H2O) and NO solutions at pH 8. The decrease in absorbance at 416 and
598 nm accompanied by an increase in absorbance at 434 and 548 nm features the
formation of the nitrosyl product, (P8+)FeII(NO+)(OH). As also observed at pH 2, the NO
binding step in basic solution is followed by the formation of (P8+)FeII(NO) as the final
reaction product. Due to insufficient stability of the initially formed {Fe–NO}6 nitrosyl
complex, characterization of its acid-base properties by spectrophotometric titration could
not be performed.
0,6
0.46
0.44
0.42
0,5
Absorbance
0.40
0,4
0.38
0.36
0.34
0.32
0.30
Absorbance
0.28
0.26
0
1
0,3
2
3
4
Time, sec
0,2
0,1
0,0
300
350
400
450
500
550
600
Wavelength, nm
Figure 5. UV-vis spectral changes resulting from binding of NO (1 × 10-3 M) to 1-OH (1
× 10-5 M) at pH 8 (temp. 10 °C). Inset shows a fit of a kinetic trace recorded at 435 nm to
a single exponential function.
However, the difference in the positions of the Soret and Q-bands in its UV-vis spectrum
(430 and 542 nm at pH 2, 434 and 548 nm at pH 8, respectively) is ascribed to the
formation of a deprotonated form of the nitrosyl complex at high pH (viz.
- 63 -
Octa-Cationic FeIII-Porphpyrin Complex
(P8+)FeII(NO+)(OH)) on the basis of analogous shifts observed for (P)FeII(NO+)(L) species
(L = H2O, OH-) formed by other water-soluble iron(III) porphyrins.4 Hence, the NO
binding to 1 at high pH is described by reaction 7.
kon
(P8+)FeIII(OH)(H2O) + NO
(P8+)FeII(NO+)(OH) + H2O
koff
(7)
Kinetic studies on the reversible binding of NO to 1-OH were performed at pH 8
according to similar experimental procedures as those employed for 1-H2O. Stopped-flow
measurements at 25 oC allowed for the determination of the rate constants kon = 1.56 × 103
M-1 s-1 and koff = 6.22 s-1 from the slope and intercept of the linear plot of kobs versus
[NO], respectively. Comparison with the corresponding values obtained at the same
temperature at pH 2 (viz. kon = 15.1 × 103 M-1 s-1 and koff = 26.3 s-1, see Table 3) shows that
both the binding and release of NO are slower for 1-OH as compared to those of 1-H2O.
Systematic measurements of kon and koff values in buffered aqueous solutions in the pH
range 2 – 8 resulted in the pH-rate profiles presented in Figure 6. The fit of the
experimental data in Figure 6a and b to a sigmoidal function resulted in pKa values of
5.11 ± 0.02 and 4.82 ± 0.02, respectively. The close similarity of both these values to pKa1
of 1 indicate that the rate of NO binding and release is controlled by the nature of axial
ligands in (P8+)FeIII(L)2 and decreases on going from 1-H2O to 1-OH.
6000
10
(a)
(b)
9
5000
8
7
6
3000
pKa = 4.8
-1
pKa = 5.1
koff, s
kon, M s
-1 -1
4000
5
4
2000
3
2
1000
1
1
2
3
4
5
6
7
8
9
pH
2
3
4
5
pH
- 64 -
6
7
8
Octa-Cationic FeIII-Porphpyrin Complex
Figure 6. pH dependence of the rate constants kon (a) and koff (b) determined from the
slopes and intercepts of linear plots of kobs versus [NO], respectively, measured in the pH
range 2.0 – 8.0. Experimental conditions [1] = 2.5 × 10-5 M, temp = 15 oC, I = 0.1 M
(adjusted with KNO3); pH 2 – 3.5: chloroacetate buffer; pH 4 – 5.5: acetate buffer; pH 6 –
7: Bis-Tris; pH 8: Tris, [Buffer] = 0.05 M
A similar trend was observed previously for other water-soluble iron(III) porphyrins4
(vide infra). Mechanistic characterization of the reaction of 1-OH with NO involved the
measurement of kon and koff as a function of temperature and hydrostatic pressure, which
provided ΔH‡, ΔS‡ and ΔV‡ values for the binding and release of NO at high pH. The
experimental results obtained in these studies are summarized in Table 3.
Table 3. Rates and Activation Parameters for the Reversible Binding of NO to 1-H2O
and 1-OH
(P8+)Fe(H2O)2
temp
(oC)
pressure
(MPa)
kon/103
(M-1s-1)
(P8+)Fe(OH)(H2O)
(M-1s-1)
koff
(s-1)
koffa
0.6 ± 0.1
1.0 ± 0.1
1.8 ± 0.1
2.9 ± 0.1
4.8 ± 0.1
kon/103
koff
(s-1)
5
10
15
20
25
1.6 ± 0.1 2.3 ± 0.1
2.8 ± 0.1 3.7 ± 0.1
5.3 ± 0.2 8.5 ± 0.2
10.5 ± 0.2 14.9 ± 0.2
15.1 ± 0.9 26.3 ± 0.5
0.43 ± 0.02
0.61 ± 0.06
0.84 ± 0.03
1.12 ± 0.05
1.56 ± 0.06
0.71 ± 0.01
1.20 ± 0.03
2.04 ± 0.02
3.52 ± 0.03
6.22 ± 0.03
5.5
1.14 ± 0.01
1.10 ± 0.08
1.06 ± 0.04
1.05 ± 0.11
77 ± 3
+94 ± 12
+1.5 ± 0.3
1.28 ± 0.05 b
1.65 ± 0.23 b
2.05 ± 0.05 b
2.55 ± 0.01 b
41 ± 1
-45 ± 2
-13.8 ± 0.4
2.14 ± 0.03 b
2.06 ± 0.02 b
1.98 ± 0.03 b
1.88 ± 0.03 b
72 ± 2
+12 ± 5
+2.6 ± 0.2
10
50
90
130
‡
ΔH , kJ/mol
ΔS‡, J/mol⋅K
ΔV‡, cm3/mol
2.2 ± 0.1
1.9 ± 0.1
1.6 ± 0.1
1.3 ± 0.1
83 ± 4
+61 ± 14
+9.3 ± 0.5
(s-1)
Data obtained by NO-trapping method with the use of [RuIII(edta)(H2O)]−
b
at 16.5 oC
a
- 65 -
72 ± 1
+10 ± 4
Octa-Cationic FeIII-Porphpyrin Complex
3.4.4. Mechanism of Reversible NO Binding to (P8+)FeIII. Comparison with Other
Water-Soluble Iron(III) Porphyrins
A. Reactivity of (P8+)FeIII(H2O)2 toward NO.
Mechanistic features of the reaction of 1-H2O with NO revealed that variable
temperature and pressure studies can be best visualized by the volume profile constructed
on the basis of ΔV‡on and ΔV‡off values, as presented in Figure 7. The positive sign of the
activation volumes observed for both “on” and “off” reactions evidences a dissociatively
activated mode of binding and release of NO from 1-H2O.
NO ‡
Partial molar volume, cm3 mol-1
H2O
FeIII
OH2
FeIII
OH2
+ NO
OH2
+ 1.5 ± 0.3
+ 9.3 ± 0.5
– 7.8 ± 0.6
NO+
FeII
+ H2O
OH2
Reactants
Transition state
Products
Reaction coordinate
Figure 7. Volume profile for the reversible binding of nitric oxide to 1-H2O.
As can be seen from the data summarized in Table 4, an analogous mechanistic picture
was revealed by previously reported studies on NO binding to other (P)Fe(H2O)2.
However, closer inspection of the data in Table 4 shows that the absolute values of ΔV‡on
and ΔV‡off, as well as the rate constants for the “on” and “off” reaction obtained for 1- 66 -
Octa-Cationic FeIII-Porphpyrin Complex
H2O, are markedly smaller than the values reported for the other porphyrins. In fact,
despite the limited number of complexes studied, clear reactivity trends can be identified
for the (P)Fe(H2O)2 species reported in Table 4.
Table 4. Rate Constants (at 298 K) and Activation Parameters for Reversible Binding of
NO to a Series of Diaqua-Ligated Water-Soluble Iron(III) Porphyrins.
1
2
3
4
5
Iron(III)
porphyrin
pKa1
(P8+)Fe b
(TMPyP4+)Fe c
(TPPS4-)Fe d
(P8-)Fe e
(TMPS4-)Fe d
5.0
5.5
7.0
9.3
6.9
Int% a
NO binding
kon /10
4
-1 -1
10
7
20
24
26
‡
ΔH on
‡
ΔS
on
‡
ΔV
on
3
(M s )
(kJ/mol)
(J/mol·K)
(cm /mol)
1.5 ± 0.1
2.9 ± 0.2
50 ± 3
82 ± 1
280 ± 20
77 ± 3
67 ± 4
69 ± 3
51 ± 1
57 ± 3
+94 ± 12
+67 ± 13
+95 ± 10
+40 ± 2
+69 ± 11
+1.5 ± 0.3
+3.9 ± 1.0
+9 ± 1
+6.1 ± 0.1
+13 ± 1
NO release
koff
‡
ΔH off
(s )
(kJ/mol)
-1
8+
b
‡
ΔS
off
(J/mol·K)
‡
ΔV
3
off
(cm /mol)
26.3 ± 0.5
83 ± 4
+61 ± 14
+9.3 ± 0.5
59 ± 4
108 ± 5
+150 ± 12 +16.6 ± 0.2
500 ± 400
76 ± 6
+60 ± 11
+18 ± 2
220 ± 2
101 ± 2
+140 ± 7
+16.8 ± 0.4
900 ± 200
84 ± 3
+94 ± 10
+17 ± 3
a
3
Contribution of the intermediate spin state (S = /2) in the spin-admixed
(P)FeIII(H2O)2 porphyrins, calculated on the basis of the empirical equation Int% =
[(80 - δ)/140] × 100, where δ is the chemical shift of the β-pyrrole 1H, compare Ref.
18; b This work; c Ref. 3a; d Ref. 30 e Ref. 4b
1
2
3
4
5
(P )Fe
(TMPyP4+)Fe c
(TPPS4-)Fe d
(P8-)Fe e
(TMPS4-)Fe d
These can be summarized as follows: (i) The rate of NO binding to (P)Fe(H2O)2
increases with increasing electron donation from the porphyrin meso substituents. The
latter is reflected by increasing contribution of the intermediate S = 3/2 spin state in the
weakly spin admixed (P)Fe(H2O)2 complexes (Int%).4b,18,28 Hence, relatively slow NO
- 67 -
Octa-Cationic FeIII-Porphpyrin Complex
coordination rates observed for 1-H2O and 2-H2O with electron-withdrawing meso
substituents become faster in (Pn-)Fe(H2O)2 species 3 – 5, for which increasing electron
donation from the porphyrin ligand (reflected by the rising Int%)29 is observed. (ii) On the
basis of the ΔV‡on values determined for the diaqua species 1 – 5, it may be concluded that
the mechanism of the “on” reaction changes from predominantly dissociative in 5-H2O to
interchange (Id or I) in 1-H2O. Notably, the reactivity trends in (i) and (ii) are analogous
to that observed for the water-exchange process on (P)Fe(H2O)2. This is in line with
earlier conclusions1a-b,5,30 that NO binding to diaqua-ligated iron(III) porphyrins is
controlled by the rate-limiting water displacement step. It is evident from (i) and (ii) that a
porphyrin-induced increase in electron density on the iron(III) center facilitates breakage
of the Fe-OH2 bond and induces a dissociative mechanism. Conversely, the presence of
electron-withdrawing meso substituents in the porphyrin periphery stabilizes the Fe–H2O
bond to such an extent that an I mechanism (without a predominant a- or d-character) for
NO coordination to the highly positively charged 1-H2O is energetically preferred over a
dissociative pathway. (iii) An increase in koff on going from (Pn+)FeIII(H2O)2 to (Pn)FeIII(H2O)2 shows that the release of NO from (P)FeII(NO+)(H2O) is facilitated by
electron-donating meso substituents, whereas positively charged meso groups apparently
stabilize the FeII–NO+ bond, making the NO dissociation step relatively slow. This
observation agrees with the recent computational and experimental results reported for a
series of {FeNO}6 porphyrin nitrosyls, which indicated that the addition of electron
density at the meso carbon positions weakens the Fe–NO bond.31 (iv) A substantially
smaller ΔV‡off observed for (P8+)FeII(NO+)(H2O) (viz. + 9.3 cm3 mol-1) as compared to the
values reported for other porphyrin nitrosyls (ΔV‡off ≈ +17 cm3 mol-1), implies a less
dissociative mode for NO release in highly positively charged (P8+)FeII(NO+)(H2O). In
general, the large positive ΔV‡off values found for NO dissociation from various porphyrin
nitrosyls were interpreted in terms of a d-activated mechanism, in which the rate-limiting
breakage of the FeII–NO+ bond is accompanied by a significant volume increase due to
the lengthening of the Fe–NO bond, spin change at the iron(III) center, and solvational
changes.1a-b,3a,30 Taking into account that NO coordination to 1-H2O follows an I
- 68 -
Octa-Cationic FeIII-Porphpyrin Complex
mechanism, the principle of microscopic reversibility requires that the reverse process,
i.e., breakage of the Fe–NO bond in (P8+)FeII(NO+)(H2O), leads to the transition state in
which the degree of bond formation with the entering water molecule is substantial. This
is supposed to offset (to some extent) other positive contributions to the observed
activation volume.
Taking into account the very different structural and electronic features of the meso
substituents in 1 – 5, it is likely that, in addition to their electronic influence, other (in
particular steric and electrostatic) factors affect (to a varying extent) the kinetics and
mechanism of NO binding and release in the individual porphyrin complexes. For
example, a somewhat smaller koff value observed for 4 as compared to those for 3 and 5
(where the electron-donating influence of the porphyrin ligand on the iron center is quite
similar to that in 4, as evidenced by similar Int% values determined for 3–5), may
possibly result from the stabilization of a bound NO ligand in (P8-)FeII(NO+)(H2O) via
through-space electrostatic interactions with –COO- groups of the flexible malonato
substituents. Furthermore, despite the slightly larger Int% value calculated for 1 as
compared to that of 2 (which suggests a longer Fe–H2O bond and, consequently, a more
labile iron center in 1), the kex and kon values measured for 1 are smaller than the
corresponding values reported for 2. This may reflect greater steric crowding around the
iron center in 1, where the presence of bulky meso substituents may impede formation of
the transition state along an interchange mechanistic pathway assigned for 1. This
contrasts the situation in the open-faced porphyrin 2, where access of the entering ligand
to the metal center is very facile. Nevertheless, although the influence of steric and
electrostatic effects should not be neglected in considering the reactivity of a given
porphyrin system toward NO, the data in Table 4 suggest that modulation of electron
density on the iron center by the porphyrin macrocycle is the main factor tuning the
dynamics of reversible NO binding to (P)FeIII(H2O)2.
- 69 -
Octa-Cationic FeIII-Porphpyrin Complex
B. Reactivity of (P8+)FeIII(OH)(H2O) toward NO.
As already stated in our earlier reports that addressed the reactivity of ferric
porphyrins toward NO as a function of pH,4 kinetic and mechanistic features of reversible
NO binding to (P)Fe(OH) differ entirely from those observed for the diaqua-ligated
forms. Although NO coordination to (P)Fe(H2O)2 is d-activated, an associative activation
mode was observed for nitrosylation of (P)Fe(OH). It was also concluded that the rate of
NO binding to (P)Fe(OH) is controlled by an Fe–NO bond-formation step rather than by
the lability of the iron center. Due to electronic reasons (see below), the formation of the
Fe–NO bond apparently exhibits a higher activation barrier in (P)Fe(OH) as compared to
that of (P)Fe(H2O)2, such that a decrease in kon (as well as in koff) is observed at a high pH.
These earlier conclusions are nicely illustrated by the experimental results obtained for
(P8+)Fe(OH)(H2O). The associative nature of NO coordination to 1-OH is evident from
the volume profile depicted in Figure 8.
OH2
Partial molar volume, cm3 mol-1
FeIII
+ NO
OH
–13.8 ± 0.4
- 16.4 ± 0.3
OH2
NO ‡
FeIII
NO+
OH
Reactants
FeII
+ 2.6 ± 0.2
OH
Transition state
Products
+ H2O
Reaction coordinate
Figure 8. Volume profile for the reversible binding of nitric oxide to 1-OH.
- 70 -
Octa-Cationic FeIII-Porphpyrin Complex
That the rate of NO coordination to 1-OH is not controlled by the lability of the
metal center is obvious from opposite effects of increasing pH on the values of kex and kon:
whereas a faster water-exchange rate at high pH indicates an increase in the lability of
coordinated water in 1-OH as compared to that in 1-H2O, the rate of NO binding
decreases at a high pH. The latter effect is ascribed to electronic and structural changes,
which determine the free energy barrier associated with the Fe–NO bond-formation step.
These mainly involve the reorganization of spin density at the iron(III) center (S =
3
/2, 5/2 → S = 0 at low pH; S = 5/2 → S = 0 at high pH) along with structural changes
accompanying this process. It was argued4 that larger spin (and structural) changes
accompanying the formation and breakage of the FeII–NO+ bond in the purely high-spin
monohydroxo-ligated species (as compared to those of the spin-admixed diaqua-ligated
forms) lead to a higher activation barrier for NO coordination and release, thus decreasing
the rate of these reactions at high pH.4 The results obtained for 1 are in line with these
earlier conclusions. In addition, although the number of monohydroxo-ligated porphyrin
complexes studied with regard to their reaction with NO is admittedly limited, the data in
Table 5 (which summarizes presently available kinetic and mechanistic information on
these reactions) allow some additional comments to be made. It is evident from the data in
Tables 4 and 5 that the rate of NO coordination and release is, in all studied cases, slower
for monohydroxo-ligated porphyrins as compared to that for (P)Fe(H2O)2, and the NO
binding is a-activated (in contrast to a d-activated mode observed at low pH).
Furthermore, the kon values measured for diaqua-ligated porphyrins vary within ca. 2
orders of magnitude and tend to correlate with the int% calculated for a given
(P)Fe(H2O)2 complex, whereas differences in the NO binding rates are relatively small for
various monohydroxo-ligated porphyrins. This again points to the different nature of the
rate-limiting step for NO binding at low and high pH. Because the lability of the metal
center is the decisive factor for the rate of NO binding at low pH, its increase (correlating
with higher int%) results in a faster NO-binding rate. On the contrary, electronic and
structural factors governing the rate-limiting Fe–NO bond formation at high pH are
- 71 -
Octa-Cationic FeIII-Porphpyrin Complex
expected to vary relatively little for different (P)Fe(OH) species, which all represent
purely high-spin porphyrin complexes.
Table 5. Rate Constants (at 298 K) and Activation Parameters for Reversible Binding of
NO to Water-Soluble Monohydroxo-Ligated Iron(III) Porphyrins
1
2
5
4
NO binding
Iron(III)
porphyrin
pKa1
(P8+)Fe a
(TMPyP4+)Fe a
(TMPS4-)Fe b
(P8-)Fe d
5.0
5.5
6.9
9.3
kon /104
ΔH
-1 -1
(M s )
‡
on
(kJ/mol)
0.16 ± 0.01
0.36 ± 0.01
1.46 ± 0.02d
5.1 ± 0.2 f
koff
-1
on
‡
ΔV
on
3
(J/mol·K)
(cm /mol)
41 ± 1
−45 ± 2
41.4 ± 0.5
−38 ± 5
28.1 ± 0.6 −128 ± 2
34.6 ± 0.4
−39 ± 1
NO release
−13.8 ± 0.4
−13.7 ± 0.6
−16.2 ± 0.4
−6.1 ± 0.2
ΔH
‡
off
(kJ/mol)
(s )
‡
ΔS
‡
ΔS
off
(J/mol·K)
‡
ΔV
3
off
(cm /mol)
(P8+)Fe a
(TMPyP4+)Fe a
(TMPS4-)Fe b
(P8-)Fe d
6.2 ± 0.1
72 ± 2
+12 ± 5
+2.6 ± 0.2
3.2 ± 0.1
78 ± 2
+25 ± 7
+9.5 ± 0.8
c
90 ± 1
+77 ± 3
+7.4 ± 1.0
10.5 ± 0.2
e
107 ± 2
+136 ± 7
+17 ± 3
11.4 ± 0.3
a
This work; b Ref. 4a; c Calculated for 298 K from the Eyring plots reported
in Ref. 4a; d Ref. 4b; e at 297 K
1
2
5
4
It follows from the data summarized in Table 6 that the relative decrease in the values of
kon and koff on going from a diaqua- to a monohydroxo-ligated (P)FeIII complex varies
considerably among the complexes studied. In particular, whereas for (TMPS)FeIII (5) the
rate constants for the “on” and “off” reactions decrease ca. 190 and 90-fold, respectively,
at high pH, much smaller changes in kon and koff are observed for 1. It can be argued that
these differences reflect different electronic and structural features of 1 and 5.
- 72 -
Octa-Cationic FeIII-Porphpyrin Complex
Table 6. Ratio of Rate Constants Observed for the NO Binding (kon\kon) and NO Release
H2O
OH
(koff\koff) for Selected Water-Soluble Iron(III) Porphyrins
H2O
OH
Iron Porphyrin
H2O
OH
H2O
OH
Int%
kon /kon
koff/koff
1 (P8+)Fe
10
9.4
4.2
2 (TMPyP4+)Fe
7
8.0
18.4
4 (P8-)Fe
24
16
19.3
5 (TMPS4-)Fe
26
192
85
Due to a relatively large contribution of the S = 3/2 spin state in 5-H2O, considerable
tetragonal distortion (manifested by short Fe–Np and elongated axial bonds) is expected
for this complex, in line with the observed lability of the iron center in 5-H2O. A
comparison of typical structural features of six-coordinate spin-admixed porphyrins26,32
with those of low-spin {FeNO}6 porphyrin nitrosyls1c (see Figure 9) also suggests that
rather small structural changes occur upon the formation (and breakage) of the Fe–NO
bond in spin-admixed 5-H2O. Thus, due to a facile substitution step and relatively small
electronic and structural changes, the energy barrier associated with reversible NO
binding to 5-H2O is expected to be small. In contrast, the reaction of NO with purely
high-spin five-coordinate 5-OH involves greater reorganization of spin density at the
metal center (S = 5/2 → S = 0, as compared to S = 3/2, 5/2 → S = 0 at low pH) and larger
structural changes (i.e., movement of the iron center into the porphyrin plane and a
change in the coordination number, and contraction of the Fe–Np bonds). As a
consequence, large differences are observed in the kinetics of the reaction of 5 with NO at
low and high pH, respectively. In the case of 1 the degree of spin admixture in diaqualigated 1-H2O is small, i.e., the complex is almost purely high spin. Furthermore, the
excessive positive charge on the porphyrin stabilizes the axial Fe–H2O bonds, making the
NO coordination at low pH relatively slow. The high-spin 1-OH complex formed at high
pH is most probably six-coordinate, with the iron(III) center placed in the porphyrin
plane.26
- 73 -
Octa-Cationic FeIII-Porphpyrin Complex
a)
NO+
b)
Fe
c)
2.27
1.65
2.00
H2O
II
1.95
Fe
0.54
2.00
2.19
9.
Typical
2.08
1.76
H2O
Figure
Fe
EtOH
structural
features
O
of
Fe(III)
porphyrins:
(a)
low-spin
(TPP)FeII(NO+)(H2O)1c (b) admixed intermediate-spin (P)Fe(EtOH)(H2O) complex (P =
tetramethylchiroporphyrin) exhibiting large S = 3/2 contribution in the ground spin state32a
(c) a five-coordinate high-spin (P)Fe-O unit of the μ-oxo bridged dimer (TPP)Fe-OFe(TPP)33
Thus, in the case of 1, electronic and structural changes upon the coordination (and
release) of NO to 1-H2O and 1-OH, respectively, are presumably quite similar, resulting
in small differences in kon and koff values at low and high pH. However, because both kon
and koff values may be influenced by various additional factors in (P)FeIII complexes (such
as steric crowding, through-space electrostatic interactions of axial ligands with charged
porphyrin substituents, etc.), the scatter in the changes in kon and koff values for complexes
reported in Table 6 is not surprising in view of their widely different steric, electronic,
and structural features.
Examination of the activation volumes for the “on” and “off” reactions of
monohydroxo-ligated complexes reported in Table 5 allows two further comments. First,
based on the differences in the ΔV‡on and ΔV‡off values among the complexes studied, it
may be concluded that the transition state in the a-activated binding of NO occurs at
different positions along the reaction coordinate. The activation volumes measured for the
“on” and “off” reactions in 4-OH, viz. ΔV‡on = – 6.1 cm3 mol-1 and ΔV‡off = + 17 cm3 mol1
, suggest an “early” transition state for this reaction, whereas the corresponding values
- 74 -
Octa-Cationic FeIII-Porphpyrin Complex
for 1-OH point to a “late” transition state. Second, the overall volume change calculated
for 1 (viz. -16.4 cm3 mol-1) is smaller than those observed for 2 – 4 (ca. – 23 cm3 mol-1).
This difference can be ascribed to the presence of a weakly bound water molecule in 1OH, which is released from the complex upon coordination of NO, partially
compensating the volume decrease associated with the formation of the FeII–NO+ bond.
Such a partial compensation is not expected for the five-coordinate (P)Fe(OH) species,
which are (presumably) the predominant forms of 2–5 at pH > pKa1.
3.5. References and Notes
1. (a) Ford, P. C.; Lorkovic, I. M. Chem. Rev. 2002, 102, 993 and references therein (b)
Ford, P. C.; Laverman, L. E.; Lorkovic, I. M. Adv. Inorg. Chem. 2003, 54, 2003 and
references therein (c) Scheidt, W. R.; Ellison, M. K. Acc. Chem. Res. 1999, 32, 350
and references therein (d) Wyllie, G. R. A.; Scheidt, W. R. Chem. Rev. 2002, 102,
1067 and references therein.
2. (a) Laverman, L. E.; Wanat, A.; Oszajca, J.; Stochel, G.; Ford, P. C.; van Eldik, R. J.
Am. Chem. Soc. 2001, 123, 285 (b) Franke, A.; Stochel, G.; Jung, Ch.; van Eldik, R. J.
Am. Chem. Soc. 2004, 126, 4181.
3. (a) Theodoridis, A.; van Eldik, R. J. Mol. Catal. A: chem. 2004, 224, 197 (b) Franke,
A.; Stochel, G.; Suzuki, N.; Higuchi, T.; Okuzono, K.; van Eldik, R. J. Am. Chem.
Soc. 2005, 127, 5360.
4. (a) Wolak, M.; van Eldik, R., J. Am. Chem. Soc. 2005, 127, 13312. (b) Jee, J-E.; Eigler,
S.; Hampel, F., Jux, N.; Wolak, M.; Zahl, A.; Stochel, G.; van Eldik, R. Inorg. Chem.
2005, 44, 7717.
5. Schneppensieper, T.; Zahl, A.; van Eldik, R. Angew. Chem., Int. Ed. 2001, 40, 1678.
6. Guldi, D. M.; Rahman, G. M. A.; Jux, N.; Balbinot, D.; Hartnagel, U.; Tagmatarchis,
N.; Prato, M. J. Am. Chem. Soc. 2005, 127, 9830.
- 75 -
Octa-Cationic FeIII-Porphpyrin Complex
7. Wanat, A.; Schneppensiepper, T.; Karocki, A.; Stochel, G.; van Eldik, R. J. Chem.
Soc., Dalton Trans., 2002, 941.
8. Spitzer, M.; Gartig, F.; van Eldik, R.; Rev. Sci. Instrum. 1988, 59, 2092.
9. Tondreau, G. A.; Wilkins, R. G. Inorg. Chem. 1986, 25, 2745.
10. Zahl, A.; Neubrand, A.; Aygen, S.; van Eldik, R. Rev. Sci. Instrum. 1994, 65, 882.
11. (a) Swift, T. J.; Connick, R. E. J. Chem. Phys. 1962, 37, 307 (b) Swift, T. J.; Connick,
R. E. J. Chem. Phys. 1964, 41, 2553 (c) Newman, K. E.; Meyer, F. K.; Merbach, A. E.
J. Am. Chem. Soc. 1979, 101, 1470.
12. Schneppensieper, T.; Seibig, S.; Zahl, A.; Tregloan, P.; van Eldik, R. Inorg. Chem.
2001, 40, 3670.
13. Zipplies, M. F.; Lee, W. A.; Bruice, T. C. J. Am. Chem. Soc.1986, 108, 4433.
14. (a) Kobayashi, N. Inorg. Chem. 1985, 24, 3324 (b) Kobayashi, N.; Koshiyama, M.;
Osa, T.; Kuwana, T. Inorg. Chem. 1983, 22, 3608.
15. Miskelly, G. M.; Webley, W. S.; Clark, Ch. R.; Buckingham, D. A. Inorg. Chem.
1988, 27, 3773.
16. El-Awady, A. A.; Wilkins, P. C.; Wilkins, R. G. Inorg. Chem. 1985, 24, 2053.
17. Walker, F. A. ‘Proton and NMR spectroscopy of paramagnetic metalloporphyrins’ In:
Kadish, K. M.; Smith, K. M.; Guilard, R. (Eds.) The Porphyrin Handbook; Kadish, K.
M., Smith, K. M., Guilard, R., Eds.; Academic Press: New York, 1999; Vol. 5.
18. Ikezaki, A.; Nakamura, M. Inorg. Chem. 2002, 41, 6225 and references therein.
19. Cheng, R-J.; Grażyński, L.; Balch, A. Inorg. Chem. 1982, 21, 2412.
20. Woon, T. C.; Shirazi, A.; Bruice, T. Inorg. Chem. 1986, 25, 3845.
21. Reed, R. A.; Rodgers, K. R.; Kushmeider, K.; Spiro, T. Inorg. Chem. 1990, 29, 2883.
22. Ivanca, M. A.; Lappin, A. G.; Scheidt, W. R. Inorg. Chem. 1991, 30, 711 and
references therein.
23. Cusanelli, A.; Frey, U.; Ritchens, D. T.; Merbach, A. E. J. Am. Chem. Soc. 1996, 118,
5265 and references therein.
24. (a) La, T.; Miskelly, G. M.; Bau, R. Inorg. Chem. 1997, 36, 5321 (b) La, T.; Miskelly,
G. M. J. Am. Chem. Soc. 1995, 117, 3613.
- 76 -
Octa-Cationic FeIII-Porphpyrin Complex
25. Evans, D. R.; Reed, Ch. A. J. Am. Chem. Soc. 2000, 122, 4660 and references therein.
26. Scheidt, W. R.; Reed, Ch. A. Chem. Rev. 1981, 81, 543.
27. Fernandez, B. O.; Lorkovic, I. M.; Ford, P. C. Inorg. Chem. 2004, 43, 5393 and
references therein.
28. Toney, G. E.; Gold, A.; Savrin, J.; Haar, L. W.; Sangaiah, R.; Hatfield, W. E. Inorg.
Chem. 1984, 23, 4350.
29. The inducing effect of porphyrin substituents is also reflected by the pKa values of
coordinated water in (P)Fe(H2O)2. However, due to the fact that the acidity of
coordinated water may easily be influenced by other factors (in particular by throughspace electrostatic interactions with charged groups in the porphiryn periphery), the
Int% value is used preferentially as a measure of the electronic influence of meso
substituents in the porphyrin systems addressed in this work.
30. Laverman, L. E.; Ford, P. C. J. Am. Chem. Soc. 2001, 123, 11614
31. (a) Linder, D. P.; Rodgers, K. R.; Banister, J.; Wyllie, G. R. A.; Ellison, M. K.;
Scheidt, W. R. J. Am. Chem. Soc. 2004, 126, 14136 (b) Linder, D. P.; Rodgers, K. R. J.
Am. Chem. Soc. 2005, 44, 1367.
32. (a) Simonato, J-P.; Pecaut, J.; Le Pape, L.; Oddou, J-L.; Jeandey, C.; Shang, M.;
Scheidt, W. R.; Wojaczyński, J.; Wołowiec, S.; Latos-Grażyński, L.; Marchon, J-C.
Inorg. Chem. 2000, 39, 3978 (b) Safo, M. K.; Schmidt, W. R.; Gupta, G. P.; Orosz, R.
D.; Reed, Ch. A. Inorg. Chim. Acta. 1991, 184, 251 (c) Cheng, B.; Scheidt, W. R. Acta
Crystallogr., Sect. C: Cryst. Struct. Commun. 1995, C57, 1271 (d) Körber, F. C. F.;
Lindsay Smith, J. R.; Prince, S.; Rizkallah, P.; Reynolds, C. D.; Shawcross, D. R. J.
Chem. Soc., Dalton Trans. 1991, 3291.
33. (a) Hofmann, A. B.; Collins, D. M.; Day, V. W.; Fleischer, E. B.; Srivastava, R. S.;
Hoard, J. L. J. Am. Chem. Soc. 1972, 94, 3620 (b) Swepston, P. N.; Ibers, J. A. Acta
Crystallogr., Sect. C. 1985, 41, 671.
- 77 -
4. Mechanistic Studies on the Nitrite-Catalyzed Reductive Nitrosylation
of Highly-Charged Anionic and Cationic FeIII-Porphyrin Complexes*
4.1. Abstract
The nitrosyl complexes formed during the binding of NO to the (Pn)FeIII(H2O)2 (n
= 8- and 8+) complexes, viz. (P8-)FeII(H2O)(NO+) and (P8+)FeII(H2O)(NO+), undergo
subsequent reductive nitrosylation reactions that were found to be catalyzed by nitrite,
which was also produced during the reaction. The effect of the nitrite concentration, pH,
temperature, and pressure on the nitrite-catalyzed reductive nitrosylation process was
studied in detail for (P8-)FeIII(H2O)2, (P8+)FeIII(H2O)2, and (P8+)FeIII(OH)(H2O), from
which rate and activation parameters were obtained. On the basis of these data, we
propose mechanistic pathways for the studied reactions. The available results favor the
operation of an inner-sphere electron-transfer process between nitrite and coordinated
NO+. By way of comparison, the cationic porphyrin complex (P8+)FeIII(L)2 (L = H2O or
OH-) was found to react with NO2- to yield the nitrite adduct (P8+)FeIII(L)(NO2-). A
detailed kinetic studied revealed that nitrite binds to (P8+)FeIII(H2O)2 according to a
dissociative mechanism, whereas nitrite binding to (P8+)FeIII(OH)(H2O) at higher pH
follows an associative mechanism, similar to that reported for the binding of NO to these
complexes.
* Joo-Eun Jee and Rudi van Eldik, Inorg. Chem. 2006, 45(16), 6523-6534
Reductive Nitrosylation for Highly Charged FeIII-Porphyrin Complexes
4.2. Introduction
In general, nitrite can interfere with the binding of NO to transition-metal
complexes, for example, as reported for aquacobalamin (vitamin B12a), where it was
found that the observed reactions were solely due to the interaction with nitrite impurities
in aqueous NO solutions.1 Nitric oxide is a versatile signaling molecule that binds
reversibly to cytochrome c oxidase (complex IV); its action ranges from hemodynamic
regulation to antiproliferative roles on vascular smooth muscle cells. NO transfer in
human hemoglobin from the heme to cysteine thiols in cys β-93 to form bioactive
nitrosothiols leads to the formation of SNO–Hb, in which reductive nitrosylation provides
a pathway for S-nitrosation.2-7 It has been proposed to be reversible and includes
considerable implications, especially in playing a major factor in vasodilation and platelet
inhibition. Redox activation of the NO molecule is needed in order for this transfer to
occur.2-7 In recent investigations on the reactions of NO, it was reported that the electronic
nature, steric effect, structure and overall charge of the porphyrin ligand bound to the
iron(III) center play a major role in determining the reactivity of NO.8-16
(P)FeIII + NO
KNO
(P)FeII(NO+) + HONO/NO2
knit
(P)FeII(NO) + NOx products
Scheme 1. General Reaction Sequence Suggested for the Nitrite-Catalyzed Reductive
Nitrosylation of (P)FeII(NO+).
The binding of NO to an Fe(III)12-14, 17 center leads to one electron reduction and
formation of (P)FeII(NO+), where P denotes a porphyrin,18 followed by reductive
nitrosylation via attack of a nucleophile, viz., a water molecule or hydroxide ion as a
general base, on the ferrous nitrosonium complex in aqueous media to produce a ferrous
- 79 -
Reductive Nitrosylation for Highly Charged FeIII-Porphyrin Complexes
nitrosyl species, viz., (P)FeII(NO). This process is referred to as reductive nitrosylation
and is summarized in reaction 1 and shown in Scheme 1.9
III
(P)Fe
+ NO
KNO
II
+
(P)Fe (NO )
+ Nu-
(P)FeII(NO-Nu)
+ NO
(1)
(P)FeII(NO) + NO-Nu
Reductive nitrosylation also occurs in nonaqueous solution, in a mixed-solvent
system of methanol and water where MeO- acts as the NO+ acceptor.19 The final iron(II)
nitrosyl porphyrin products were characterized by various spectroscopic methods, such as
UV-vis, EPR, and MCD, and electrochemical methods, such as cyclic voltammetry.20
Several groups have systematically studied the reductive nitrosylation of water-soluble
ferric porphyrins such as TPPS,7,12 TMPyP12-14 and hemoproteins such as ferro- and21
ferric-cytochrome22 and met-hemoglobin23 and met-myoglobin24 in buffered aqueous
solution at various pH conditions; several groups have also investigated the roles of a
general base buffer, the hydroxide ion, and nitrite catalysis.24 A surprising fact is that the
reduction of ferric nitrosyl porphyrins, (P)FeII(NO+), by NO is catalyzed by nitrite, which
is always present as an impurity in deoxygenated aqueous NO solutions. Two plausible
mechanisms
have
been
suggested
to
account
for
nitrite-catalyzed
reductive
nitrosylation.12,13 The observed reaction can proceed via direct nucleophilic attack of
nitrite on the electrophilic site of coordinated NO+ to give (P)FeII-(N2O3), which would be
a key intermediate species that decomposes to the five-coordinate (P)FeII complex and
N2O3. N2O3 then rapidly hydrolyzes to nitrite, and the five-coordinate ferrous complex
(P)FeII rapidly binds NO to yield (P)FeII(NO). The reported rate constant for the binding
of NO to ferrous porphyrins and proteins is around 3 orders of magnitude faster than for
ferric porphyrins.25 An alternative pathway involves an outersphere electron-transfer
mechanism that occurs between nitrite and coordinated NO+ on (P)FeII(NO+) to form the
ferrous nitrosyl compound (P)FeII(NO) plus an NO2 radical that rapidly binds NO to
produce N2O3, followed by hydrolysis to nitrite. Importantly, nitrite is not only present as
- 80 -
Reductive Nitrosylation for Highly Charged FeIII-Porphyrin Complexes
an impurity in the aqueous NO solution in such a catalytic cycle but is also a reaction
product so that the system is in principle autocatalytic. These results led us to further
investigate reactions with nitrite and the reaction with NO in the presence of nitrite
impurities at various pH for the series of highly water soluble porphyrin complexes
studied before.10, 12, 13
In this context, we studied the reaction of (P8+)FeIII(H2O)2, [5,10,15,20-tetrakis-(4’tert-butyl-2’,6’-bis(4-tert-butylpyridine)phenyl)porphinato]iron(III),
with
NO2-
at
different pH values, temperatures and pressures. We also studied the reductive
nitrosylation reaction following the binding of NO to the highly positively charged ferric
porphyrin (P8+)FeIII(H2O)2 and the highly negatively charged ferric porphyrin (P8)FeIII(H2O)2,
[54,104,154,204-tetra-tert-butyl-52,56,152,156-tetrakis-(2,2-biscarboxylato-
ethyl)-5,10,15,20-tetraphenyl-porphyrin]iron(III),
to
produce
the
ferrous
nitrosyl
porphyrins (P8-)FeII(NO) and (P8+)FeII(NO), respectively. The reductive nitrosylation
reactions were studied at pH 2.0 and 4.0 for (P8+)FeIII(H2O)2, at pH 8.0 for
(P8+)FeIII(OH)(H2O), and at pH 7.0 for (P8-)FeIII(H2O)2. The differently charged iron(III)
porphyrins were used to compare the reaction properties and reactivity induced by
variation of the porphyrin ligand. Variable pH, temperature and pressure measurements
provided detailed kinetic and mechanistic information on the reductive nitrosylation
process. The results are compared with kinetic data and mechanistic information reported
for other water soluble iron(III) porphyrins. A feasible mechanism that accounts for the
observed reactivity patterns within the series of complexes studied is proposed.
4.3. Experimental section
Materials. The water-soluble porphyrin complexes Na7[(P8-)FeIII]10b and [(P8+)FeIII]Br910c
were synthesized and characterized as described in previous papers.10 Bis-tris, Tris,
Hepes26 and NaOAc/HOAc were used as buffer solutions and were purchased from
Sigma-Aldrich. All chemicals used in this study were of analytical grade reagent. The NO
gas was purified from impurities of nitrogen oxides such as NO2 and N2O3, by passing
- 81 -
Reductive Nitrosylation for Highly Charged FeIII-Porphyrin Complexes
through an Ascarite II column (NaOH on silica gel from Sigma-Aldrich) and concentrated
NaOH solution, and was obtained from Riessner Gase or Linde 93 in a purity of at least
99.5%.
Solution preparation. All solutions were prepared under strict oxygen-free conditions
with Milli-Q water and handled in gastight glassware because of the high oxygen
sensitivity of NO and the nitrosyl complexes. Oxygen-free Ar and N2 were used to
prepare deoxygenated solutions. Buffered solutions were prepared with 0.05 M Bis-tris,
Tris, Hepes and NaOAc/HOAc, and solutions at pH 2.0 were prepared with HNO3. The
ionic strength was kept at 0.1 M with NaClO4 and KNO3.
Measurements. pH measurements were carried out on a Metrohm 623 pH meter
equipped with a Sigma glass electrode. UV-vis spectra were recorded on a Shimadzu UV2100 spectrophotometer equipped with a thermostated cell compartment CDS-260. UVvis spectra at pressures up to 150 MPa were recorded in a custom-built high pressure
optical cell.27 Stopped-flow kinetic measurements on the reaction of nitrite and NO with
(P8+)FeIII(H2O)2 were carried out using an Applied Photophysics SX-18MV stopped-flow
spectrometer. Deoxygenated water solutions of (P8+)FeIII(H2O)2 were rapidly mixed with
nitrite and NO solutions containing a certain concentration of nitrite. The observed rate
constants and activation parameters for the reactions with nitrite and NO/nitrite were
monitored at 435 nm, where the change in absorbance is at a maximum. We measured the
rate constants for the conversion (P8+)FeII(H2O)(NO+), generated from NO and
(P8+)FeIII(H2O)2, to (P8+)FeII(NO). All kinetic experiments were performed under pseudofirst-order conditions, i.e., with at least a 10-fold excess of nitrite and NO. Reported rate
constants are mean values of at least five kinetics runs and the uncertainties are quoted on
the basis of the standard deviation. High-pressure stopped-flow studies were performed
on a custom-built instrument (from 10 to 130 MPa).28 Kinetic traces were recorded on an
IBM-compatible computer and analyzed with the OLIS KINFIT (Bogart, GA) set of
programs.
- 82 -
Reductive Nitrosylation for Highly Charged FeIII-Porphyrin Complexes
4.4. Results and Discussion
4.4.1. Reaction of (P8+)FeIII(H2O)2 with Nitrite
To understand the effect of nitrite on the reactions of the highly charged anionic
and cationic FeIII porphyrins with NO, we first studied the reactions of these complexes
with nitrite in the absence of NO. Preliminary investigations showed that the cationic
complex reacts efficiently with nitrite but that the anionic complex does not bind nitrite at
all. For that reason, we focused our studies on the binding of nitrite to the cationic
complex. The reaction of different concentrations of HONO/NO2- with (P8+)FeIII(H2O)2
led to characteristic spectral changes. The spectrum of (P8+)FeIII(H2O)2 shows absorption
maxima at 402 nm (ε = 8.6 × 104 M-1 cm-1) and 529 nm (ε = 7.4 × 103 M-1 cm-1) in aqueous
solution at pH 2.0 and at 406 nm (ε = 8.9 × 104 M-1 cm-1) and 529 nm (ε = 7.4 × 103 M-1
cm-1) at pH 4.0 in a 0.05 M Hepes buffer solution.10a
1,8
1,4
1,3
(a) pH 2.0
1,2
1,2
1,6
1,6
(b) pH 4.0
1,5
1,1
1,0
0,9
1,2
Absorbance
0,8
0,7
Absorbance
Absorbance
Absorbance
1,4
1,4
1,0
0,8
0,6
0,000
0,002
0,004
0,006
0,008
0,010
0,012
0,014
-
[NO 2 ], M
0,6
1,3
1,2
1,1
1,0
1,0
0,0000
0,0005
0,0010
0,0015
0,0020
-
[NO 2 ], M
0,8
0,6
0,4
0,4
0,2
0,0
300
0,2
350
400
450
500
550
600
650
700
0,0
300
Wavelength, nm
350
400
450
500
550
600
650
700
Wavelength, nm
Figure 1. Spectral changes observed during the binding of nitrite at 298 K: (a) to
(P8+)FeIII(H2O)2, pH 2.0, [(P8+)FeIII]9+ = 1.0 × 10-5 M, [NaNO2] = 1 to 15 mM, inset: plot
of absorbance versus nitrite concentration from which the equilibrium constant KHONO =
(9.9 ± 0.4) × 103 M-1 was calculated; (b) to (P8+)FeIII(H2O)2, pH 4.0 (Hepes buffer),
[(P8+)FeIII]9+ = 1.4 × 10-5 M, [NaNO2] = 0.5 to 2 mM, inset: plot of absorbance versus
nitrite concentration from which KNO2- = (8.8 ± 0.4) × 104 M-1 was calculated;
- 83 -
Reductive Nitrosylation for Highly Charged FeIII-Porphyrin Complexes
Figure 1. shows the reaction of (P8+)FeIII(H2O)2 with various concentrations of
HONO/NO2-, which leads to a red shift to 424 and 548 nm at pH 2.0 and 4.0, respectively,
with several isosbestic points at 416, 480, 535, and 590 nm, indicating that only two
complex species exist in equilibrium and that the same product is formed at both pH
levels. The spectral changes observed for the formation of the stable nitrite complex
(P8+)FeIII(H2O)(NO2-)
differ
from
those
observed
for
the
formation
of
(P8+)FeII(H2O)(NO+). In the present case, coordinated water is substituted by nitrite with
no change in the oxidation state of the metal center, as given in reaction 2.29,30 For the
reaction with NO, FT-IR and Raman data suggest that charge transfer occurs during the
binding of NO to form (P8+)FeII(H2O)(NO+). The apparent equilibrium constants (KHONO
and KNO2-) for the binding of HONO/NO2- to (P8+)FeIII(H2O)2 at pH 2 and 4, respectively,
were calculated from the spectral changes (see insets in Figure 2), and found to be KHONO
= (9.9 ± 0.4) × 103 M-1 at pH 2.0 and KNO2- = (8.8 ± 0.4) × 104 M-1 at pH 4.0. These values
show that NO2- is a much stronger nucleophile than HONO as expected.
(P8+)FeIII(H2O)2 + NO2-
k2
k-2
(P8+)FeIII(H2O)(NO2-) + H2O
(2)
The kinetics of the observed reaction was studied under pseudo-first-order conditions, i.e.,
at least a 10-fold excess of nitrite, as a function of nitrite concentration, pH, temperature,
and pressure. As can be seen from Figure 2, plots of kobs vs [total nitrite] are linear, with a
slope equal to the rate constant for the formation of (P8+)FeIII(H2O)(NO2-) and an intercept
close to zero, i.e., kobs = k2[NO2-] + k-2 ≈ k2[NO2-]. The observed second-order rate
constant for nitrite binding to (P8+)FeIII(H2O)2, k2 = (9.0 ± 0.2) × 103 M-1 s-1 at pH 2.0, is
10-fold lower than the value found at pH 4.0, k2 = (85 ± 4) × 103 M-1 s-1, which accounts
for the difference in the overall equilibrium constants reported above.
- 84 -
Reductive Nitrosylation for Highly Charged FeIII-Porphyrin Complexes
280
100
90
(a)
240
(b)
80
-1
60
kobs, s
kobs, s
-1
200
70
50
160
120
40
80
30
20
40
10
0
0
0,000
0,002
0,004
0,006
[total nitrite], M
0,008
0,010
0,012
0,0000
0,0005
0,0010
0,0015
0,0020
0,0025
0,0030
[total nitrite], M
Figure 2. Plot of kobs versus [total nitrite] for nitrite binding to (P8+)FeIII(H2O)2 at (a) pH
2.0 and (b) pH 4.0 Experimental conditions: [(P8+)FeIII]9+ = 7.5 × 10-6 M, λdet = 430 nm,
temp = 25.0 oC, I = 0.1 M (with KNO3)
This trend can be accounted for in terms of the lower nucleophilicity of HONO mainly
present at pH 2.0, as compared to nitrite mainly present at pH 4.0. A more systematic pH
dependence of k2 = kobs/[total NO2-] was studied in the range 1.8 – 4.5, for which the
results are presented in Figure 3. It can be seen from the data that the rate constant for
nitrite binding to (P8+)FeIII(H2O)2 decreases and becomes slower on going to lower pH
and tends to zero at pH ≤ 1. On the basis of reactions 3 – 5, we can express the rate law
for the pH dependence of the reaction with nitrite as given in 6.
HONO
Ka
NO2- + H+
k1
(P8+)FeIII + HONO
k2
(P8+)FeIII + NO2kobs
k1[H+] + k2Ka
+
Ka + [H ]
Product
(4)
Product
(5)
[total NO2-]
- 85 -
(3)
(6)
Reductive Nitrosylation for Highly Charged FeIII-Porphyrin Complexes
[total NO2-]
kobs
=
1
k2
[H+]
+
(7)
k2Ka
This rate law can be simplified by using the fact that k2 >> k1 (where k1 has the value of
k2 measured at pH = 2.0), as shown by the data in Figure 3 and rewritten as given in
reaction 7. The plot of [total NO2-]/kobs vs [H+] (Figure 3b) is linear with a slope of 1/k2Ka
and an intercept of 1/k2, from which it follows that k2 = 1.14 × 105 M-1 s-1 and Ka = 8.06 ×
10-4 M-1, i.e., pKa = 3.09. The latter value is in close agreement with that determined from
a spectrophotometric titration of nitrous acid, viz. pKa = 3.27 in 0.1 M KNO3.31 By
combining the values of k1 and k2 with the apparent equilibrium constants KHONO and
KNO2-, the rate constants for the dissociation of nitrite (k-1 and k-2) were calculated to be
0.83 ± 0.01 and 0.96 ± 0.21 s-1 at 25 °C, respectively. Thus the release of nitrite does not
significantly depend on pH in the studied range.
90000
0,0014
(a)
(b)
0,0012
60000
-
kobs / [NO2 ], M s
-1 -1
[NO2 ] / kobs , M s
75000
0,0008
-
45000
0,0010
0,0006
30000
0,0004
15000
0,0002
0
0,0000
0
1
2
3
4
5
pH
0,00
0,02
0,04
+
0,06
0,08
0,10
0,12
[H ], M
Figure 3. (a) Plot of kobs/[nitrite] versus pH for nitrite binding to (P8+)FeIII(H2O)2 in
buffered aqueous solutions in the pH range 1.0 – 4.5, observed pKa = 3.10 ± 0.06. (b) Plot
of [nitrite]/kobs versus [H+] to determine Ka and k2. Experimental conditions: [(P8+)FeIII]9+
= 2.0 × 10-5 M, λdet = 430 nm, temp = 25.0 oC, I = 0.1 M (KNO3)
- 86 -
Reductive Nitrosylation for Highly Charged FeIII-Porphyrin Complexes
The temperature dependence of the reaction was studied over the range 5 – 25 oC at pH
2.0 and 4.0, and the corresponding Eyring plots resulted in ΔH‡ and ΔS‡ values of 73 ± 1
kJ mol-1 and +75 ± 6 J mol-1 K-1 at pH 2.0, respectively, and 76 ± 13 kJ mol-1 and +103 ±
5 J mol-1 K-1 at pH 4.0, respectively. The effect of pressure on the reaction of nitrite with
(P8+)FeIII(H2O)2 was investigated at pH 2.0 over the pressure range 10 – 130 MPa. A
summary of all rate and activation parameters is given in Table 1.
4.4.2. Reaction of (P8+)FeIII(OH)(H2O) with Nitrite
(P8+)FeIII(H2O)2 has a pKa value of 5.0, indicating that the formation of a hydroxo
ligated complex with a weakly bound water molecule, (P8+)FeIII(OH)(H2O), occurs at >
pH 5.0.10a As can be seen in Figure 4, the spectra of (P8+)FeIII(OH)(H2O) at 416 nm (ε =
9.2 × 104 M-1 cm-1) and 595 nm (ε = 7.2 × 103 M-1 cm-1) on reaction with nitrite at pH 8.0
to yield (P8+)FeIII(OH)(NO2-) changes to 425 nm (ε = 1.2 × 105 M-1 cm-1) and 537 nm (ε =
6.4 × 103 M-1 cm-1), respectively.
0,8
0,7
pH 8.0
0,65
0,60
Absorbance
0,55
Absorbance
0,6
0,5
0,50
0,45
0,40
0,4
-0,005
0,000
0,005
0,010
0,015
0,020
0,025
0,030
0,035
0,040
-
[NO 2 ], M
0,3
0,2
0,1
0,0
350
400
450
500
550
600
650
700
Wavelength, nm
Figure 4. Spectral changes observed during the binding of nitrite at 298 K: (c) to
(P8+)FeIII(OH)(H2O), pH 8.0 (Tris buffer), [(P8+)FeIII]9+ = 6.0 × 10-6 M, [NaNO2] = 0.5 to
35 mM, inset: plot of absorbance versus nitrite concentration from which KNO2– = (9.6 ±
1.5) × 102 M-1 was calculated
- 87 -
Reductive Nitrosylation for Highly Charged FeIII-Porphyrin Complexes
Kinetic data for the reaction with nitrite at pH 8.0 were obtained as for pH 2.0 and 4.0
and are reported in Figure 5.
100
90
80
kobs, s
-1
70
60
50
40
30
20
10
0
0,00
0,02
0,04
0,06
0,08
0,10
0,12
-
[NO2 ], M
Figure 5. Nitrite concentration dependence of the reaction of (P8+)FeIII(OH)(H2O) with
NO2-. Experimental conditions: [(P8+)FeIII]9+ = 8.5 × 10-6 M, [NO] = 1 mM, λdet = 435 nm,
temp = 25.0 oC, I = 0.1 M (KNO3), pH 8.0.
The results for reaction 8 fit the rate law kobs = k3[NO2-] + k-3 ≈ k3[NO2-] according to the
data in Figure 5, from which it follows that k3 = (8.5 ± 0.2) × 102 M-1 s-1 at 25 °C. The
equilibrium constant KNO2- (= k3/k-3) at pH 8 calculated from the spectral changes
observed in Figure 4 has the value (9.6 ± 1.5) × 102 M-1, from which it follows that k-3 =
0.89 ± 0.04 s-1, which is very close to the dissociation rate constants found at pH 2 and 4.
(P8+)FeIII(OH)(H2O) + NO2-
k3
k-3
(P8+)FeIII(OH)(NO2-) + H2O
(8)
The temperature and pressure dependence of the reaction was studied at pH 8.0, and the
activation parameters are summarized in Table 1. The data in Table 1 can be interpreted
in a similar way as that reported for the pH dependence of the binding of NO to
(P8+)FeIII(H2O)2 in our earlier study.10a
- 88 -
Table 1. Temperature and Pressure Dependence of the Reaction of Nitrite with (P8+)FeIII(H2O)2 and (P8+)FeIII(H2O)(OH)
(P8+)FeIII
temp
pressure
(oC)
(MPa)
pH 1.0a
kobs
(s-1)
pH 2.0b
kobs
(s-1)
pH 4.0c
kobs
(s-1)
pH 8.0d
kobs
(s-1)
2.5
0.1
2.8 ± 0.3
4.9 ± 0.4
6.2 ± 0.3
3.9 ± 0.3
5
0.1
4.0 ± 0.4
5.7 ± 0.3
7.8 ± 0.4
5.0 ± 0.5
10
0.1
7±1
10 ± 1
15 ± 2
8±1
15
0.1
13 ± 2
18 ± 1
27 ± 3
12 ± 2
20
0.1
28 ± 2
32 ± 3
46 ± 3
20 ± 2
25
0.1
53 ± 3
54 ± 2
81 ± 1
28 ± 3
-1 -1
3
3
kobs/[NO2 ] at 25°C, (M s )
(9.0 ± 0.2) × 10 (81 ± 1) × 10 (0.93 ± 0.04) × 103
kon at 25oC e, (M-1 s-1)
(8.3 ± 0.5) × 103 (85 ± 1) × 103 (0.85 ± 0.02) × 103
2.5
10
4.7 ± 0.2
3.6 ± 0.3
50
4.1 ± 0.4
3.8 ± 0.1
90
3.6 ± 0.3
4.0 ± 0.3
130
3.2 ± 0.3
4.2 ± 0.1
‡
88 ± 3
73 ± 1
76 ± 13
57 ± 1
ΔHon (kJ/mol)
‡
+109 ± 11
+75 ± 6
+103 ± 5
6±5
ΔSon (J/mol·K)
3
‡
f
+7.3 ± 0.4
3.0 ± 0.1
ΔVon (cm /mol)
a
b
c
[NO2 ] = 40 mM, λdet = 430 nm. [NO2 ] = 6 mM, λdet = 435 nm. [NO2 ] = 1 mM, λdet = 435 nm.
d
[NO2-] = 30 mM, λdet = 435 nm were used. e values calculated from the slope of kobs versus [NO2-].
f
It was impossible to measure the activation volume due to the reaction being too fast.
- 89 -
Reductive Nitrosylation for Highly Charged FeIII-Porphyrin Complexes
A comparison of the data is presented in Table 2 for the reactions at pH 2 and 8. At low
pH, the reaction with nitrite follows a dissociative interchange (Id) mechanism as
supported by the positive ΔV‡ value, which is in good agreement with that found for the
water-exchange reaction and the binding of NO.10a The activation volume is larger than
that found for the reaction with NO, which can be ascribed to the neutralization of charge
(i.e., decrease in electrostriction) during the formation of (P8+)FeIII(H2O)(NO2-). At pH
8.0, the binding of nitrite also follows an associative interchange (Ia) pathway with
(P8+)FeIII(OH)(H2O) as judged from the negative activation volume, which is in
agreement with that found for the binding of NO.10a The more positive (less negative)
value can again be ascribed to charge neutralization during the formation of
(P8+)FeIII(OH)(NO2-) which results in a decrease in electrostriction that is accompanied by
a volume increase.
Table 2. Comparison of the Rate Constants and Activation Parameters for the Binding of
NO2- and NO to (P8+)FeIII(H2O)2 at pH 2.0 and (P8+)FeIII(OH)(H2O) at pH 8.0 and 25 oC
Porphyrin
pH
kNO2-/103 (M-1 s-1)
(P8+)Fe + NO22
8
8.3 ± 0.5
kNO/103 (M-1 s-1)
‡
ΔH (kJ/mol)
73 ± 1
‡
+75 ± 6
ΔS (J/mol·K)
‡
3
+7.3 ± 0.4
ΔV (cm /mol)
a
data taken from ref. 10(a).
(P8+)Fe + NO a
2
8
0.85 ± 0.02
57 ± 1
+6±5
-3.0 ± 0.1
15.1 ± 0.9 1.56 ± 0.06
77 ± 3
41 ± 1
+94 ± 12
-45 ± 2
+1.5 ± 0.3 -13.8 ± 0.4
4.4.3. Spontaneous Reductive Nitrosylation of (P8-)FeIII and (P8+)FeIII
Exposure of an aqueous solution of (P8-)FeIII(H2O)2 to NO results in rapid
characteristic spectral changes and the formation of a Soret band at 427 nm (ε = 1.5 × 105
M-1 cm-1) and Q-band at 540 nm at pH = 7.0 in 0.05 M bis-tris buffer (I = 0.1 M),10a
indicating the formation of (P8-)FeII(H2O)(NO+). This species undergoes a subsequent
- 90 -
Reductive Nitrosylation for Highly Charged FeIII-Porphyrin Complexes
slow reaction with the formation of a Soret band at 414 nm (ε = 9.6 × 104 M-1 cm-1) and
Q-band at 610 nm with clean isosbestic points at 300, 345, 523, and 556 nm as shown in
Figure 6.
1,0
0.9
0.8
Absorbance
0,8
0.7
0.6
Absorbance
0.5
0,6
0.4
-2000
0
2000
4000
6000
80 00
10000 12000 14000 16000
Time, sec
0,4
0,2
0,0
250
300
350
400
450
500
550
600
650
700
750
Wavelength, nm
Figure 6. Spectral changes observed following the binding of NO to (P8-)FeIII(H2O)2.
Inset: kinetic trace of absorbance at 426 nm vs time fitted to a single exponential function.
Experimental conditions: [(P8-)FeIII]7- = 1×10-5 M, [NO] = 1 mM, [added NO2-] = 0, temp
= 25.0 oC, I = 0.1 M with NaClO4, pH = 7.0, The first 10 spectra were taken every 6 min,
the next 10 spectra every 9 min, and the rest every 15 min.
The final nitrosyl product appeared to be very stable and irreversible on bubbling Ar or
N2 through the solution. This final spectrum is identical to that of the five-coordinate
nitrosyl ferrous adduct (P8-)FeII(NO) formed by the addition of NO to the reduced ferrous
porphyrin, (P8-)FeII, which suggests that (P8-)FeII(H2O)(NO+) is converted to (P8-)FeII(NO)
via reduction as outlined in reactions 9 – 11.
(P8-)FeIII(H2O)2 + NO
KNO
(P8-)FeII(H2O)(NO+) + H2O
kred
(P8-)FeII(H2O) + NO2- + 2H+
+ H 2O
fast
(P8-)FeII(H2O) + NO
(P8-)FeII(NO) + H2O
(P8-)FeII(H2O)(NO+)
- 91 -
(9)
(10)
(11)
Reductive Nitrosylation for Highly Charged FeIII-Porphyrin Complexes
The concentration of trace impurities of nitrite in aqueous NO solutions is too low to
induce the observed reductive nitrosylation reactions (see the following section on nitritecatalyzed reductive nitrosylation).
The rapid reaction of (P8-)FeIII(H2O)2 with NO in reaction 9 leads to an equilibrium
mixture of (P8-)FeIII(H2O)2 and (P8-)FeII(H2O)(NO+); the reaction is followed by the slow
nucleophilic attack of water on coordinated NO+ in reaction 10 to produce nitrite and (P8)FeII(H2O), which rapidly binds NO in reaction 11 to yield (P8-)FeII(NO) (the NO binding
constant for ferrous porphyrins ≈ 109 M-1 s-1)25. The observed spectral changes are
analogous to that reported in the case of (TPPS)FeIII for the formation of the ferrous
analogue (TPPS)FeII(NO) from the ferric nitrosyl porphyrin (TPPS)FeIII(NO).12 The
observed kinetic trace can be fitted with a single-exponential function, as shown in the
inset of Figure 6. The [NO] dependence of the observed rate constant for the reductive
nitrosylation of (P8-)FeII(H2O)(NO+) at pH 7.0 and 25 oC is reported in Figure 7.
-4
2,8x10
-4
2,4x10
-4
2,0x10
-4
kobs, s
-1
1,6x10
-4
1,2x10
-5
8,0x10
-5
4,0x10
0,0
0,0000
0,0002
0,0004
0,0006
0,0008
0,0010
0,0012
0,0014
[NO], M
Figure 7. Plot of kobs vs [NO] for the reductive nitrosylation of (P8-)FeII(H2O)(NO+).
Experimental conditions: [FeIII(P8-)]7- = 2.0 ×10-5 M, [NO] in the range from 0 to 1.2 mM,
temp = 25.0 oC, λdet = 430 nm, I = 0.1 M (with NaClO4), pH 7.0.
- 92 -
Reductive Nitrosylation for Highly Charged FeIII-Porphyrin Complexes
The observed rate constant increases with increasing [NO] and reaches a limiting value at
high [NO]. This is typical for a reaction scheme that consists of a rapid pre-equilibrium
followed by a rate-determining step as outlined in reactions 9 – 11. According to the
corresponding rate law in eq 12, a plot of (kobs)-1 vs [NO]-1 should be linear with a slope
(KNOkred)-1 and an intercept (kred)-1, from which KNO = (3.9 ± 0.3) × 103 M-1 and kred = (2.8
± 0.2) × 10-4 s-1 were obtained. The calculated equilibrium constant is in close agreement
with the corresponding value KNO = kon/koff = (3.8 ± 0.2) × 103 M-1 (at 24 oC) determined
from laser flash photolysis measurements.10b The value of kred is also in a good agreement
with that measured directly, viz. kred = 2.2 × 10-4 s-1, under high [NO] conditions.
kred KNO[NO]
kobs =
(12)
1 + KNO[NO]
The (P8+)FeIII(H2O)2 complex undergoes a similar reaction at pH 2.0 following the
binding of NO, but the reduction occurs more rapidly than for (P8-)FeIII(H2O)2, such that
the spectral changes for the subsequent reaction were recorded using a rapid-scan
technique. After the mixing of (P8+)FeIII(H2O)2 with NO, new bands appear at 422 nm (ε
= 9.5 × 104 M-1 cm-1) and 553 nm at pH 2.0 that indicate the subsequent formation of
(P8+)FeII(NO) as shown in Figure 8a. At pH 4.0, the reaction proceeded in the same
manner as at pH 2.0, and new bands are formed at 422 nm (ε = 9.9 × 104 M-1 cm-1) and
553 nm, as shown in Figure 8b. The observed kinetic traces can be fitted with a singleexponential function, as shown in the insets of panels a and b of Figure 8.
0,6
0,44
(a)
0,42
0,40
0,5
Absorbance
0,38
0,4
0,36
0,34
Absorbance
0,32
0,30
0,28
0,3
0
100
200
300
400
Time, sec
0,2
0,1
0,0
300
350
400
450
500
Wavelength, nm
- 93 -
550
600
650
Reductive Nitrosylation for Highly Charged FeIII-Porphyrin Complexes
0,7
(b)
0,48
0,46
Absorbance
0,6
0,5
0,44
0,42
Absorbance
0,40
0,4
0,38
0,36
0
50
0,3
100
150
200
250
300
Time, sec
0,2
0,1
0,0
300
400
500
600
700
Wavelength, nm
Figure 8. (a) Repetitive scan spectra recorded following the binding of NO to
(P8+)FeIII(H2O)2 at pH = 2.0. Inset: Kinetic trace at 420 nm fitted with a single exponential
function. Experimental conditions: [(P8+)FeIII]9+ = 4.1 × 10-6 M, [NO] = 0.6 mM, λdet =
422 nm, temp = 25.0 oC, I = 0.1 M (with KNO3). Spectra were recorded every 3 sec (b)
Spectral changes recorded following the binding of NO to (P8+)FeIII(H2O)2 at pH 4.0.
Conditions: [(P8+)FeIII]9+ = 4.8 × 10-6 M, [NO] = 1 mM, temp = 25.0 oC. Spectra were
recorded every 5 s. Inset: Observed kinetic trace is observed at 432 nm.
Kinetic data for the reductive nitrosylation of (P8+)FeII(H2O)(NO+) were measured
as a function of [NO] at pH 2.0, for which the results are reported in Figure 9. The results
are very similar to those reported for (P8-)FeIII(H2O)2 in Figure 7 and can be interpreted in
the same way in terms of reactions 9 – 11. A fit of the data according to eq 12 results in
KNO = (5.4 ± 0.4) × 102 M-1 and kred = (4.0 ± 0.2) × 10-2 s-1, from which it follows that KNO
is in close agreement with the corresponding value KNO = kon/koff = (5.7 ± 0.4) × 102 M-1
estimated from the kinetic data at 25 oC.10a The value of kred is approximately 1 × 102
larger for the reduction of the positively charged ferrous nitrosyl porphyrin than for the
negatively charged porphyrin.
- 94 -
Reductive Nitrosylation for Highly Charged FeIII-Porphyrin Complexes
0,018
0,016
0,014
kobs, s
-1
0,012
0,010
0,008
0,006
0,004
0,002
0,000
0,0000
0,0002
0,0004
0,0006
0,0008
0,0010
0,0012
[NO], M
Figure 9. Plot of kobs vs [NO] for the reductive nitrosylation of (P8+)FeII(H2O)(NO+).
Experimental conditions: [FeIII(P8+)]9+ = 3.0 ×10-5 M, [NO] in the range from 0 – 1.0 mM,
temp = 25.0 oC, λdet = 431 nm, I = 0.1 M (with KNO3), pH 2.0.
This is consistent with earlier data reported for the reductive nitrosylation of the
corresponding nitrosyl complexes of (TMPyP4+)FeIII and (TPPS4-)FeIII.12 These
differences must be related to the electrophilicity of the Fe(III) center that is affected by
the porphyrin environment, which in turn determines the stability of the ferrous
nitrosonium intermediate, (P)FeII(NO+), and controls the rate of the subsequent reductive
nitrosylation reaction. Once (P)FeII(NO+) is formed, the subsequent reduction of the
nitrosyl ligand can be accelerated by the positively charged electron-withdrawing meso
substituents in (P8+)FeII(NO+) as compared to the electron-donating substituents in (P8)FeII(NO+) (see Scheme 2a, b). A similar effect was observed for the cationic complex on
going to higher pH where (P8+)FeII(OH)(NO+) undergoes a subsequent reductive
nitrosylation reaction.
Figure 10 shows the spectral changes that result from mixing (P8+)FeIII(OH)(H2O)
with an aqueous NO solution at pH 8. The decrease in absorbance at 416 and 598 nm is
accompanied by an increase in absorbance at 422 nm (ε = 1.1 × 105 M-1 cm-1) and 553 nm
and features the formation of (P8+)FeII(NO). Hence, the coordination of NO and the
- 95 -
Reductive Nitrosylation for Highly Charged FeIII-Porphyrin Complexes
subsequent reductive nitrosylation of (P8+)FeII(OH)(NO+) are suggested to occur
according to reactions 13 – 15 in a manner similar to that outlined in reactions 9 – 11.
0,5
0,38
0,36
0,4
Absorbance
Absorbance
0,34
0,32
0,30
0,3
0,28
0,26
0
500
1000
1500
2000
2500
3000
3500
4000
Time, sec
0,2
0,1
0,0
300
350
400
450
500
550
600
650
700
Wavelength, nm
Figure 10. Repetitive scan spectra recorded following the binding of NO to
(P8+)FeIII(H2O)(OH) at pH = 8.0. Inset: Kinetic trace of absorbance vs time at 422 nm
fitted with a single exponential function. Experimental conditions: [(P8+)FeIII]9+ = 7.5 ×
10-6 M, [NO] = 1 mM, temp = 25.0 oC, pH 8.0, I = 0.1 M (KNO3). The spectra were
recorded every 25 s.
(P8+)FeIII(OH)(H2O) + NO
(P8+)FeII(OH)(NO+)
(P8+)FeII(OH) + NO
KNO
(P8+)FeII(OH)(NO+) + H2O
kred
(P8+)FeII(OH) + NO2- + H+
OHfast
(P8+)FeII(NO) + OH-
(13)
(14)
(15)
The [NO] dependence of the reductive nitrosylation of (P8+)FeII(OH)(NO+) was studied,
and the results are displayed in Figure 11. A fit of the data to equation 12 resulted in KNO
= (2.8 ± 0.3) × 102 M-1 and kred = (4.4 ± 0.7) × 10-3 s-1. The obtained value for KNO is in a
good agreement with the corresponding value KNO = kon/koff = (2.5 ± 0.3) × 102 M-1
obtained from stopped-flow measurements at 25 oC.10a
- 96 -
Reductive Nitrosylation for Highly Charged FeIII-Porphyrin Complexes
0,0012
kobs, s
-1
0,0009
0,0006
0,0003
0,0000
0,0000
0,0002
0,0004
0,0006
0,0008
0,0010
0,0012
[NO], M
Figure 11. Plot of kobs vs [NO] for the reductive nitrosylation of (P8+)FeII(OH)(NO+).
Experimental conditions: [FeIII(P8+)]9+ = 7.0 × 10-6 M, [NO] in the range from 0 to 1.0
mM, temp = 25.0 oC, λdet = 422 nm, I = 0.1 M (KNO3), pH 8.0.
The measured kred value for (P8+)FeII(OH)(NO+) at pH 8.0 indicates that the reaction
is 10 times slower than for (P8+)FeII(H2O)(NO+) under similar experimental conditions,
which can be accounted for in terms of the effect of the OH- group in the position trans to
NO+ that will induce electron density onto the nitrosyl ligand and reduce its electrophilic
character.
A pictorial presentation of the suggested electronic effects is presented in Scheme 2, in
which (a) shows the stabilization of the nitrosyl complex by the increased electron density
at the Fe(II) center of (P8-)FeII(H2O)(NO+) and the influence of the negatively charged
porphyrin substituents; (b) indicates the opposite effect caused by the influence of
positively charged substituents in (P8+)FeII(H2O)(NO+); and (c) shows the trans-labilizing
effect caused by the OH- ligand in (P8+)FeII(OH)(NO+).
- 97 -
Reductive Nitrosylation for Highly Charged FeIII-Porphyrin Complexes
Scheme 2
CO2Θ
O
(a)
⊕
N
Θ
2OC
O
(b)
⊕
N
⊕
⊕
FeII
FeII
OH2
OH2
O
N
N
(c)
⊕
N
⊕
⊕
N
N
FeII
OH
4.4.4. Nitrite-Catalyzed Reductive Nitrosylation of (P8-)FeIII and (P8+)FeIII
The spontaneous reductive nitrosylation reactions outlined in reaction schemes 9 –
11 and 13 – 15 suggest that the produced nitrite could subsequently catalyze the reductive
nitrosylation reactions. To investigate this further, we performed a detailed study of the
nitrite-catalyzed reductive nitrosylation process by introducing larger concentrations of
nitrite into the solutions. Reductive nitrosylation of (P8-)FeII(H2O)(NO+) and
(P8+)FeII(H2O)(NO+) is accelerated significantly on addition of nitrite and depends on the
selected pH, as illustrated by the kinetic data reported in Figures 12 and 13, respectively.
It should be noted that trace impurities of nitrite in aqueous solutions of NO are too low to
induce reductive nitrosylation of (P8-)FeII(H2O)(NO+) under these conditions. The
observed rate constant for nitrite-induced reductive nitrosylation of (P8-)FeII(H2O)(NO+)
at pH 7.0 depends linearly on the nitrite concentration with an observed second-order rate
constant of 1.6 ± 0.1 M-1 s-1 (Figure 12). Similar dependencies were found for the reaction
with (P8+)FeII(H2O)(NO+) in the pH range 2 – 8 (Figure 13).
- 98 -
Reductive Nitrosylation for Highly Charged FeIII-Porphyrin Complexes
0,030
0,025
kobs , s
-1
0,020
0,015
0,010
0,005
0,000
0,000
0,005
0,010
0,015
0,020
-
[NO2 ], M
Figure 12. Nitrite concentration dependence of the reductive nitrosylation of (P8)FeII(H2O)(NO+). Experimental conditions: [FeIII(P8-)]7- = 2.0 × 10-5 M, [NO] = 1 mM,
temp = 25.0 oC, λdet = 426 nm, I = 0.1 M (with NaClO4), pH 7.0.
0,20
kobs, s
-1
0,16
pH2
pH3
pH4
pH8
0,12
0,08
0,04
0,00
0,0000
0,0004
0,0008
0,0012
0,0016
0,0020
-
[NO2 ], M
Figure 13. Nitrite concentration dependence of the reductive nitrosylation of
(P8+)FeII(H2O)(NO+) and (P8+)FeII(OH)(NO+) at pH 2.0, 3.0, 4.0, and 8.0. Experimental
conditions: Nitric acid used for pH 2.0, 0.05 M Hepes buffer for pH 3.0 and 4.0, and Tris
buffer for pH 8.0; [(P8+)FeIII]9+ = 7.5 × 10-6 M, [NO] = 1 mM, temp = 25.0 oC, λdet = 431
nm, I = 0.1 M (with KNO3).
- 99 -
Reductive Nitrosylation for Highly Charged FeIII-Porphyrin Complexes
In these experiments the indicated nitrite concentration includes the concentration of
nitrite impurities present in aqueous NO solutions. The observed second-order rate
constant has values of 55 ± 3 and 85 ± 1 M-1 s-1 at pH 2 and 4, respectively, whereas the
value at pH 8 is much smaller. The increase in rate constant with increasing pH was
studied in more detail in the range 1.8 – 4.5. The plot of kobs/[nitrite] vs pH presented in
Figure 14 clearly fits a sigmoidal function corresponding to a pKa value of 3.00 ± 0.07,
which is very close to the pKa value of HONO in 0.1 M KNO3.31 From a fit of the data, it
follows that nitrite has approximately two times the catalytic activity of HONO and that
both contribute in the studied pH range. The decrease in the catalytic effect observed at
pH 8 must be related to the influence of the hydroxy ligand that reduces the electrophilic
character of the trans coordinated NO+.
95
90
kobs/[nitrite], M s
-1 -1
85
80
75
70
65
60
55
50
45
40
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
4,0
4,5
5,0
pH
Figure
14. Plot of kobs/[nitrite] vs pH for the reductive nitrosylation of
(P8+)FeII(H2O)(NO+) in buffered aqueous solutions in the pH range 1.8 – 4.5.
Experimental conditions: [(P8+)FeIII]9+ = 2.0 × 10-5 M, [NO] = 1 mM, temp = 25.0 oC, λdet
= 431 nm, I = 0.1 M (with KNO3). Determined pKa = 3.00 ± 0.07.
According to the reaction sequence proposed for the reductive nitrosylation process
in Scheme 1, the observed rate constant can be expressed as shown in eq 16. Because KNO
- 100 -
Reductive Nitrosylation for Highly Charged FeIII-Porphyrin Complexes
and [NO] are known under the selected experimental conditions, the experimental kobs
values could be converted to the corresponding knit values, summarized in Table 3 as a
function of temperature and pressure for the studied complexes at different pH values.
The activation parameters were estimated in the usual way, for which the corresponding
straight Eyring plots are obtained.
kobs =
knitKNO[NO2-][NO]
1 + KNO[NO]
(16)
4.4.5. Suggested Mechanism and Comparison of Iron(III) Porphyrins
The results of this study clearly demonstrate the important role of the overall
charge on the porphyrin ligand and the metal center in controlling the rate of the reductive
nitrosylation reaction. In the case of the (P8-)FeII(H2O)(NO+) complex, the overall
negative charge on the porphyrin strongly reduces the positive charge on the nitrosyl
ligand, which also shows up in the relatively high pKa value of the (P8-)FeIII(H2O)2
complex. In contrast, reductive nitrosylation of (P8+)FeII(H2O)(NO+) is 1 × 102 faster and
is ascribed to the higher electrophilicity of the coordinated NO+. This also shows up in the
much lower pKa value of (P8+)FeIII(H2O)2. In the case of (P8+)FeII(H2O)(NO+), the reaction
is sensitive to the nature of the reactant, viz. HONO or NO2-, and deprotonation of the
coordinated water molecule results in a lowering of the electrophilicity of the coordinated
NO+ and a slower reaction. These effects suggest that HONO and NO2- catalyze the
reductive nitrosylation reaction through direct nucleophilic attack on coordinated NO+ in
a rate-determining step to form an intermediate FeII-N2O3 complex, which subsequently
dissociates N2O3 and binds NO in a ligand exchange process to form the final
(Pn)FeII(NO) complex. The released N2O3 undergoes rapid hydrolysis to form HONO or
NO2- depending on the pH of the solution. The mechanistic details are shown in Scheme
3. The suggested mechanism is in agreement with the rate law in eq 16, and the reported
activation parameters for knit (summarized in Table 4) can now be used to reveal further
mechanistic details of the catalytic process.
- 101 -
Table 3. Temperature and Pressure Dependence of the Nitrite-Catalyzed Reductive Nitrosylation Reactions of
(P8-)FeII(H2O)(NO+), (P8+)FeII(H2O)(NO+) and (P8+)FeII(OH)(NO+) at 25 oC
pH
temp pressure
(oC)
(MPa)
(P8-)FeII(H2O)(NO+)
(P8+)FeII(H2O)(NO+)
(P8+)FeII(H2O)(NO+)
7.0
2.0
4.0 a
3
kobs×10
(s-1)
knit
(M-1 s-1)
3
kobs×10
(s-1)
knit
(M-1 s-1)
3
kobs×10
(s-1)
(P8+)FeII(OH)(NO+)
8.0
knit
(M-1 s-1)
3
kobs×10
(s-1)
knit
(M-1 s-1)
0.1
5±1
12 ± 2
6±1
31 ± 4
0.1
9±1
23 ± 2
11 ± 1
52 ± 5
0.1
1.2 ± 0.2
0.9 ± 0.1
17 ± 1
46 ± 2
18 ± 1
92 ± 5
0.1
1.8 ± 0.3
1.4 ± 0.2
38 ± 2
99 ± 5
31 ± 2
163 ± 9
0.1
2.4 ± 0.2
2.1 ± 0.2
58 ± 2
161 ± 6
45 ± 2
251 ± 10
4.3 ± 0.6
26 ± 3
0.1
3.4 ± 0.4
3.2 ± 0.3
7.6 ± 0.3
39 ± 2
0.1
4.6 ± 0.4
5.1 ± 0.5
11.5 ± 0.9
59 ± 4
0.1
15.6 ± 0.8
81 ± 5
b
c
10
0.9 ± 0.1
59 ± 2
165 ± 6
47 ± 3
263 ± 11 2.5 ± 0.2
44 ± 3
0.6 ± 0.1
b
c
50
1.0 ± 0.1
52 ± 3
146 ± 9
40 ± 2
223 ± 9
2.4 ± 0.1
43 ± 2
0.7 ± 0.1
b
c
90
1.2 ± 0.1
48 ± 3
134 ± 8
32 ± 2
177 ± 10 2.3 ± 0.2
41 ± 3
1.0 ± 0.1
b
c
130
1.5 ± 0.2
1.5 ± 0.2
42 ± 2
116 ± 6
26 ± 2
147 ± 10 2.2 ± 0.2
40 ± 3
‡
ΔH (kJ/mol)
60 ± 2
90 ± 3
73 ± 2
68 ± 1
‡
ΔS (J/mol·K)
–36 ± 7
+99 ± 10
+92 ± 7
+5 ± 2
‡
3
–8.6 ± 0.4
+7.2 ± 0.5
+12.3 ± 0.7
+2.2 ± 0.2
ΔV (cm /mol)
8II
+
8+
II
+
For (P )Fe (H2O)(NO ) and (P )Fe (H2O)(NO ), [NO] = 1mM and [NO2 ] = 1 mM for both temperature and
pressure dependent experiments. a[NO2-] = 0.5 mM. b measured at 15 oC. c[NO] = 0.5 mM, [NO2-] = 0.5 mM. All
kinetic traces were recorded at 431 nm.
5
10
15
20
25
30
35
40
25
- 102 -
Reductive Nitrosylation for Highly Charged FeIII-Porphyrin Complexes
O
OH2
FeIII
+ NO
⊕N
KNO
FeII
OH2
OH2
•N
NO2-
knit
O
FeII
+ NO
FeII
+ N2O3
+ H2O
OH2
+ H2O
-
2NO2 + 2H+
Scheme 3. Suggested Mechanism for the Nitrite-Catalyzed Reductive Nitrosylation of
Nitrosyl Complex [(P)FeII(NO+)] According to an Innersphere Mechanism
Thermal and pressure activation parameters for the reductive nitrosylation process
were determined at pH 7 for (P8-)FeII(H2O)(NO+), and at pH 2, 4, and 8 for
(P8+)FeII(H2O)(NO+) and (P8-)FeII(OH)(NO+). The activation entropy and volume data for
reductive nitrosylation of (P8-)FeII(H2O)(NO+) are significantly negative and in line with
rate-determining bond formation between coordinated NO+ and NO2-. The bondformation process is expected to be accompanied by some charge neutralization, which
will cause a decrease in electrostriction and partially offset the negative intrinsic entropy
and volume contributions. As argued above, the electrophilicity of coordinated NO+ in
this complex is not expected to be that high because of the overall negative charge on the
porphyrin, such that the decrease in electrostriction that accompanies bond formation can
only partially compensate for the intrinsic contributions arising from bond formation. In
the case of the (P8+)FeII(H2O)(NO+) complex, however, these activation parameters show
large positive values at both pH 2 and 4, indicating that a major decrease in
electrostriction during bond formation between coordinated NO+ and HONO/NO2- must
- 103 -
Reductive Nitrosylation for Highly Charged FeIII-Porphyrin Complexes
be responsible for these values. The electrophilic character of coordinated NO+ in this
complex is expected to be much higher than in the case of the anionic complex, with the
result that charge neutralization during the interaction with HONO and NO2- is expected
to play a major role. This trend is also seen clearly when comparing the activation entropy
and volume values found at pH 2 and 4 for the reductive nitrosylation by HONO and
NO2-, respectively. The more positive values at pH 4 can be ascribed to a larger
contribution coming from a decrease in electrostriction that will be accompanied by the
release of more solvent molecules, i.e., an increase in entropy and volume, attributable to
a more effective charge-neutralization process. On going to the data for the same reaction
at pH 8, both the activation entropy and volume are much smaller (both close to zero) and
demonstrate that the decrease in electrostriction almost fully compensates the intrinsic
entropy and volume contributions expected for the bond-formation process. This is fully
in line with arguments presented above in that the presence of the hydroxo ligand clearly
decreases the electrophilicity of coordinated NO+. Thus the reported activation parameters
are fully in line with the mechanistic interpretation offered on the basis of the trends in the
observed rate constants for the reductive nitrosylation process as a function of overall
charge on the porphyrin and the pH of the solution by which coordinated water is
deprotonated in the case of the cationic complex. The subsequent reactions in Scheme 3
are all proposed to be non-rate-determining steps and as such do not affect the reported
rate and activation parameters.
The results of the present study are compared to those reported for related systems
in Table 4. The values of knit decrease steadily along the series of complexes (P8+)FeIII >
(TMPyP4+)FeIII > (TPPS4-)FeIII > (P8-)FeIII, i.e., with decreasing overall charge on the
porphyrin. Electron-donating substituents on the porphyrin slow the reductive
nitrosylation process, whereas electron-withdrawing substituents accelerate the reaction,
suggesting that they induce the formation of (P)FeII(H2O)(NO+) and increase the
electrophilicity of coordinated NO+. Activation entropies and activation volumes for
reductive nitrosylation of cationic complexes of the type (Pn+)FeII(H2O)(NO+) are all
significantly positive, whereas those for the anionic complex (P8-)FeII(H2O)(NO+) are
- 104 -
Reductive Nitrosylation for Highly Charged FeIII-Porphyrin Complexes
significantly negative. This supports the trends discussed above and can be correlated
with the electrophilicity of coordinated NO+, which in turn controls the contribution of
changes in electrostriction that accompanies bond formation between coordinated NO+
and HONO/NO2-. The consistency within the series of available data further supports the
validity of the suggested mechanism as outlined in Scheme 3. The results of the present
study clearly support the operation of an innersphere electron-transfer mechanism, which
is favored over the alternative outersphere electron-transfer mechanism discussed for a
number of systems as recently reviewed by Ford and co-workers.9,12
- 105 -
Table 4. Comparison of Rte and Activation Parameters for the Nitrite-Catalyzed Reductive Nitrosylation Reaction of a
Series of (P)FeII(H2O)(NO+) Complexes
Porphyrin
type
(P8+)Fe
Porphyrin
charge
+8
a
pKa
pH
slopeb
(M-1 s-1)
‡
knit, 25 oC
ΔH
(M s )
(kJ/mol)
-1 -1
‡
‡
ΔS
ΔV
(J/mol·K)
(cm /mol)
3
2
55 ± 3
155 ± 8
90 ± 3
+99 ± 10
+7.2 ± 0.5
4
89 ± 1
242 ± 3
73 ± 2
+92 ± 7
+12.3 ± 0.7
8
4.3 ± 0.1
22 ± 1
68 ± 1
+5 ± 2
+2.2 ± 0.2
4+
c
(TMPyP )Fe
+4
5.5
4
15.0 ± 0.1
42 ± 1
88 ± 2
+92 ± 6
+8.8 ± 0.1
4+
d
+4
5
(TMPyP )Fe
25 ± 1
83 ± 3
4d
(TPPS )Fe
7.0
5
3.1
−4
2.2 ± 0.1
89.2
7
(P )Fe
−8
1.6 ± 0.1
2.1 ± 0.2
60 ± 2
-36 ± 7
–8.6 ± 0.4
a
b
pKa value of the corresponding di-aqua complex. value calculated from kobs versus [NO2 ] at constant [NO]. c
measured at 15 °C taken from ref. 13. d taken from ref. 12.
5.0
- 106 -
Reductive Nitrosylation for Highly Charged FeIII-Porphyrin Complexes
4.5. References and Notes
1. (a) Wolak, M.; Stochel, G.; Hamza, M.; van Eldik, R. Inorg. Chem. 2000, 39, 2018. (b)
Firth, R. A.; Hill, H. A. O.; Pratt, J. M.; Williams, R. J. P.; Jackson, W. R.
Biochemistry 1967, 6, 2178.
2. (a) Weichsel, A.; Maes, E. M.; Andersen, J. F.; Valenzuela, J. G.; Shokhireva, T. K.;
Walker, F. A.; Montfort, W. R. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 594. (b)
Gladwin, M. T.; Lancaster Jr, J. R.; Freeman, B. A.; Schechter, A. N. Nat. Med. 2003,
9, 496.
3. Luchsinger, B. P.; Rich, E. N.; Gow, A. J.; Williams, E. M.; Stamler, J. S.; Singel, D. J.
Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 461.
4. Beltran, B.; Orsi, A.; Clementi, E.; Moncade, S. Br. J. Pharmacol. 2000, 129, 953.
5. (a) Stamler, J. S.; Simon, D. I.; Osborne, J. A.; Mullins, M. E.; Jaraki, O.; Michel, T. ;
Singel, D. J.; Loscalzo, J. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 444. (b) Stamler, J.
S.; Jaraki, O.; Osborne, J.; Simon, D. I.; Keaney, J.; Vita, J.; Singel, D.; Valeri, R.;
Loscalzo. J. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 7674.
6. Stamler, J. S.; Jia, L.; Eu, J. P.; Mcmahon, T. J.; Demchenko, I. T.; Bonaventura, J.;
Gernert, K.; Piantadosi, C. A. Science. 1997, 276, 2034.
7. Tran, D.; Skelton, B. W.; White, A. H.; Lavermann, L. E.; Ford, P. C. Inorg. Chem.
1998, 37, 2505.
8. Lim, M. D.; Lorkovic, I. M.; Ford, P. C. J. Inorg. Biochem. 2005, 99, 151.
9. Ford, P. C.; Fernandez, B. O.; Lim, M. D. Chem. Rev. 2005, 105, 2439.
10. (a) Jee, J.-E.; Wolak, M.; Balbinot, D.; Jux, N.; Zahl, A.; van Eldik, R. Inorg. Chem.
2006, 45, 1326. (b) Jee, J.-E.; Eigler, S.; Jux, N.; Wolak, M.; Zahl, A.; Stochel, G.;
van Eldik, R. Inorg. Chem. 2005, 44, 7717. (c) Guldi, D. M.; Rahman, G. M. A.; Jux,
N.; Balbinot, D.; Hartnagel, U.; Tagmatarchis, N.; Maurizio, P. J. Am. Chem. Soc.
2005, 127, 9830.
11. Yoshimura, T.; Suzuki, S.; Nakahara, A.; Iwasaki, H.; Masuko, M.; Matsubara, T.
Biochemistry. 1986, 25, 2436.
- 107 -
Reductive Nitrosylation for Highly Charged FeIII-Porphyrin Complexes
12. Fernandez, B. O.; Lorkobic, I. M.; Ford, P. C. Inorg. Chem. 2004, 43, 5393.
13. Theodoridis, A.; van Eldik, R. J. Mol. Catal. A: Chem 2004, 24, 197.
14. Trofimova, N. S.; Safronov, A. Y.; Ikeda, S. Inorg. Chem. 2003, 42, 1945.
15. Vilhena, F. S. D. S.; Louro, S. R. W. J. Inorg. Biochem. 2004, 98, 459.
16. Bohle D. S.; Hung, C.-H. J. Am. Chem. Soc. 1995, 117, 9584.
17. (a) Han, T. H.; Fukuto, J. M.; Liao, J. C. Nitric Oxide 2004, 10, 74. (b) Tsuge, K.;
DeRosa, F.; Lim, M. D.; Ford, P. C. J. Am. Chem. Soc. 2004, 126, 6564. (c) Selcuki,
C.; van Eldik, R.; Clark, T. Inorg. Chem. 2004, 43, 2828.
18. Enemark, J. H.; Feltham, R. D. Coord. Chem. Rev. 1974, 13, 339.
19. Wayland, B. B.; Olsen, L. W. J. Am. Chem. Soc. 1974, 96, 6037.
20. (a) Barley, M. H.; Takeuchi, K. J.; Meyer, T. J. J. Am. Chem. Soc. 1986, 108, 5876.
(b) Barley, M. H.; Rhodes, M. R.; Meyer, T. J. Inorg. Chem. 1987, 26, 1746.
21. Yoshimura, T.; Suzuki, S.; Nakahara, A.; Iwasaki, H.; Masuko, M.; Matsubara, T.
Biochim. Biophys. Acta 1985, 831, 267.
22. Yoshimura, T.; Suzuki, S. Inorg. Chim. Acta. 1988, 152, 241.
23. (a) Gow, A. J.; Luchsinger, B. P.; Pawloski, J. R.; Singel, D. J.; Stamler, J. S. Proc.
Natl. Acad. Sci. U.S.A. 1999, 96, 9027. (b) Keilin, D.; Hartree, E. F. Nature (London).
1937, 139, 548 (c) Chien, J. C. W. J. Am. Chem. Soc. 1969, 91, 2166.
24. (a) Hoshino, M.; Maeda, M.; Onischi, R.; Seki, H.; Ford, P. C. J. Am. Chem. Soc.
1996, 118, 5702. (b) Miranda, K. M.; Nims, R. W.; Thomas, D. D.; Espey, M. G.;
Citrin, D.; Bartberger, M. D.; Paolocci, N.; Fukuto, J. M.; Feelisch, M.; Wink, D. A. J.
Inorg. Biochem. 2003, 93, 52.
25. (a) Laverman, L. E.; Ford, P. C. J. Am. Chem. Soc. 2001, 123, 11614 (b) Hoshino, M.;
Laverman, L.; Ford, P. C. Coord. Chem. Rev. 1999, 187, 75 and references therein.
26. Medzon, E. L.; Gedies, A. Canadian J. Microbiol. 1971, 17, 651.
27. Spitzer, M.; Gartig, F.; van Eldik, R. Rev. Sci. Instrum. 1988, 59, 2092.
28. van Eldik, R.; Gaede, W.; Wieland, S.; Kraft, J.; Spitzer, M.; Palmer, D. A. Rev. Sci.
Instrum. 1993, 64, 1355.
- 108 -
Reductive Nitrosylation for Highly Charged FeIII-Porphyrin Complexes
29. Wanat, A.; Gdula-Argasinska, J.; Rutkowska-Zbik, D.; Witko, M.; Stochel, G.;
van Eldik, R. J. Biol. Inorg. Chem. 2002, 7, 165.
30. (a) Munro, O. Q.; Scheidt, W. R. Inorg. Chem. 1998, 37, 2308 (b) Nasri, H.; Goodwin,
J. A.; Scheidt, W. R. Inorg. Chem. 1990, 29, 185.
31. Riordan, E.; Minogue, N.; Healy, D.; O`Driscoll, P.; Sodeau, J. R. J. Phys. Chem. A.
2005, 109, 779, and references therein. An often quoted literature value is pKa = 3.27
- 109 -
5. Influence of an Extremely Negatively Charged Porphyrin
on the Reversible Binding Kinetics of NO to Fe(III)
and the Subsequent Reductive Nitrosylation*
5.1 Abstract
The polyanionic, water-soluble, and non-μ-oxo dimer-forming iron porphyrin
(hexadecasodium
iron
54,104,154,204-tetra-t-butyl-52,56,102,106,152,156,202,206-
octakis[2,2-bis(carboxylato)ethyl]-5,10,15,20-tetraphenylporphyrin), (P16−)FeIII, with 16
negatively charged meso substituents on the porphyrin was synthesized and fully
characterized by UV-vis and 1H NMR spectroscopy. A single pKa1 value of 9.90 ± 0.01
was determined for the deprotonation of coordinated water in the six-coordinate
(P16−)FeIII(H2O)2 and as attributed to the formation of the five-coordinate monohydroxoligated form, (P16−)FeIII(OH). The porphyrin complex reversibly binds NO in aqueous
solution to yield the nitric oxide adduct, (P16−)FeII(NO+)(L), where L = H2O or OH–. The
kinetics for the reversible binding of NO were studied as a function of pH, temperature,
and pressure using the stopped-flow technique. The data for the binding of NO to the
diaqua complex are consistent with the operation of a dissociative mechanism on the basis
of the significantly positive values of ΔS‡ and ΔV‡, whereas the monohydroxo complex
favors an associatively activated mechanism as determined from the corresponding
negative activation parameters. The rate constant, kon = 3.1 × 104 M-1 s-1 at 25 °C,
determined for the NO binding to (P16−)FeIII(OH) at higher pH, is significantly lower than
the corresponding value measured for (P16−)FeIII(H2O)2 at lower pH, namely, kon = 11.3 ×
105 M-1 s-1 at 25 °C. This decrease in the reactivity is analogous to that reported for other
diaqua- and monohydroxo-ligated ferric porphyrin complexes, and is accounted for in
terms of a mechanistic changeover observed for (P16−)FeIII(H2O)2 and (P16−)FeIII(OH). The
formed nitrosyl complex, (P16−)FeII(NO+)(H2O), undergoes subsequent reductive
nitrosylation to produce (P16−)FeII(NO), which is catalyzed by nitrite produced during the
reaction. Concentration-, pH-, temperature-, and pressure-dependent kinetic data are
Influence of an Extremely Negatively Charged FeIII-porphyrin
reported for this reaction. Data for the reversible binding of NO and the subsequent
reductive nitrosylation reaction are discussed in reference to that available for other
iron(III) porphyrins in terms of the influence of the porphyrin periphery.
*Joo-Eun Jee, Siegfried Eigler, Norbert Jux, Achim Zahl, and Rudi van Eldik, Inorg. Chem. 2007,
46(8), 3336-3352
5.2 Introduction
Nitric oxide (NO) is a versatile signaling molecule and its interaction with metalcentered proteins plays an important role. In the human system, NO is associated with
biological functions ranging from vasodilatation of vascular smooth muscle cells to
neurotransmission, cytotoxic immune response, inflammation and regulation of cell
death.1 It is generated in vivo by NO synthase in various organs from arginine with the
aid of molecular oxygen and NADPH.1,2 Nitric oxide is very reactive to Fe(II) and Fe(III),
for which different electronic and structural factors of the iron center influence the
chemical properties of the resulting nitrosyl product as well as the NO binding and
dissociation rate constants. The reactivity of iron porphyrins is finely regulated by a
variety of structural and electronic features, for example, the nature of the axial ligands,
the type of substituents on the porphyrin periphery, the polarity of the reaction medium,
and other factors. To investigate the role of these factors, numerous spectroscopic,
structural, and mechanistic studies are reported on the interaction of NO with synthetic
model complexes.3
Mechanistic studies on the reactions of NO with heme proteins or model ferric
porphyrins have been performed by the application of laser flash photolysis and stoppedflow techniques. Studies on the reversible binding of NO to synthetic iron(II) and iron(III)
porphyrins,4,5 and iron(III) heme proteins6-10 such as cytochrome P450 and metmyoglobin, have been performed in which distinctive features of the active site of heme
proteins were used to develop useful bio-mimetic model systems.
- 111 -
Influence of an Extremely Negatively Charged FeIII-porphyrin
The binding of NO to an oxidizing metal (Mn+) induces intramolecular electron
transfer, leading to the formation of (P)M(n-1)+−NO+, followed by nitrosylation of a
nucleophile (Nu−) to produce (P)M(n−1)+ and Nu−−NO+. The observed reaction involves
reductive nitrosylation as expressed in reaction 1. Recent reports have supported that
reductive nitrosylation is proposed as a viable pathway in which hemoglobin binds NO to
the cys β−93 residue and forms S-nitrosohemoglobin in the transport and metabolism of
NO.11,12
(P)Mn+ + NO
KNO
(P)M(n−1)+−NO+
+Nu−
(P)M(n-1)+ + Nu−−NO+
(1)
On the basis of spectroscopic and kinetic data, the NO reduction of ferric-heme
proteins (viz., ferric cytochrome c, metmyoglobin and methemoglobin)13-15 and synthetic
model ferric porphyrins such as TPPS (tetra-(4-sulfonatophenyl)-porphyrinato), TMPyP
(meso-tetrakis(N-methyl-4-pyridyl) porphyrinato),6,16,17 P8+ (5,10,15,20-tetrakis-(4’-tbutyl-2’,6’-bis(4-t-butylpyridine)phenyl)- porphyrinato),18 and P8- (54,104,154,204-tetra-tbutyl-52,56,152,156-tetrakis-(2,2-bis-carboxylato-ethyl)-5,10,15,20tetraphenylporphyrinato)18 were systematically studied in aqueous medium, in which the
resulting nitrosyl adducts ((P)FeIII + NO Æ (P)FeII−NO+) interact with a nucleophile,
namely, OH− and NO2−, to yield ferroheme proteins. Recent studies performed in our
laboratories clearly revealed that the observed rate constant for NO reduction depends on
the concentration of NO and OH− and suggested that the porphyrin environment involving
oppositely charged substituents has a crucial influence on the observed rate constants and
mechanistic features of nitrite-catalyzed reductive nitrosylation.18
The goal of these investigations is to understand the influence of the iron porphyrin
micro-environment on the reactivity with NO and the stability of the resulting
(P)FeII(NO+) species toward subsequent reaction in aqueous solution. In this context, the
reported studies were undertaken to investigate the systematic influence of the porphyrin
environment on the properties and reactivity of water-soluble ferric porphyrins.3-7 We
now report the synthesis and spectroscopic characterization of an extremely negatively
- 112 -
Influence of an Extremely Negatively Charged FeIII-porphyrin
charged iron(III) porphyrin, (P16−)FeIII(L)2 (hexadecasodium iron 54,104,154,204-tetra-tbutyl-52,56,102,106,152,156,202,206-octakis[2,2-bis(carboxylato)ethyl)]-5,10,15,20tetraphenylporphyrin) (Figure 1), and evaluate the detailed kinetics of its interaction with
NO. In the latter context, variable pH-, temperature-, and pressure-dependent stoppedflow measurements provided a detailed kinetic and mechanistic description of the
reversible binding of NO to (P16−)FeIII(H2O)2 and (P16−)FeIII(OH) present in aqueous
solution at low and high pH, respectively. The subsequent reductive nitrosylation was also
studied as a function of concentration, temperature, and pressure. The results are
discussed in reference to kinetic data and mechanistic information reported for other
water-soluble iron(III) porphyrins.
O
O
O
O
L
N
R
O
O
O
O
O
O
O
N Fe
O
O
O
O
O
R
N
R=
N
O
O
L
O
O
O
16 Na
O
O
O
Figure 1. Structure of (P16–)FeIII(L)2 where L = H2O or OH–
5.3. Experimental section
Synthesis and Characterization of (P16−)FeIII. The chemicals and solvents employed for
the synthesis of (P16−)FeIII were used as received unless otherwise noted. Solvents were
dried using standard procedures. Column chromatography was performed on silica gel 60,
32–63 μm, 60 Å (MP Biomedicals). Standard 1H and 13C NMR spectra were recorded on
a Bruker Avance 300 spectrometer. FAB mass spectrometry was performed with
Micromass Zabspec. Standard UV-vis spectra were recorded on a Shimadzu UV-3102 PC
UV-vis NIR scanning spectrophotometer. IR spectra (KBr pellets) were recorded with a
- 113 -
Influence of an Extremely Negatively Charged FeIII-porphyrin
FT-IR IFS 88 infrared spectrometer (Bruker Analytische Messtechnik GmbH). Elemental
analyses
were
carried
out
on
a
CHN-Mikroautomat
(Heraeus).
Thin-layer
chromatography (TLC) was carried out on E. Merck silica gel 60 F254 plates. Zinc(II)54,104,154,204-tetra-t-butyl-52,56,102,106,152,156,202,206-octabromo-5,10,15,20tetraphenylporphyrin (2) was synthesized as described previously.19
Zinc(II)-54,104,154,204-tetra-t-butyl-52,56,102,106,152,156,202,206-octakis[2,2bis(methoxycarbonyl)ethyl)]-5,10,15,20-tetraphenylporphyrin (3): KH (24 mg, 0.60
mmol) was suspended in DMF (20 mL) under a N2-atmosphere. Dimethyl malonate (160
mg, 1.20 mmol in 20 mL DMF) was added dropwise at room temperature (rt). After an
additional hour, porphyrin 2 (100 mg, 0.061 mmol) was added, and the solution was
stirred for 4 h at rt. The reaction mixture was poured onto an ice/water mixture to
precipitate the product. The solid was filtered and washed thoroughly with water. The
purple residue was dissolved in CH2Cl2, dried over MgSO4, and concentrated in vacuum.
The product was further purified by column chromatography (SiO2, CH2Cl2/EtOAc 10:1)
and was obtained as a purple solid. Yield: 104 mg (83%, 0.050 mmol). 1H NMR (400
MHz, CDCl3, rt): δ 8.64 (s, 8H, β-pyrr.-H), 7.43 (s, 8H, o-Ar–H), 3.18 (s, 48H, CH3), 3.07
(t, 8H, 3J=8.3 Hz, CH), 2.79 (d, 16H, 3J=8.3 Hz, Ar-CH2), 1.52 (s, 36H, CH3); 13C NMR
(75 MHz, CDCl3, rt): δ 169.0, 151.2, 150.1, 139.3, 139.0, 131.4, 124.6, 115.7, 52.2, 51.9,
34.8, 34.1, 31.6 ppm; MS (FAB, NBA): m/z 2055 (M+.); IR (KBr): ν [cm-1] 2957, 2907,
2872, 1737, 1436, 1332, 1285, 1227, 1200, 1150, 1065, 1027, 992, 883, 799, 725; UV-vis
(CH2Cl2): λ [nm] (ε [l mol-1 cm-1]) 430 (3.94 × 105), 561 (1.27 × 104), 599 (1.1 × 103).
54,104,154,204-Tetra-t-butyl-52,56,102,106,152,156,202,206-octakis[2,2bis(methoxycarbonyl)ethyl)]-5,10,15,20-tetraphenylporphyrin (4): HCl (6M, 50 mL)
was added to a solution of 3 (80 mg, 0.039 mmol) in CH2Cl2 (50 mL), and the two layers
were shaken vigorously. The green (organic) layer was shaken once again with 2 M HCl
(50 mL) and twice with water (50 mL each). After neutralization with a saturated
NaHCO3 solution (50 mL) and a final wash with brine (50 mL), the organic layer was
dried with MgSO4, and the solvent was removed under reduced pressure. The compound
was further cleaned by column chromatography (silica gel, CH2Cl2/ethyl acetate 9:1) and
- 114 -
Influence of an Extremely Negatively Charged FeIII-porphyrin
was obtained as a purple powder. Yield: 76 mg (98%, 0.038 mmol). 1H NMR (300 MHz,
CDCl3, rt): δ 8.71 (s, 8H, β-pyrr.-H), 7.45 (s, 8H, o-Ar-H), 3.16 (t, 8H, 3J=8.2 Hz, CH),
3.12 (s, 48H, CH3), 2.79 (d, 16H, 3J=8.2 Hz, Ar–CH2), 1.53 (s, 36H, CH3), -2.51 (s, 2H,
NH);
C NMR (75 MHz, CDCl3, rt): δ 168.6, 151.7, 139.1, 137.8, 124.4, 115.7, 51.9,
13
51.8, 34.8, 33.7, 31.5. MS (FAB, NBA): m/z 1991 (M+.), 1932 (M+.-COOCH3); IR (KBr):
ν [cm-1] 2957, 2907, 2872, 1733, 1606, 1559, 1436, 1343, 1278, 1227, 1197, 1150, 1061,
1027, 965, 887, 868, 803, 737; UV-vis (CH2Cl2): λ [nm] (ε [l mol–1 cm–1]) 422 (4.31 ×
105), 517 (1.67 × 104), 550 (2.3 × 103), 593 (3.7 × 103), 646 (1.0 × 103).
Chloroiron(III)
54,104,154,204-Tetra-t-butyl-52,56,102,106,152,156,202,206-octakis[2,2-
bis(methoxycarbonyl)ethyl)]-5,10,15,20-5,10,15,20-tetraphenylporphyrin (5): FeCl2
(230 mg, 1.81 mmol) was added to a solution of 4 (230 mg, 0.12 mmol) in THF (30 mL)
and the mixture was heated under reflux for 24 h. The solvent was evaporated, and the
residue was dissolved in CH2Cl2 and washed with 6 M HCl. The organic layer was
separated and washed twice with water. After it was dried over MgSO4, the compound
was precipitated to give a dark brown powder. Yield: 189 mg (73%, 0.088 mmol). 1H
NMR (300 MHz, CDCl3, rt): δ 82.6 (br s, β-pyrr.-H), 16.6, 14.6 (br s, aryl-H); MS (FAB,
NBA): m/z 2046 (M+-Cl); IR (KBr): ν [cm-1] 2957, 2907, 2872, 1733, 1606, 1436, 1227,
1197, 1150, 1031, 995, 883, 837, 802, 725. UV-vis (CH2Cl2): λ [nm] (ε [mol-1 cm-1]) 391
(5.85 × 105), 427 (9.39 × 105), 517 (1.38 × 104), 579 (3.8 × 103), 692 (2.7 × 103).
Hexadecasodium
hydroxoiron(III)
54,104,154,204-tetra-t-butyl-
52,56,102,106,152,156,202,206-octakis[2,2-bis(carboxylato)ethyl)]-5,10,15,20tetraphenylporphyrin (1-OH): NaOH (1.50 g, 37.5 mmol) was added to a solution of 5
(150 mg, 0.072 mmol) in ethanol (20 mL), and the reaction mixture was heated under
reflux for 2 h. After the mixture was cooled to room temperature, the precipitate was
filtered, washed with ethanol (200 mL), and dried under reduced pressure. Gel permeation
chromatography (Sephadex LH20) in methanol and the subsequent precipitation with
diethyl ether gave a dark brown powder which, according to the microanalysis, contains
sodium hydroxide in the lattice. Yield: 230 mg (86%, 0.061 mmol, based on formula
- 115 -
Influence of an Extremely Negatively Charged FeIII-porphyrin
obtained by microanalysis). 1H NMR (300 MHz, pD = 7, rt): δ 48.6 (br s, β-pyrr.-H),
9.71, 3.75, 2.81; IR (KBr): ν [cm-1] 2964, 2876, 1563, 1405, 1359, 1332, 1290, 1065, 999,
876, 837, 802; UV-vis (H2O, unbuffered): λ [nm] (ε [mol-1 cm-1]) 324 (2.4 × 104), 401
(7.5 × 104), 510 (1.0 × 104). Anal. calcd for C92H77FeN4Na16O33 38NaOH: C, 29.78; H,
3.12; N, 1.51. Found: C, 29.48; H, 3.24; N, 1.27.
Materials. Mes, Bis-Tris, Taps, sodium borate, Caps, and NaOH were used as buffer
solutions and were purchased from Sigma-Aldrich. All chemicals used in this study were
of analytical grade reagent. The NO gas (Messer Griesheim or Riessner Gase, ≥ 99.5 vol
%) was purified from impurities of nitrogen oxides such as NO2 and N2O3 by being
passed through an Ascarite II column (NaOH on silica gel from Sigma-Aldrich) and
concentrated NaOH solution.
Solution Preparation. All solutions were prepared under strict oxygen-free conditions
with Milli-Q water and handled in gastight glassware because of the high oxygensensitivity of NO and the nitrosyl complexes. Oxygen-free Ar and N2 were used to
prepare deoxygenated solutions. Buffered solutions of the appropriate pH for stoppedflow measurements were prepared with Mes, Bis-Tris, Taps, Sodium borate, and Caps
buffers at a concentration of 0.05M. The pH of the solution was controlled with HClO4
and NaOH. The ionic strength was kept constant at 0.1 M with NaClO4.
Measurements. pH measurements were carried out on a Metrohm 623 pH meter
equipped with a Sigma glass electrode. A NO electrode (World Precision Instruments
isolated nitric oxide meter, model ISO-NO) was used to determine the concentration of
NO gas in aqueous solution. The NO electrode was calibrated with a freshly prepared
KNO2 solution according to the method suggested by the manufacturer. UV–vis spectra
were recorded in gastight cuvettes on a Shimadzu UV–2100 spectrophotometer equipped
with a thermostated cell compartment CDS-260. UV–vis spectra at pressures up to 150
MPa were recorded in a custom-built high-pressure optical cell.20
Stopped-flow kinetic measurements on the reaction of NO with (P16−)FeIII at pH 6.5
and 12.7 were carried out with an Applied Photophysics SX–18MV stopped-flow
spectrophotometer. Deoxygenated aqueous solutions of (P16−)FeIII were rapidly mixed in
- 116 -
Influence of an Extremely Negatively Charged FeIII-porphyrin
varying volume ratios with a saturated NO solution in a gastight syringe to obtain the
appropriate NO concentration (0.2 – 2 mM). The kinetics of the reaction were monitored
at 430 and 435 nm where the change in absorbance is a maximum for the nitrosyl adducts
at pH 6.5 and 12.7, respectively. The rates of NO binding (kon) and release (koff) were
determined from the slope and intercept of linear plots of kobs versus [NO], respectively,
as described in more detail in the Results and Discussion. More accurate NO dissociation
rates at different temperatures and pressures were measured directly by the NO-trapping
method, in which [RuIII(edta)(H2O)]– was used as an efficient NO scavenger. This
involved rapid mixing of a (P16−)FeII(NO+)(L), L = H2O or OH–, solution (2 × 10-5 M, L =
H2O and OH– at pH 6.5 and 12.7, respectively) containing a small excess of NO with
aqueous solutions of [RuIII(edta)(H2O)]– (1 – 2 mM) to give [RuIII(edta)(NO)]– and
(P16−)FeIII(L)2, as shown by the observed spectral change. The kinetics of NO release was
followed in the stopped-flow spectrophotometer at 430 (pH 6.5) or 435 nm (pH 12.7). We
also measured the rate constants for the conversion of (P16−)FeII(H2O)(NO+) to
(P16−)FeII(NO). The observed rate constants and activation parameters for the reactions
with NO/nitrite were monitored at 431 nm where the change in absorbance is at a
maximum. All kinetic experiments were performed under pseudo-first-order conditions,
that is, with at least a 10-fold excess of NO over the iron porphyrin. Reported rate
constants are the mean values of at least five kinetics runs, and the quoted uncertainties
are based on the standard deviation. High-pressure stopped-flow studies were performed
on a custom-built instrument (from 10 to 130 MPa).21 Kinetic traces were recorded on an
IBM-compatible computer and analyzed with the OLIS KINFIT (Bogart, GA) set of
programs.
17
O NMR Water Exchange Measurements. Rate constants for water exchange on the
paramagnetic (P16−)FeIII complex and the corresponding activation parameters, ΔH‡ex,
ΔS‡ex, and ΔV‡ex were measured at pH = 6.5 and 12.7 using a 17O NMR line broadening
technique. Aqueous solutions of (P16−)FeIII (20 mM) were prepared at pH 6.5 (Mes buffer
solution adjusted with HClO4) and 12.7 (with NaOH). Both solutions were kept at 0.1 M
NaClO4 for the approximate ionic strength, in which 10% of the total sample volume of
- 117 -
Influence of an Extremely Negatively Charged FeIII-porphyrin
enriched 17O-labeled water (normalized 19.2% 17O H2O, D-Chem Ltd) was added to each
solution. A sample containing the same components without the (P16−)FeIII complex was
used as a reference. Variable-temperature and -pressure Fourier transform
17
O NMR
spectra were recorded at a frequency of 54.24 MHz on a Bruker Advance DRX 400WB
spectrometer. The temperature dependence of the
17
OH2 line broadening was studied in
the range of 278 – 353 K. A homemade high-pressure probe22 was used for the variablepressure experiments performed at the selected temperature (at 293 K at pH 6.5) and in
the pressure range of 1 – 150 MPa. The sample was placed in a standard 5 mm NMR tube
cut to a length of 45 mm. Hydrostatic pressure was transmitted to the sample by a
movable macor piston, and the temperature was controlled as described elsewhere.22 The
reduced transverse relaxation times (1/T2r) were calculated for each temperature and
pressure from the difference in the line widths observed in the presence and absence of
the metal complex, (Δνobs – Δνsolvent). The reduced transverse relaxation time is related to
the exchange rate constant kex = 1/τm (where τm is the mean coordinated solvent lifetime)
and to the NMR parameters by the Swift and Connick equation (2),22-24 as described
previous section 2.
1
T2r
= π
1
Pm
(Δνobs – Δνsolvent) =
1
T2m-2 + (T2mτm)-1 + Δω2m
τm
(T2m-1 + τm-1)2 + Δω2m
+
1
T2os
(2)
In analogous temperature-dependent 17O NMR measurements performed for the (P16–)FeIII
complex at pH 12.7 (0.05M NaOH), the line-width differences in the presence and
absence of the metal complex were very small, indicating the absence of a significant
water-exchange process for the (P16-)FeIII form present at higher pH. Because of the small
observed differences, the data obtained in the variable-temperature study did not enable a
reliable fit to the Swift and Connick equation.
Electrochemical Measurement. Cyclic voltammetric measurement was carried out using
an Autolab PGSTAT 30 device (Eco Chemie). A conventional three-electrode
arrangement was employed consisting of a gold disk working electrode (geometric are =
0.07cm2), a platinum wire auxiliary electrode, and a Ag/AgCl, NaCl (3M) reference
- 118 -
Influence of an Extremely Negatively Charged FeIII-porphyrin
electrode, manufactured from Metrohm. The measurements in aqueous solution were
performed in 0.1M Bis-tris electrolyte and 25oC. Complex concentration was 1mM. The
solution was initially thoroughly degassed with purified nitrogen (15min), and a stream of
N2 gas was passed over the sample solutions during the measurements.
5.4. Results and Discussion
5.4.1. Synthesis of (P16-)FeIII (1).
We choose porphyrin 2 as a starting material19 because its bromomethyl groups
can be substituted easily with potassium malonates to yield the malonate ester porphyrin 3.
Demetalation with concentrated hydrochloric acid transformed 3 into the free-base
porphyrin 4 as shown in Scheme 1. Subsequent reaction with ferrous chloride in THF
gave the neutral iron(III) porphyrin octa-malonic ester system 5. 1H NMR and UV–vis
spectroscopic data clearly show that 5 is a paramagnetic iron(III) porphyrin with a single
chloro axial ligand. The β-pyrrole 1H resonance at 82.6 ppm (half-width of ~ 300 Hz)
indicates the overall C4v symmetry of the porphyrin and proves the S = 5/2 spin state.25
R
R1
R
R
R
N
1. HCl, CH2Cl2
2. FeCl2, THF
N
Zn
N
R
R1
N
N
R1
R1
R1
R
R
R
Cl
Fe
N
N
N
R1
R1
R1
R = Br
O
NaOH, EtOH
(2)
(5)
R2
R2
R2
R2
OH
N
Fe
N
N
N
R2
R2
R2
R2
O
R2 =
(1-OH)
Scheme 1. Synthesis of (P16-)FeIII
- 119 -
NaO
O
ONa
R1 =
O
O
O
Influence of an Extremely Negatively Charged FeIII-porphyrin
Saponification of the malonic esters of 5 with sodium hydroxide in ethanol leads to
precipitation of a brownish material that is soluble in water but totally insoluble in apolar
solvents. Gel permeation chromatography (Sephadex LH20) in methanol and subsequent
precipitation with diethyl ether gave a solid material containing about 75% of 1 (the
impurities being NaOH and H2O) as was found earlier for (P8–)FeIII in a similar way.5(a)
Standard NMR spectra measured in unbuffered D2O have shown that under these
conditions 1 exists as a high-spin monohydroxo complex (P16–)FeIII(OH) (β-pyrrole 1H at
80.0 ppm, see also further text).
Two structural details which are important with regard to the investigations presented
in this paper can be extracted from the previously shown crystal structure of the zinc
precursor of (P8–)FeIII crystallized from THF.5(a) First, the malonate groups are located
above and below the porphyrin plane, thus not allowing for the formation of μ-oxo-dimer
formation for steric and electrostatic reasons (as confirmed by in-depth spectroscopic
studies described below). Second, the malonate groups cannot coordinate to the metal
center in an intramolecular fashion without extreme distortion of the molecule (the
average distance of the malonate oxygen atoms from the zinc atom is ~7 Å, and the
closest distance is ~4 Å). Nevertheless, ester or carboxylate groups of malonate
substituents may interact with axial ligands coordinated to the metal center.
5.4.2. Studies on (P16–)FeIII
The speciation of (P16–)FeIII as a function of pH in aqueous medium was studied by
UV–vis, 1H NMR, and
17
O NMR techniques. A spectrophotometric titration of a (P16–
)FeIII solution in the pH range of 1 – 13 at 0.1 M ionic strength (adjusted with NaClO4)
resulted in the spectral changes presented in Figure 2. The spectral changes are separated
into two parts, and the corresponding plot of absorbance at 420 nm versus pH is shown in
the inset of Figure 2b. In the pH range of 1 – 6, the absorbance continuously increases
over the whole spectral range as a result of the deprotonation of the carboxylic acid
groups upon increasing the pH which is accompanied by an increase in the solubility of
the porphyrin complex (see Figure 2a).
- 120 -
Influence of an Extremely Negatively Charged FeIII-porphyrin
0,8
0,7
1,0
(a)
1,0
(b)
0,9
0,8
0,8
0,7
0,5
0,4
0,3
0,2
Absorbance
Absorbance
Absorbance
0,6
0,6
0,6
0,5
0,4
0,3
0,2
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
pH
0,4
0,2
0,1
0,0
300
400
500
600
700
0,0
250
300
350
400
450
500
550
600
650
700
Wavelength, nm
Wavelength, nm
Figure 2. UV-vis spectral changes observed for aqueous solutions of (P16−)FeIII in the pH
ranges of (a) 1 − 6 and (b) 6 − 13. The inset shows a plot of the absorbance at 420 nm
versus the pH in the range of 1 – 13. Experimental conditions: [(P16−)FeIII] = 1.1 × 10-4 M,
temp = 25 °C, I = 0.1 M (with NaClO4).
As can be seen in Figure 2b, an increase in pH from 6 to 13 leads to a gradual shift of the
peaks at 398 (ε = 6.4 × 104 M–1 cm–1) and 532 nm (ε = 1.04 × 104 M–1 cm–1) to 418 (ε = 7.2
× 104 M–1 cm–1) and 606 nm (ε = 7.1 × 103 M–1 cm–1). The observed pattern is similar to
that reported for other water-soluble iron(III) porphyrins and typical for the formation of
monohydroxo-ligated species from the corresponding diaqua-ligated (P)FeIII(H2O)2.25,26
No further spectral changes occurred in solution at high pH upon extended standing, and
the observed spectral changes in the pH range of 6 – 13 were fully reversible. This
indicates rapid interconversion of species present at high and low pH upon changing the
pH and suggests that the possible formation of a μ-oxo dimer does not occur in the
present system. This interpretation is further supported by the NMR data reported below.
Two pKa values were determined from the plot in Figure 2b, from which the first pKa of ~
2 in the range of 1 < pH < 6 is attributed to the deprotonation of the carboxylic acid
groups (reaction 3) on the modified porphyrin in reference to the pKa values for
benzylmalonic acid of 2.56 and 5.22.5 The second pKa1 = 9.90 ± 0.01 in the pH range 6 –
13 is attributed to the deprotonation of coordinated water as described by reaction 4.
- 121 -
Influence of an Extremely Negatively Charged FeIII-porphyrin
(H16P)FeIII(H2O)2
(P16–)FeIII(H2O)2 + 16 H+
1 < pKa(RCOOH) < 6
(3)
(P16–)FeIII(H2O)2
(P16–)FeIII(H2O)(OH–) + H+
pKa1 = 9.90 ± 0.01
(4)
On the basis of the data summarized in Table 1, the pKa1 value for (P16–)FeIII(H2O)2
is the highest value reported for water-soluble iron(III) porphyrins so far and must be
related to the extremely negative charge on the porphyrin substituents, which increases
the electron density on the iron(III) center in (P16–)FeIII(H2O)2 and, as a result, increases
the pKa1 value. Another reason for the high pKa1 value can be related to the through-space
interaction of deprotonated carboxylate groups of the flexible malonate substituents on
the porphyrin with the coordinated water molecules stabilized by hydrogen bonding, as
reported for other negatively charged water-soluble ferric porphyrins, namely, (P8–
)FeIII(H2O)2 and (TanP4–)FeIII(H2O)2.5a,27
1
H NMR measurements were carried out for (P16–)FeIII(H2O)2 and (P16–
)FeIII(H2O)(OH–) at pD values of 6.5 and 12.7. 1H NMR spectra of complexes of the type
(P)FeIII(L)n (n = 1 and 2) have extensively been analyzed and serve as an excellent means
of identification of the product being formed,28 in which the β-pyrrole proton signal is
sensitive to the spin and ligation state of the iron center by hyperfine interaction specially
for paramagnetic iron(III) porphyrins. The chemical shift of the β-pyrrole resonance in
the purely high spin state (S = 5/2) iron(III) porphyrin has a value of approximately +80
ppm, characteristic for the (P)Fe–X type (X = OH–, Cl–) complex,25 whereas in the case of
pure intermediate spin state (S = 3/2) iron(III) porphyrins the β-pyrrole 1H resonance
moves upfield to approximately –60 ppm. The β-pyrrole resonance in the intermediate
spin admixed state (S = 3/2, 5/2) is in between +80 and –60 ppm. Our 1H NMR data
indicates a β-pyrrole resonance at +45.6 ppm for pD = 6.5 that shifts downfield to +80
ppm at pD = 12.7. The broad β-pyrrole resonance at +80 ppm at high pH is diagnostic for
a monomeric high spin, monohydroxo-ligated iron(III) porphyrin, consistent with the
other reported water-soluble ferric porphyrins.4,5,25
- 122 -
Table 1. pKa1 Values and β-pyrrole 1H NMR Chemical Shifts of Synthetic Water-Soluble Iron(III) Porphyrins
Iron(III) porphyrina
(P
16−
)Fed
8
(P −)Fee
(TMPS4−)Fef (TPPS4−)Fe (4-TMPyP4+)Fe
8+
(P )Feh
OOC
OOC
COO
COO
SO3
N
N
SO3
meso phenyl substituents
COO
COO
OOC
OOC
pKa1b
β-pyrrole
1
H (ppm)c
N
9.9
9.3
6.9
7.0
5.5
5.0h
(P)FeIII(H2O)2
45.6
45.6, 46.7
43
52g
70g
66
(P)FeIII(OH)
80
82
82
NAg
NAg
83
a
The quoted charge represents the overall charge on the meso substituents in a given porphyrin ligand;
Referenced to TMPS; d This work; e Ref 5a; f Ref 4; g Ref 28a, NA = not assigned; h Ref 5b.
- 123 -
b
Ref 36;
c
Influence of an Extremely Negatively Charged FeIII-porphyrin
The absence of a β-pyrrole resonance at ∼ 13 ppm, characteristic for the ((P)FeIII)2O
dimer, excludes the formation of the μ-oxo-bridged binuclear species at high pH. The
observed signal for the diaqua-ligated iron(III) porphyrins exhibits variable δβ-py values in
the range from 43 – 70 ppm, which indicate a varying contribution of S = 3/2 in the spinadmixed mixture (S = 3/2, 5/2) from which it follows that Int % is 24.5 for (P16–)Fe(H2O)2,
similar to that found for other (Pn–)Fe(H2O)2. The observed data for (Pn+)FeIII(H2O)2
shows a marked downfield shift of the β-pyrrole resonance toward the high-spin value
with the increasing electron-withdrawing capability of the porphyrin substituents.
Temperature- and pressure-dependent 17O NMR measurements were performed at
pH 6.5 and 12.7. Detailed variable-temperature and -pressure measurements gave the
‡
‡
‡
water exchange rate constants (kex) and activation parameters (ΔH ex, ΔS ex, and ΔV ex).
The positive values of ΔS
‡
ex
= +66 ± 10 J mol–1 K–1 and ΔV
‡
ex
= +6.5 ± 0.3 cm3 mol–1 at
pH = 6.5 support the operation of a dissociative interchange (Id) mechanism for the waterexchange process on (P16–)FeIII(H2O)2, which is similar to that found for other diaqualigated porphyrins (Id or D).5, 7c From the analysis of the data, the obtained rate constant
for water exchange, kex = 4.2 × 106 s-1 at pH 6.5 and 25 °C, is in the similar range of kex
values reported for complexes with negatively charged meso substituents on the
porphyrin environment. Conversely, the smaller kex values determined for the positively
charged iron porphyrins of TMPyP4+ and P8+ suggest that the lability of the metal center is
decreased by the influence of the positive porphyrin periphery, thus, stabilizing the
FeIII−H2O bond through an inductive effect. Although there is a trend of increased lability
for the P16– and P8– complexes as compared to the TMPS4– complex, the water exchange
process for the P16– and P8– complexes is slower than for the TMPS4– complex, which
could be attributed to the steric hindrance of the bulky negative carboxylate substituents
that prevent the exchange of coordinated water in a dissociative manner. The data
obtained for (P16–)FeIII(H2O)2 and related results for other iron(III) porphyrins are
summarized in Table 2.
- 124 -
Influence of an Extremely Negatively Charged FeIII-porphyrin
Table 2. Rate Constants (at 298 K) and Activation Parameters for Water Exchange
Reactions of (P)FeIII(H2O)2 Complexes
Iron(III)
porphyrin
(P16-)Feb
Int%a
kex
ΔH
‡
ex
‡
ΔS
ex
‡
ΔV
ex
(s )
-1
(kJ mol-1)
(J mol-1 K-1)
(cm mol-1)
24.5
(42 ± 5) × 105
55 ± 3
+66 ± 10
+6.5 ± 0.3
(P8-)Fec
24
(77 ± 1) × 105
61 ± 6
+91 ± 23
+7.4 ± 0.4
(TPPS4-)Fed
20
(20 ± 1) × 105
67 ± 2
+99 ± 10
+7.9 ± 0.2
26
(210 ± 10) × 105
61 ± 1
+100 ± 5
+11.9 ± 0.3
7
(4.5 ± 0.1) × 105
71 ± 2
+100 ± 6
+7.4 ± 0.4
4-
e
(TMPS )Fe
4+
d
(TMPyP )Fe
8+
3
f
53 ± 3
+28 ± 9
+1.5 ± 0.2
(P )Fe
10
(5.5 ± 0.1) × 104
a
3
Contribution of the intermediate spin state (S = /2) in the spin-admixed (P)FeIII(H2O)2
porphyrins, compare Ref 4b; b This work; c Ref 5a; d Ref 7c; e Ref 4; f Ref 5b.
In analogous 17O NMR measurements performed at pH 12.7, the bulk 17OH2 signal as a
function of temperature resulted in only small changes in the line width in the presence
and absence of the (P16–)FeIII, which suggests that (P16–)FeIII at high pH forms a fivecoordinate monohydroxo-ligated iron porphyrin, (P16–)FeIII(OH), that exhibits no
measurable water exchange reaction.
Earlier literature reports showed that high-spin, monohydroxo bound iron(III)
porphyrins may exist as five-coordinate (P)FeIII(OH) species in non-coordinating solvents
or as six-coordinate (P)FeIII(H2O)(OH) species in aqueous solution as supported by
17
O
NMR measurements.4 17O NMR water exchange studies indicated that no water exchange
reaction was observed for iron porphyrins with negatively charged substituents, although
another water molecule could bind to the complex to form (Pn–)FeIII(H2O)(OH) in
aqueous solution. The observations can be explained in terms of the presence of fivecoordinate (Pn–)FeIII(OH) complexes under such conditions as a result of the strong
electron-donating substituents in the porphyrin meso position and the trans labilizing
effect of coordinated hydroxide that results in an electron-rich character of the iron(III)
center. In contrast, however, (Pn+)FeIII(OH) complexes with positively charged
substituents tend to exist as six-coordinate (Pn+)FeIII(H2O)(OH) species such that line- 125 -
Influence of an Extremely Negatively Charged FeIII-porphyrin
broadening could be observed in the
17
O NMR experiments. The latter data are
summarized later in the text in Table 6.
It is well established from the 1H and 17O NMR data summarized in Table 2 that
the rates of water exchange correlate with the contribution of the intermediate spin state S
= 3/2 (Int %) in the spin admixed state (S = 3/2, 5/2) of ferric porphyrins, where the
contribution of S = 3/2 was estimated from the proton β-pyrrole signal.29 The results show
that the calculated Int % value increases with the increasing lability of the water molecule
coordinated to the iron(III) center, which is consistent with an increasing water-exchange
rate (kex) resulting from the lengthening of the FeIII–OH2 bond. The population of the S =
3
/2 spin state arises from destabilization of the dx2-y2 orbital, resulting in an increased
electron density on the nitrogens of porphyrin in (Pn–)FeIII(H2O)2 as compared to that in
(Pn+)FeIII(H2O)2. The observed effects also affect the reactivity of the complexes towards
NO as will be discussed in more detail later.
Inspection of the structural features reported for 5-coordinate high-spin, 6coordinate spin-admixed, and 6-coordinate low spin (P)FeIII(L) systems,8 suggest that
minor structural changes are demanded for the formation of (P)FeII(H2O)(NO+) from 6coordinate (P)FeIII(H2O)230 in which in both cases the FeIII centers remain in the porphyrin
plane. However, NO binding to 5-coordinate (P)FeIII(OH) complexes requires some
structural changes as a result of a change in coordination number, which involves
movement of the FeIII center from out-of-plane into the porphyrin plane to yield
(P)FeII(OH)(NO+).
16
III
On the basis of the reported spectroscopic results, (P –)Fe (H2O)2 represents the
six-coordinate spin-admixed (S = 3/2, 5/2) species in the range 6.0 < pH < 9.9, whereas
(P16–)FeIII(OH) is predominantly present as a high-spin (S = 5/2) five-coordinate complex
at pH > 9.9 with an out-of plane metal center toward the OH– ligand.31 No indication for
the formation of a μ-oxo-bridged dimer or dihydroxo species was found in the pH range
studied.
- 126 -
Influence of an Extremely Negatively Charged FeIII-porphyrin
5.4.3. Reaction of (P16–)FeIII(H2O)2 with Nitric Oxide
When gaseous NO is added to a solution of (P16–)FeIII(H2O)2, rapid spectral
changes occur as shown in Figure 3, indicating the formation of a typical low-spin iron
nitrosyl complex in which electron transfer from NO to Fe(III) produces (P16–
)FeII(H2O)(NO+). The maximum of the Soret- and Q-bands shifts from 398 and 532 nm to
428 (ε = 9.8 × 104 M–1 cm–1) and 541nm (ε = 1.3 × 104 M–1 cm–1), respectively, during
formation of the final product according to reaction 5.
kon
(P16–)FeIII(H2O)2 + NO
koff
(P16–)FeII(H2O)(NO+) + H2O
KNO = kon/koff
(5)
The binding of NO appears to be reversible as determined from an experiment in which
the bubbling of an inert gas through the product shifted the equilibrium back to the
reactant side. The thermodynamic equilibrium constant, KNO = (1.7 ± 0.2) × 105 M–1 was
determined from the spectral changes observed for various NO concentrations measured
in solution with an NO-electrode, as shown in the inset of Figure 3.5
1,50
16
14
1,25
12
10
(Ao-A)
-1
Absorbance
1,00
8
6
0,75
4
-5
0
5
10
15
20
25
-1
30
35
40
45
50
55
-1
[NO] , mM
0,50
0,25
0,00
300
350
400
450
500
550
600
650
700
Wavelength, nm
Figure 3. Spectral changes recorded for the binding of NO to (P16−)FeIII(H2O)2. The inset
shows a plot of (A − A0)−1 versus [NO]−1 (where A0 = absorbance at 398 nm at [NO] = 0
- 127 -
Influence of an Extremely Negatively Charged FeIII-porphyrin
and A= absorbance at 398 nm at a given NO concentration). Experimental conditions:
[(P16–)FeIII] = 1.34 × 10–5 M, pH = 6.5 (0.05M Mes), temp = 25 °C, I = 0.1 M (with
NaClO4)
The kinetics for the reversible binding of NO were investigated by stopped-flow
technique for the “on” and “off” reaction, respectively. The observed reaction proved to
be follow pseudo-first-order kinetics with an at least 10-fold excess of NO, and the
observed rate constant, kobs, is expressed by eq 6.
kobs = kon[NO] + koff
(6)
Accordingly, kobs depends linearly on [NO] with a slope of kon = (33.6 ± 0.5) × 104 M-1 s-1
and an intercept of koff = 4 ± 3 s-1 at 14.7 °C. The values of koff are small and subjected to
large error limits when determined by extrapolation of plots of kobs versus [NO] to [NO] =
0. More reliable koff values were obtained in a direct way by using an NO trap, namely,
[RuIII(edta)(H2O)]–, an efficient scavenger for NO. Fast NO trapping by an excess of
scavenger from (P16–)FeII(H2O)(NO+) gives the original complex (P16–)FeIII(H2O)2 and
[RuIII(edta)(NO)]–, as indicated by the observed spectral changes. The kinetic traces were
recorded at 430 nm and could be fitted with a single-exponential function. The rate
constant for NO release is the rate-limiting step under the selected conditions (see
reaction 7) since the observed rate constant does not depend on the concentration of
[RuIII(edta)(H2O)]– employed since it is a much faster reaction than either kon or koff. The
values of koff determined in this way differ considerably from those extrapolated from the
data in Figure 4 as shown in Table 3.
(P16–)FeII(H2O)(NO+) + H2O
koff
kon
(P16–)FeIII(H2O)2 + NO
[RuIII(edta)(H2O)]–
[RuIII(edta)(NO)]– + H2O
- 128 -
(7)
Influence of an Extremely Negatively Charged FeIII-porphyrin
350
14.7
300
12.5
250
200
kobs, s
-1
9.7
150
6.3
100
4.7
50
0
0,0000
0,0002
0,0004
0,0006
0,0008
0,0010
[NO], M
Figure 4. Plots of kobs versus [NO] for the reaction of (P16-)FeIII(OH2)2 with NO in the
restricted temperature range of 4.7 − 14.7 °C measured with the stopped-flow technique.
Experimental conditions: [(P16−)FeIII] = 2.0 × 10−5 M, pH 6.5 (0.05 M Mes), λdet = 430 nm,
I = 0.1 M (with NaClO4).
The overall equilibrium constant obtained from the kinetic data, KNO = kon/koff = (1.7 ±
0.1) × 105 M-1 at 25 °C, is in an excellent agreement with the thermodynamic value of KNO
determined from spectral changes as a function of [NO], namely, (1.7 ± 0.2) × 105 M-1.
The binding (kon) and release of NO (koff) rate constants were determined as a
function of temperature in the restricted range of 4.7 – 14.7 °C because of limitations of
the stopped-flow technique (see Table 3 and Figure 4). Eyring plots were constructed
‡
‡
from which the activation parameters, ΔH and ΔS for the “on” and “off” reactions, were
determined. Despite the large difference in the koff values, the calculated activation
parameters, ΔH
‡
off
and ΔS‡off, obtained with the stopped-flow and NO-trapping techniques
are rather similar and do not affect the mechanistic interpretation.
- 129 -
Influence of an Extremely Negatively Charged FeIII-porphyrin
Table 3. Rate and Activation Parameters for the Binding and Release of NO to (P16)FeIII(H2O)2 at pH 6.5 by Stopped-Flow and NO-Trapping Methods
(×104 M-1 s-1)
koff
(s-1)
koff a
0.1
0.1
0.1
0.1
0.1
0.1
0.1
9.6 ± 0.2
12.2 ± 0.4
18.3 ± 0.3
25.9 ± 0.3
33.6 ± 0.5
0.6 ± 0.4
0.9 ± 0.5
2±1
3±2
4±3
2.3 ± 0.2
3.2 ± 0.2
6.1 ± 0.1
10.1 ± 0.1
13.7 ± 0.3
31.4 ± 0.2
65.5 ± 0.5
10
50
90
130
10.5 ± 0.3
8.6 ± 0.4
7.0 ± 0.4
5.9 ± 0.5
temp
pressure
(°C)
(MPa)
4.7
6.3
9.7
12.5
14.7
20
25
2.5
kon
(s-1)
1.7 ± 0.1
1.3 ± 0.1
1.0 ± 0.1
0.7 ± 0.1
ΔH‡ (kJ mol-1)
80 ± 1
117 ± 13
110 ± 2
‡
-1 -1
ΔS (J mol K )
+138 ± 4
+173 ± 24 +161 ± 8
‡
3
-1
ΔV (cm mol )
+10.8 ± 0.2
+16.9 ± 0.3
a
Data obtained by the NO-trapping method with the use of
[RuIII(edta)(H2O)]–
For a better understanding of the underlying reaction mechanism, activation volumes
were determined from the effect of pressure up to 130 MPa on kon and koff and are
included in Table 3. The reported activation volumes were used to construct a volume
profile for reaction 5 as shown in Figure 5. The obtained rate and activation parameters
are summarized in comparison with other (P)FeIII(H2O)2 complexes in Table 4.
- 130 -
Influence of an Extremely Negatively Charged FeIII-porphyrin
Partial molar volume, cm3 mol-1
H2O
NO ‡
FeIII
OH2
OH2
FeIII
+ NO + 10.8 ± 0.2
OH2
+ 16.9 ± 0.3
⊕NO
– 6.9 ± 0.3
FeII
+ H2O
OH2
Reactants
Transition state
Products
Reaction coordinate
Figure 5. Volume profile for the reversible binding of nitric oxide to (P16−)FeIII(H2O)2
according to reaction 5.
5.4.4. Reaction of (P16–)FeIII(OH) with Nitric Oxide
As reported in our earlier papers,5 spectroscopic and kinetic studies on the reaction
of NO with iron(III) porphyrins at pH >> pKa indicate that the nature of the resulting
nitrosyl species and the NO binding mechanism differ from those observed for the
diaqua-ligated iron porphyrins. Figure 6 shows the spectral changes that accompany the
reaction of (P16–)FeIII(OH) with NO at pH 12.7, where the decrease in the absorbance
maximum at 418 nm is accompanied by the appearance of new bands at 433 (ε = 9.7 ×
104 M–1 cm–1) and 547 nm (ε = 1.2 × 104 M–1 cm–1) for the formation of the nitrosyl
product, (P16–)FeII(OH)(NO+). The latter UV–vis spectrum differs from that observed for
the corresponding reaction at low pH (viz., 428 and 541 nm at pH 6.5) and points to the
formation of a deprotonated form of the nitrosyl product at high pH as given in eq 8.
(P16–)FeIII(OH) + NO
kon
koff
(P16–)FeII(OH)(NO+)
- 131 -
KNO = kon/koff
(8)
Influence of an Extremely Negatively Charged FeIII-porphyrin
Table 4. Rate Constants (at 298 K) and Activation Parameters for Reversible Binding
of NO to a Sseries of Diaqua-Ligated Water Soluble Iron(III) Porphyrins
NO binding
Iron(III)
porphyrin
pKa1
(P16-)Fea
9.8
24.5
113 ± 5 f
80 ± 1
+138 ± 4
+11 ± 1
8-
ΔH
kon
(×104 M-1 s-1)
b
‡
on
-1
(kJ mol )
‡
ΔS
‡
on
-1 -1
(J mol K )
ΔV
on
3
-1
(cm mol )
9.3
24
82 ± 1
51 ± 1
+40 ± 2
+6.1 ± 0.1
4-
c
6.9
26
280 ± 20
57 ± 3
+69 ± 11
+13 ± 1
4-
c
7.0
20
50 ± 3
69 ± 3
+95 ± 10
+9 ± 1
5.5
7
2.9 ± 0.2
67 ± 4
+67 ± 13
+3.9 ± 1.0
5.0
10
1.5 ± 0.1
77 ± 3
+94 ± 12
+1.5 ± 0.3
(P )Fe
(TMPS )Fe
(TPPS )Fe
(TMPyP4+)F
8+
Int%
d
e
(P )Fe
NO release
koff
ΔH
-1
(s )
(P16-)Fea
-1
on
-1
‡
-1
ΔV
3
on
-1
(cm mol )
(J mol K )
22 ± 3
117 ± 13
+173 ± 24
g
g
110 ± 2
+161 ± 8g
+17 ± 1g
220 ± 2
101 ± 2
+140 ± 7
+16.8 ± 0.4
4-
c
900 ± 200
84 ± 3
+94 ± 10
+17 ± 3
4-
c
500 ± 400
76 ± 6
+60 ± 11
+18 ± 2
59 ± 4
108 ± 5
+150 ± 12
+16.6 ± 0.2
(TMPS )Fe
(TPPS )Fe
(TMPyP4+)F
8+
‡
ΔS
(kJ mol )
66 ± 1
(P8-)Feb
‡
on
d
e
(P )Fe
26.3 ± 0.5
83 ± 4
+61 ± 14
+9.3 ± 0.5
a
b
c
d
e
f
This work; Ref 5a; Ref 3d; Ref 6a; Ref 5b; Calculated for 298K from Eyring
plots based on data from Table 3; g Data measured with [RuIII(edta)(H2O)]– as NO
scavenger.
- 132 -
Influence of an Extremely Negatively Charged FeIII-porphyrin
For the reversible binding of NO to (P16–)FeIII(OH) at pH 12.7, KNO = (4.1 ± 0.4) ×
103 M-1 at 25 °C was determined from the spectral changes as a function of the [NO]
shown in Figure 6.4
1,50
3,6
3,4
3,2
1,25
3,0
(A-A0)
-1
2,8
Absorbance
1,00
2,6
2,4
2,2
2,0
1,8
0,75
1,6
1,4
1
2
3
4
5
-1
6
7
8
-1
[NO] , mM
0,50
0,25
0,00
300
350
400
450
500
550
600
650
700
Wavelength, nm
Figure 6. Spectral changes resulting from the binding of NO to (P16−)FeIII(OH). The inset
shows a plot of (A − A0)−1 versus [NO]−1 (where A0 = absorbance at 435 nm at [NO] = 0
and A = absorbance at 435 nm at a given NO concentration). Experimental conditions:
[(P16−)FeIII] = 1.38 × 10−5 M, pH = 12.7 (0.05M NaOH), temp = 25 °C, I = 0.1 M (with
NaClO4).
Detailed kinetic measurements for the reversible binding of NO to (P16–)FeIII(OH) were
carried out in a manner similar to that employed for (P16–)FeIII(H2O)2. kon = (31.5 ± 0.2) ×
103 M-1 s-1 and koff = 8.0 ± 0.1 s-1 at 25 °C were obtained from the slope and intercept of a
linear plot of kobs versus [NO], respectively.
The thermodynamic equilibrium constant is close to the value calculated from the
kinetic data, namely, KNO = kon/koff = (3.9 ± 0.2) × 103 M-1 at 25 °C. Variable-temperature
and -pressure experiments were performed to determine the activation parameters in a
manner similar to that for the diaqua complex. The results are reported in Figures 7 and
Table 5 and are compared with a series of related systems in Table 6.
- 133 -
Influence of an Extremely Negatively Charged FeIII-porphyrin
6
(a)
kobs, s
-1
30
5
10
15
20
25
(b)
kon
4
ln(k/T)
40
20
-4
10
koff
-6
0
0,0000
-8
0,0002
0,0004
0,0006
0,0008
0,0010
0,00335
0,00340
[NO], M
0,00345
0,00350
0,00355
0,00360
1/T
Figure 7. (a) Plots of kobs versus [NO] for the reaction of (P16−)FeIII(OH) with nitric oxide
in the temperature range of 5 − 25 °C measured with the stopped-flow technique. (b)
Corresponding Eyring plots for the on and off reactions. Experimental conditions:
[(P16−)FeIII] = 2.0 × 10−5 M, pH 12.7 (0.05 M NaOH), λdet = 435 nm, I = 0.1 M
Table 5. Rates and Activation Parameters for the Binding and Release of NO for
(P16-)FeIII(OH) at pH 12.7 by Stopped-Flow and NO-Trapping Method
temp
pressure
(°C)
(MPa)
5
10
15
20
25
0.1
0.1
0.1
0.1
0.1
(×103 M-1 s-1)
koff
(s-1)
koff a
14.8 ± 0.2
19.1 ± 0.9
21.8 ± 0.5
26.3 ± 1.2
31.5 ± 0.2
0.32 ± 0.04
1.1 ± 0.1
2.0 ± 0.3
4.1 ± 0.6
8.0 ± 0.1
0.6 ± 0.1
1.0 ± 0.1
1.8 ± 0.1
2.9 ± 0.1
4.8 ± 0.1
kon
(s-1)
10
4.5 ± 0.2
0.33 ± 0.04
50
5.3 ± 0.3
0.25 ± 0.03
90
6.3 ± 0.5
0.19 ± 0.02
130
7.4 ± 0.4
0.15 ± 0.03
‡
-1
ΔH (kJ mol )
23 ± 1
108 ± 7
84 ± 1
‡
-1 -1
– 82 ± 4
+136 ± 19
+53 ± 4
ΔS (J mol K )
‡
3
-1
– 9.4 ± 0.2
+15 ± 1
ΔV (cm mol )
a
Data obtained by the NO-trapping method with the use of [RuIII(edta)(H2O)]–
2.5
- 134 -
Influence of an Extremely Negatively Charged FeIII-porphyrin
Table 6. Rate Constants (at 298 K) and Activation Parameters for Water Exchange and
Reversible Binding of NO to Monohydroxo-ligated Iron(III) Porphyrins
Iron(III)
kex
porphyrin
-1
(×105 s )
NO binding
kon
4
-1 -1
ΔH
‡
on
-1
‡
ΔS
on
-1
‡
-1
(×10 M s )
(kJ mol )
(J mol K )
ΔV
on
3
-1
(cm mol )
(P16-)Fea
e
3.1 ± 0.4
23 ± 1
–82 ± 4
–9.4 ± 0.2
(P8-)Feb
e
5.1 ± 0.2
34.6 ± 0.4
–39 ± 1
–6.1 ± 0.2
(TMPS4-)Fec
f
1.46 ± 0.02
28.1 ± 0.6
–128 ± 2
–16.2 ± 0.4
(TMPyP4+)Fed
g
0.36 ± 0.01
41.4 ± 0.5
–38 ± 5
–13.7 ± 0.6
24 ± 6
0.16 ± 0.01
41 ± 1
–45 ± 2
NO release
–13.8 ± 0.4
(P8+)Fed
koff
ΔH
‡
off
‡
ΔS
off
‡
ΔV
off
-1
(s )
-1
(kJ mol )
-1 -1
(J mol K )
8.0 ± 0.1
108 ± 7
+136 ± 19
7.1 ± 0.1h
84 ± 1h
+53 ± 4h
(P8-)Feb
11.4 ± 0.3i
107 ± 2
+136 ± 7
+17 ± 3
(TMPS4-)Fec
10.5 ± 0.2j
90 ± 1
+77 ± 3
+7.4 ± 1.0
(TMPyP4+)Fed
3.2 ± 0.1
78 ± 2
+25 ± 7
+9.5 ± 0.8
(P8+)Fed
6.2 ± 0.1
72 ± 2
+12 ± 5
+2.6 ± 0.2
(P16-)Fea
a
3
-1
(cm mol )
+15 ± 1h
This work; b Ref 5a; c Ref 4; d Ref 5b; e No water exchange process was detected,
compare Ref 5a; f Although the effect of (TMPS4-)FeIII(OH) on the bulk water line
width is observable in temperature dependent 17O NMR studies, it was too small to
determine kex and the corresponding activation parameters for the water exchange
reaction, compare Ref 4; g Formation of a μ-oxo dimer at porphyrin concentrations
required for NMR measurements precludes reliable studies on the water exchange
process; h Data measured with [RuIII(edta)(H2O)]– as a scavenger for NO; i at 297 K;
j
Calculated for 298K from the Eyring plots, compare Ref 5b.
- 135 -
Influence of an Extremely Negatively Charged FeIII-porphyrin
The reported activation volumes for the “on” and “off” reactions were used to construct
the volume profile reported in Figure 8.
Partial molar volume, cm3 mol-1
FeIII
+ NO
OH
NO
- 9.4 ± 0.2
‡
FeIII
OH
– 24.4 ± 0.6
⊕NO
+ 15 ± 1
Reactants
Transition state
Reaction coordinate
FeII
OH
Products
Figure 8. Volume profile for the reversible binding of nitric oxide to (P16−)FeIII(OH)
according to reaction 8.
As mentioned above, the spectra reported in Figure 9a show differences for the
nitrosyl product formed at pH 6.5 and 12.7. The plot of absorbance at 434 nm versus pH
in Figure 9b, fitted with a sigmoidal function, resulted in pKa(NO) = 9.68 ± 0.03 for the
deprotonation of a coordinated water molecule in the nitrosyl product (P16–
)FeII(NO+)(H2O) as given in reaction 9. The observed reactions can be summarized as
outlined in Scheme 2.
(P16–)FeII(NO+)(H2O)
(P16–)FeII(NO+)(OH–) + H+
- 136 -
pKa(NO) = 9.68
(9)
Influence of an Extremely Negatively Charged FeIII-porphyrin
1,7
2,00
1,75
(a)
at pH 6.5
at pH 12.7
(b)
1,6
1,5
1,50
Absorbance
Absorbance
1,4
1,25
1,00
0,75
1,3
pKa = 9.68 ± 0.03
1,2
1,1
1,0
0,50
0,9
0,25
0,8
0,00
300
350
400
450
500
550
600
650
6
700
7
8
9
10
11
12
13
pH
Wavelength, nm
Figure 9. (a) Spectral changes accompanying the reaction of (P16−)FeIII with NO at pH 6.5
and 12.7. (b) A plot of absorbance at 434 nm versus pH in buffered aqueous solutions in
the pH range of 6.5 − 12.7. Experimental conditions: [(P16−)FeIII] = 7 × 10-5 M, [NO] = 1
mM, temp = 25 °C, I = 0.1 M (with NaClO4). Buffers: pH 6.5, 0.05 M Mes; pH 7, 0.05 M
Bis-Tris; pH 7.5 − 8.8, 0.05 M Taps; pH 9.1, 0.05 M borate; pH 9.5 − 11.6, 0.05 M Caps;
pH 12.0 − 12.7, NaOH.
Scheme 2.
⊕NO
OH2
FeIII
+ NO
OH2
+ H3O+
kon
koff
+ NO
kon
koff
+
pKa = 9.68
⊕NO
FeII
OH
OH
- 137 -
H2O
OH2
+ H+
pKa1 = 9.90
FeIII
FeII
Influence of an Extremely Negatively Charged FeIII-porphyrin
In comparison to the binding and release rate constants at pH 6.5 (viz., 1.84 × 105 M–
1
s–1 and 6.1 s–1, at 10 °C, see Table 3) the corresponding values at pH 12.7 and 10 °C are
1.91 × 104 M–1 s–1 and 1.0 s–1, respectively, indicating that both NO binding and release
rates for (P16–)FeIII(OH) are significantly slower than those for (P16–)FeIII(H2O)2. A
comparison of these rate constants along with those for a series of complexes studied
before is given in Table 7.
Table 7. Ratio of Rate Constants Observed for the NO Binding (kon / kon) and
H2O
H2O
OH
Release (koff / koff) for Selected Water-soluble Iron(III) Porphyrins
OH
(P)FeIII
H2O
Int%
OH
kon / kon
H2O
OH
koff / koff
(P16-)Fe
(P8-)Fe
24.5
24
(TMPS4-)Fe
26
192
(TMPyP4+)Fe
7
8.0
18.4
(P8+)Fe
10
9.4
4.2
36
16
9.1
19.3
85
From the data shown in Figure 10a, it can be seen that the rates of NO binding as a
function of pH in the range of 6.5 – 12.7 resulted in kon values that decrease with
increasing pH (see Figure 10b). A fit of the data resulted in a pKa value of 9.83 ± 0.03,
which is similar to that determined from a spectrophotometric titration (viz., pKa1 = 9.90),
suggesting that the rate of NO binding is controlled by the axial ligand bound to the metal
center and reflects differences in the kinetics of NO binding at low and high pH. In the
case of NO dissociation rates, the koff values are rather small and include large errors
when extrapolated from plots of kobs versus [NO]. More reliable values were obtained for
koff using the NO-trapping method mentioned above. A similar observation was shown
previously for other water-soluble iron(III) porphyrins.
- 138 -
Influence of an Extremely Negatively Charged FeIII-porphyrin
180
kobs, s
-1
100
80
60
120000
100000
-1
120
80000
pKa = 9.83 ± 0.03
60000
40000
40
20000
20
0
0,0000
(b)
140000
-1
140
160000
(a)
kon, M s
160
6.5
7.6
8.4
8.9
9.3
9.7
9.9
10.2
10.7
11.7
12.7
0
0,0002
0,0004
0,0006
0,0008
0,0010
6
7
8
9
10
11
12
13
pH
[NO], M
Figure 10. (a) pH dependence of the rate constants for the reaction of NO with (P16−)FeIII.
(b) Plots of kon versus pH, where kon was determined from the slopes of kobs versus [NO]
plots measured in the pH range of 6.5−12.7. Experimental conditions: [(P16−)FeIII] = 2.0 ×
10-5 M, temp = 10 °C, λdet = 435 nm, I = 0.1 M (with NaClO4).
5.4.5. Spectroscopic and Kinetic Studies on the Subsequent Reactions.
The nitrosyl complex (P16–)FeII(H2O)(NO+) at pH 6.5 undergoes a subsequent
reaction on a longer time scale in which the characteristic bands at 428 and 541 nm
disappear with a concomitant appearance of new bands at 416 and 612 nm, respectively
(see Figure 11). The obtained kinetic trace for this reaction can be fitted with a singleexponential function as shown in the inset of Figure 11.
- 139 -
Influence of an Extremely Negatively Charged FeIII-porphyrin
0,8
0,8
Absorbance
0,7
Absorbance
0,6
0,6
0,5
0,4
0,3
0
0,4
5000
10000
15000
20000
25000
30000
35000
Time, sec
0,2
0,0
300
350
400
450
500
550
600
650
700
Wavelength, nm
Figure 11. Spectral changes observed during the subsequent reaction following the
binding of NO to (P16−)FeIII(H2O)2. The inset shows the kinetic trace of absorbance versus
time fitted to a single-exponential function. Experimental conditions: [(P16−)FeIII] = 1 ×
10-5 M, [NO] = 1 mM, pH = 6.5 (0.05M Mes), λdet = 431 nm, temp = 25 °C, I = 0.1 M
with NaClO4. The first ten spectra were recorded every 6 min, the next 10 spectra every 9
min, and the rest every 15 min to give kobs = 1.56 × 10-4 s-1.
The final spectrum resembles that of a five-coordinate ferrous nitrosyl product and is the
same as that observed for the product formed in the reaction of NO with reduced (P16–
)FeII. This suggests that (P16–)FeII(H2O)(NO+) is converted to (P16–)FeII(NO) by a slow
redox process as outlined in reactions 10 – 12. The observed spectral changes are similar
to that reported for other negatively charged water-soluble iron(III) porphyrins.17,18
(P16–)FeIII(H2O)2 + NO
(P16–)FeII(H2O)(NO+)
(P16–)FeII(H2O) + NO
KNO
kred
+ H2O
fast
(P16–)FeII(H2O)(NO+) + H2O
(10)
(P16–)FeII(H2O) + NO2- + 2H+
(11)
(P16–)FeII(NO) + H2O
(12)
- 140 -
Influence of an Extremely Negatively Charged FeIII-porphyrin
The effect of the NO concentration on the observed reductive nitrosylation reaction
at pH 6.5 and 25 °C is reported in Figure 12a, from which it follows that kobs increases
with increasing [NO] in a nonlinear way and reaches a constant value at high [NO].
0,00018
11000
(a)
(b)
10500
0,00016
10000
9500
9000
kobs , s
0,00012
-1
kobs, s
-1
0,00014
8500
8000
7500
0,00010
7000
6500
0,00008
6000
0,00006
0,0000
5500
0,0002
0,0004
0,0006
0,0008
0,0010
0,0012
0
[NO], M
2000
4000
6000
-1
8000
10000
12000
-1
[NO] , M
Figure 12. (a) Plot of kobs versus [NO] for the reductive nitrosylation of
(P16−)FeII(H2O)(NO+). (b) Plot of kobs−1 versus [NO]−1. Experimental conditions:
[(P16−)FeIII] = 2.0 × 10-5 M, pH 6.5 (0.05 M Mes), temp. = 25 °C, λdet = 431 nm, I = 0.1 M
NaClO4
In agreement with the rate law (eq 13) for the above given reaction scheme, the plot of
kobs–1 vs [NO] –1 is linear as displayed in Figure 12b, from which the values for KNO and
kred were calculated to be (1.4 ± 0.2) × 105 M–1 and (1.7 ± 0.3) × 10-4 s–1, respectively.
This value for KNO is close to the equilibrium constant calculated from the kinetic data,
namely, KNO = kon/koff = (1.7 ± 0.1) × 105 M–1 at 25 °C, and the value of kred is in a good
agreement with that measured directly at high [NO], namely, kobs = 1.56 × 10-4 s–1.
kobs =
kred KNO[NO]
1 + KNO[NO]
(13)
The observed value for kred for (P16–)FeII(H2O)(NO+) is the smallest among the
series of water-soluble iron porphyrins (P)FeIII as summarized in Table 9. In general, slow
- 141 -
Influence of an Extremely Negatively Charged FeIII-porphyrin
reduction rates are observed for the negatively charged complexes with values for kred of
approximately 10–4 s–1 at 25 °C. In contrast, the positively charged porphyrins show a
much faster reductive nitrosylation, which suggests that the nucleophilicity of coordinated
NO+ on the FeII center is affected by the porphyrin environment in controlling the stability
of the intermediate (Pn)FeII(H2O)(NO+), which affects the rate of the subsequent redox
reaction.
The observed reductive nitrosylation reaction is catalyzed by nitrite and was
systematically studied as a function of pH, nitrite concentration, temperature, and
pressure. The suggested sequence for the nitrite-catalyzed reaction is given in reaction 14,
for which the observed rate constant can be expressed as shown in eq 15.
(P16–)FeIII + NO
KNO
–
(P16–)FeII(NO+) + NO2
knit
(14)
(P16–)FeII(NO) + other products
kobs =
knitKNO[NO2-][NO]
1 + KNO[NO]
(15)
The observed rate constant shows a linear dependence on the nitrite concentration
as shown in Figure 13 for which the second-order rate constant is (1.3 ± 0.2) × 10–2 M–1 s–
1
, which presents knitKNO[NO]/(1 + KNO[NO]). Since KNO and [NO] are known under the
selected experimental conditions, the values of kobs could be converted to the
corresponding knit values (summarized in Table 8) as a function of temperature and
pressure at pH 6.5. The activation parameters were estimated in the usual way and the
results are summarized along with the rate constants and activation parameters for the
other investigated complexes in Table 9. On the basis of these data, the nitrite-induced
reductive nitrosylation is the slowest for (P16–)FeII(H2O)(NO+) in comparison to all other
studied porphyrins, suggesting that the overall negative charge reduces the electrophilicity
of the coordinated NO+ and slows down the direct binding of NO2–.
- 142 -
Influence of an Extremely Negatively Charged FeIII-porphyrin
0,35
0,30
0,20
3
kobs x 10 , s
-1
0,25
0,15
0,10
0,05
0,00
0,000
0,005
0,010
0,015
0,020
0,025
-
[NO2 ], M
Figure 13. Nitrite concentration dependence of the reductive nitrosylation of
(P16−)FeII(H2O)(NO+). Experimental conditions: [(P16−)FeIII] = 3.0 × 10-5 M, [NO] = 1
mM, temp. = 25 °C, λdet = 431 nm, pH 6.5 (0.05M Mes), I = 0.1 M (with NaClO4).
Table 8. Effect of Temperature and Pressure on the Nitrite-Induced Reductive
Nitrosylation Reaction of (P16−)FeII(H2O)(NO+) with Nitrite Ion at pH 6.5a
knit
pressure
(°C)
(MPa)
(× 10 s )
25
30
35
40
45
0.1
0.1
0.1
0.1
0.1
0.22 ± 0.02
0.37 ± 0.04
0.58 ± 0.05
1.09 ± 0.05
1.53 ± 0.07
1.4 ± 0.1
2.5 ± 0.3
4.1 ± 0.4
8.1 ± 0.5
11.9 ± 0.5
10
50
90
130
‡
-1
ΔH (kJ mol )
‡
ΔS (J mol-1 K-1)
‡
3
ΔV (cm mol-1)
0.33 ± 0.06
0.39 ± 0.05
0.45 ± 0.06
0.54 ± 0.07
2.2 ± 0.3
2.7 ± 0.5
3.1 ± 0.4
3.6 ± 0.7
80.4 ± 1.4
–10.8 ± 3.1
– 9.8 ± 0.4
27
a
kobs
temp
3 -1
[NO2–] = 16 mM
- 143 -
2
(× 10 M-1 s-1)
Table 9. Rate and activation parameters for nitrite-induced reductive nitrosylation of (Pn)FeII(H2O)(NO+) and
(Pn)FeII(OH)(NO+) at 25 °C
Iron(III)
pKa
porphyrin
(P16-)Fec
9.9
8d
(P )Fe
9.2
4e
(TPPS )Fe
7.0
4+
f
(TMPyP )Fe 5.5
(P8+)Fed
5.0
pH
kreda
(× 10-4 s-1)
slopeb
knit
-1 -1
(M-1 s-1)
(M s )
ΔH
‡
-1
(kJ mol )
‡
ΔS
-1 -1
(J mol K )
‡
ΔV
3
-1
(cm mol )
6.5
80 ± 1
–11 ± 3
–9.8 ± 0.4
1.7 ± 0.2
(1.3 ± 0.2) ×10-2 (1.4 ± 0.2) ×10-2
7
2.8 ± 0.2
1.6 ± 0.1
2.1 ± 0.2
60 ± 2
–36 ± 7
–8.6 ± 0.4
3.1
5
2.2 ± 0.1
4.6 ± 0.4
g
4.5
70
15.0 ± 0.1
42 ± 3
88 ± 2
+92 ± 6
+8.8 ± 0.1
2
400 ± 20
55 ± 3
155 ± 5
90 ± 3
+99 ± 10 +7.2 ± 0.5
4
89 ± 1
242 ± 7
73 ± 2
+92 ± 7
+12.3 ± 0.7
a
b
Calculated values using eq12 when no nitrite was added; Value calculated from kobs versus [NO2 ] at constant [NO]; c This
work; d Ref 18; e Measured at 15oC, ref 17; f Ref 6a; g Observed rate constant (kobs) for the studied reaction.
- 144 -
Influence of an Extremely Negatively Charged FeIII-porphyrin
The observed reaction is also catalyzed by hydroxide ions. The [OH–] dependence of
the reductive nitrosylation reaction is reported in Figure 14, in which the observed
reaction was studied in the pH range of 6.0 – 8.4 where a water molecule is still bound to
the center. Since the plot gives a straight line with a nonzero intercept, kobs can be
formulated as given in eq 16.
kobs = kOH[OH–] + kH2O
(16)
The obtained slope and intercept of the plot are kOH = 1.0 × 103 M–1 s–1 and kH2O =
1.1 × 10–4 s–1, respectively. This value of kOH is consistent with that measured for
metalloproteins and ferric heme complex, (P8–)FeII(H2O)(NO+), for which analogous
measurements resulted in kOH = 4.5 × 103 M–1 s–1 and kH2O = 1.1 × 10–4 s–1, respectively.3,6
This means that OH– and H2O, as well as NO2–, contribute to the reductive nitrosylation
of (P16–)FeII(H2O)(NO+) to yield (P16–)FeII species. The base-catalyzed reaction is much
more effective than the nitrite-catalyzed reaction as shown by kOH = 1.0 × 103 M–1 s–1 and
knit = (1.3 ± 0.2) × 10–2 M–1 s–1, respectively.
3,0
2,5
1,5
3
10 kred , s
-1
2,0
1,0
0,5
0,0
0
5
10
7
15
20
25
30
10 [OH], M
Figure 14. Hydroxide concentration dependence for the reductive nitrosylation of (P16)FeIII(H2O)2 in the pH range 6.0 – 8.4. Experimental conditions: [(P16-)FeIII] = 1.3 × 10-5
M, temp = 25 oC, λdet = 428nm, I = 0.1 M with NaClO4
- 145 -
Influence of an Extremely Negatively Charged FeIII-porphyrin
The overall reactions is accompanied by an electron-transfer process in which (P16–
)FeII(NO) is formed from (P16–)FeII(NO+). Potential measurements using cyclic
voltammetry resulted in an irreversible ΔE1/2((P16-)FeII−NO+)/(P16-)FeII−NO) of 0.20V
versus Ag/AgCl at pH 6.5. This value fits excellently to the linear free-energy relationship
(LFER) reported for the nucleophilic addition (OH–) rate constant and the redox potential
for a series of trans-tetrapyridine ruthenium nitrosyl complexes.32 For the kOH = 1.0 × 103
M–1 s–1 value found for the present complex, the reported correlation predicts a redox
potential of 0.21V, which is in perfect agreement with our experimental value. According
to the data referenced, the ruthenium complexes have lower rate constant because of the
steric hindrance of pyridine ligands which are free to rotate. In the present case, the
malonate substituents on the porphyrin cause sufficient steric hindrance such that (P16)FeII(H2O)(NO+) complex also fits the reported LFER.
5.4.6. Suggested Mechanisms and Comparison with Other Iron(III) Porphyrins
A. Reactivity of (P16-)FeIII(H2O)2 and (P16-)FeIII(OH) toward NO
Mechanistic information on the “on” and “off” reactions of NO with (P16–
)FeIII(H2O)2 can best be obtained from the volume profile presented in Figure 5. The
positive activation volumes for the binding and release of NO favor a dissociative
mechanism which is analogous to that reported for a series of diaqua-ligated porphyrins
summarized in Table 4. The overall volume change observed for the reaction is not only
the result of displacement of water by NO but also of a change in spin state from S =
3
/2,5/2 for the diaqua complex to S = 0 for the FeII nitrosyl complex. The data in Table 4
show that the rates for NO binding correlate to some extent with the contribution of the
intermediate spin state (Int %) in the spin-admixed state (S = 3/2, 5/2). As mentioned
above, increasing Int % correlates with increasing lability of the ligand on the metal
center as influenced by the increasing electron-donating properties of the meso
substituents on the porphyrin. The trend in ΔV
‡
on
along the series of complexes suggests a
gradual changeover from a dissociative mechanism for the anionic complexes to a
- 146 -
Influence of an Extremely Negatively Charged FeIII-porphyrin
dissociative interchange mechanism for the cationic complexes. Almost similar trends are
observed for the water exchange reactions for the series of (P)FeIII(H2O)2 complexes
summarized in Table 4. The magnitude of the water exchange rate constant and the close
agreement between the volumes of activation for the water exchange process and the
binding of NO, suggest that the rate and mechanism of the latter process is controlled by
the water exchange process of the diaqua complexes.4,5,7c,33 Thus, electron-donating
substituents on the porphyrin induce a dissociative mechanism by weakening the Fe–OH2,
whereas electron-withdrawing substituents strengthen the Fe–OH2 bond and tend to favor
a dissociative interchange mechanism.
The rate constants for the dissociation of NO from the nitrosyl complex
(P)FeII(H2O)(NO+) summarized in Table 4 show for some of the complexes the trend that
the nitrosyl complex is stabilized by positively charged as compared to negatively
charged substituents on the porphyrin. This observation is in agreement with results from
Raman Resonance and DFT studies.34 In general, the value of ΔV
of ΔV
‡
on since
‡
off
is more positive than
the “off” reaction involves bond cleavage, formal oxidation of FeII to FeIII,
accompanied by a spin-state change (S = 0 to S = 3/2, 5/2) and solvent reorganization
resulting from the charge neutralization during FeII–NO+ bond cleavage.3
The reversible binding of NO at higher pH revealed a different kinetic behavior for
the monohydroxo-ligated (P16–)FeIII(OH) complex compared to that of the (P16–
)FeIII(H2O)2 complex, as indicated by the negative activation parameters, namely, ΔS
– 82 ± 4 J mol–1 K–1 and ΔV
‡
on =
‡
on
=
3
– 9.4 ± 0.2 cm mol-1. It was in general concluded that
the slow rate constant for NO binding to complexes of the type (P)FeIII(OH) is mainly
controlled by the FeII–NO+ bond-formation step and the accompanying electronic and
structural changes, rather than the lability of the metal center.4,5 Electronic changes for
the formation of the FeII–NO+ bond are accompanied by reorganization of the spin
arrangement in the iron(III) center from high spin (S = 5/2) to low spin (diamagnetic, S =
0). In addition, structural changes as a result of the iron center that moves from out-ofplane to in-plane during bond formation are also expected.5(b) These conclusions suggest
that the larger spin and structural changes for the monohydroxo-ligated species
- 147 -
Influence of an Extremely Negatively Charged FeIII-porphyrin
(P)FeIII(OH) demand a higher activation barrier for NO binding and release, such that
lower reaction rates were observed. The observed results are consistent with the earlier
findings for related complexes summarized in Table 6.5 The rate-determining step for NO
binding to (P16–)FeIII(OH) via an associative addition mechanism is mainly controlled by
electronic changes on the FeIII center and the accompanying structural rearrangement.
Larger spin and structural changes during the formation and breakage of the FeII–NO+
bond accompanied by higher activation barriers account for the slower reactions in the
case of the monohydroxo ligated species (see rate data in Table 6).
We propose in the mechanistic scheme outlined in reaction 16 that NO binding to
five-coordinated (P16–)FeIII(OH) involves the formation of a diffusion-controlled
encounter complex {(P16–)FeIII(OH) || NO}, followed by the activation step for the
formation of the FeII–NO+ bond, similar to that suggested for high-spin 5-coordinate
monohydroxo ligated iron(III) porphyrins.35
(P16–)FeIII(OH) + NO
kD
k-D
{(P16–)FeIII(OH) || NO}
k
[(P16–)FeIII(OH)⋅⋅⋅⋅NO]
‡
(16)
(P16-)FeII(OH)(NO+)
On the basis of the results summarized in Tables 4 and 6, (P)FeIII(OH) complexes
follow an associatively activated addition mechanism for NO binding and show slower
binding rates in contrast to a dissociatively activated mode observed for (P)FeIII(OH2)2
complexes. Furthermore, the reported values for kon vary by a factor of approximately 102
for the diaqua-ligated porphyrins and appear to correlate with the contribution of the
intermediate spin state, S = 3/2. In comparison, however, differences in the NO binding
rate constants are rather small for all studied (P)FeIII(OH) complexes. This difference is
because the lability of the FeIII complex controls the rate of NO binding to the sixcoordinate (P)FeIII(OH2)2 complexes, which does not play a role in the case of the fivecoordinate (P)FeIII(OH) complexes.
- 148 -
Influence of an Extremely Negatively Charged FeIII-porphyrin
The volume profile for the binding of NO to (P16–)FeIII(OH) is shown in Figure 8,
from which it can clearly be seen that the overall reaction volume (ΔV = ΔV
‡
on
– ΔV
‡
off)
has a value of – 24.4 ± 0.6 cm3 mol-1, which is close to a value of approximately – 23
cm3 mol–1 found for several other hydroxo complexes (except for (P8+)FeIII, see data in
Table 6). This large overall volume collapse for the binding of NO is partially caused by
the formation of the Fe–NO bond and the change in spin state from a five-coordinate,
high spin hydroxo reactant to a six-coordinate, diamagnetic (low-spin) nitrosyl product.
The activation volumes reported for (P16–)FeIII(OH) and (P8−)FeIII(OH) suggest an “early”
transition state for the on reaction and a “late” transition state for the off reaction,
compared to the opposite trend for the other complexes cited in Table 6. This is also in the
value of KNO in terms of the more effective binding of NO.
From the data summarized in Table 7, it is clearly seen how the rate constants for
the binding and release of NO decrease on going from (P)FeIII(OH2)2 to (P)FeIII(OH) for
the series of complexes. This is throughout ascribed to the role of the electronic barriers
involved in the reactions with the (P)FeIII(OH) complexes. These different decreases arise
from the characteristic electronic and structural properties among the complexes studied
(P)FeIII(OH2)2. In purely high-spin five-coordinate (P)FeIII(OH) complexes, it normally
demands larger reorganization of the spin state of the metal center and structural changes
with rather small variation in konOH for the studied complexes. While larger ratios for the
NO binding rate constants (konH2O/konOH) are observed for (Pn−)FeIII complexes, resulting
from mainly high kon values, smaller ratios are observed for the (Pn+)FeIII complexes.
B. Subsequent Reductive Nitrosylation of (P16–)FeII(H2O)(NO+)
Our report18 on the reactivity of ferric nitrosyl complexes as a function of the
nature of the porphyrin substituents, indicates that kinetic and mechanistic features of the
subsequent reductive nitrosylation reaction of negatively charged porphyrins,
(Pn−)FeII(H2O)(NO+), differs from that found for positively charged porphyrins,
(Pn+)FeII(H2O)(NO+). The observed reduction of coordinated NO+ is catalyzed by the
addition of nitrite to the reaction solution. It was further concluded that the rate of nitrite- 149 -
Influence of an Extremely Negatively Charged FeIII-porphyrin
catalyzed reduction of (P)FeII(H2O)(NO+) depends on the electrophilicity of coordinated
NO+ and the nature of the reactant, namely, NO2– or HONO. The available results (see
Table 9) favor the operation of an inner-sphere electron-transfer process between nitrite
and coordinated NO+. Charge neutralization during bond formation between NO2– and
NO+ on positively charged (Pn+)FeII(H2O)(NO+) complexes largely accounts for the
positive activation entropies and volumes associated with this process. In contrast,
negative values for these activation parameters observed for the reaction with the
negatively charged (Pn−)FeII(H2O)(NO+) complexes suggest that bond-formation and
charge concentration account for the observed data. These conclusions are in good
agreement with the experimental data found for the nitrite- induced reductive
nitrosylation of (P16−)FeII(H2O)(NO+) as shown in Table 9. The rate of NO2– coordination
(knit) to the nitrosyl complex is faster for (Pn+)FeII(H2O)(NO+) than that for
(Pn−)FeII(H2O)(NO+). For example, the observed rate constants for the binding of NO2– to
(P16−)FeII(H2O)(NO+) decrease by a factor of 104 compared to that observed for
(P8+)FeII(H2O)(NO+), and are even 102 times slower than those for (P8−)FeII(H2O)(NO+).
This again indicates that the crucial factor that controls the rate of reductive nitrosylation
is the overall negative charge on the porphyrin that increases the electron density on metal
center and reduces the electrophilicity of coordinated NO+. Thus direct nucleophilic
attack of nitrite on coordinated NO+ is suggested as rate-determining step to form a
(P)FeII–N2O3 intermediate that subsequently releases N2O3 and rapidly reacts with NO to
form (P)FeII–NO. From the data in Table 9, an increase in activation volume for nitrite
binding to (P)FeII(H2O)(NO+) on going from (P16−)FeIII to (P8+)FeIII clearly shows that the
contribution of bond formation is facilitated by negatively charged meso substituents,
while a decreasing electrostriction prevails over bond-formation in porphyrins containing
electron-donating substituents.
- 150 -
Influence of an Extremely Negatively Charged FeIII-porphyrin
5.5 References
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Mar, G. N.; Eaton, G. R.; Holm, R. H.; Walker, F. A. J. Am. Chem. Soc. 1973, 95, 63.
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- 154 -
6. P450 model complex on the Reversible Binding Kinetics of NO
6.1 Introduction
Heme-thiolate proteins are the recommended collective name for a class of
hemeproteins in which the heme iron’s fifth ligand is a thiolate group (typically of a
cysteine residue). A distinctive feature of heme-thiolate proteins is a Soret absorption
band at around 450 nm for the CO complex of the reduced form. This class includes
families of complexes such as, heme chloroperoxidase that catalyzes the halogenation of a
number of aliphatic substrates, nitric oxide synthase (NOS) that catalyzes the oxidation of
a guanidine nitrogen of L-arginine to produce NO, and P450 enzymes which are
distributed in bacteria, fungi, plants and animals and catalyze mono-oxygenation reactions,
i.e., insertion of one atom of oxygen into the substrate, whereas the other oxygen atom is
reduced to water. In general, they act as terminal oxidases in multi-component electron
transfer chains, referred to as P450 containing mono-oxygenase systems. In addition,
P450nor is a unique enzyme that catalyzes reductive dimerization of NO to N2O.
Nitric oxide produced endogeneously, fulfills important roles in mammalian
biology as an intracellular signaling agent for a variety of biological functions, such as
vasodilation, neurotransmission, bronchodilation, and cytotoxic immune response.1 Much
of the biochemistry of this molecule involves heme proteins, including the biosynthesis of
nitric oxide and signal transduction mediated by NO. In this context, recent studies have
been concerned with the preparation and mechanistic evaluation of synthetic iron(III/II)
porphyrin complexes as useful biomimetic model systems in simulating the mechanism of
heme ligand binding and activation and, thereby, determining the distinctive features of
the active site of heme proteins. Thus, studies on the binding of NO by synthetic iron
porphyrins should aid our understanding of how NO interacts with heme-containing
biomolecules. A synthetic NO-heme-thiolate complex is also expected to be useful as a
model for nitric oxide synthase (NOS) and P450nor.
P450 model complex
The sulfur bound iron(III) porphyrin complex, SR(FeIII) (see Scheme 1) is known
to have a reactivity similar to that of cytochrome P450. Furthermore, due to the
introduction of bulky groups on the RS− coordination face of the porphyrin molecule, the
thiolate complex is unique in that it retains its axial thiolate to be stable during catalytic
oxidation. It was also established that the thiolate ligand plays an important role in the
characteristic oxidizing ability of the SR(FeIII) complex, which has a marked influence on
the reactivity of the high valent iron-oxo porphyrin intermediate.2,3 Recently,
spectroscopic and electrochemical studies revealed that the nitric oxide complex of
SR(FeIII) appeared to be diamagnetic (EPR silent), whereas the parent SR(FeIII) complex
showed a typical low-spin signal for a FeIII-porphyrin-thiolate complex.4 It is, however,
important to note that the SR(FeIII) complex is insoluble in water and can only be studied
in organic solvents.
R
H
O
Fe
ROH
H2O
III
H
H
O
Fe
Fe
H2O
S
S
RH
RH
III
III
S
e-
O
+•
O
Fe
S
RH
2H+, e-
IV
O
Fe
H2O
RH
RH
III
S
Fe
O2
II
S
Scheme 1. Catalytic schematic diagram for Cytochrome P450
Our studies concentrated on the interaction of NO with synthetic iron(III)
porphyrin models to investigate the influence of the porphyrin microenvironment in a
given FeIII system on the rate and mechanism of the binding and release of NO, and on the
stability of the resulting (P)FeII(NO+) species toward subsequent redox reactions in
solution. We now report the speciation of a water-soluble thiolate ligated and positively
- 156 -
P450 model complex
charged iron(III) porphyrin, (RSP4+)FeIII (Figure 1) in aqueous medium and present the
results of mechanistic studies on its reactivity toward NO at different pH. The results are
compared with those of thiolate-heme enzymes. It is important to note that this is the first
water soluble synthetic model for P450.
N
N
S
N
N
Fe
O
N
N
R
N
H
H
N
O
N
O
SH
NH
N
Figure 1. The synthetic heme-thiolate complex, (RSP4+)FeIII
6.2 Experimental Section
Materials. The iron(III) porphyrin-thiolate complex (RSP4+)FeIII was synthesized and
characterized by the group of Dr. Norbert Jux. NO gas was purchased from Riessner Gase
or Linde 93 in a purity of at least 99.5 vol % and cleaned from trace amounts of higher
nitrogen oxides like N2O3 and NO2 by passing it though an Ascarite II column (NaOH on
silica gel, Sigma-Aldrich) via vacuum line techniques. All other chemicals used
throughout this study were of analytical grade agent.
Solution Preparation. All solutions were prepared from de-ionized water and handled in
gastight glassware under strict exclusion of air oxygen due to the oxygen sensitivity of
NO and (RSP4+)FeIII(NO) nitrosyl complexes. Oxygen-free nitrogen was used to
deoxygenate the solutions. Tris buffer (0.05 M) was used to prepare solutions of desired
- 157 -
P450 model complex
pH, which was adjusted by the addition of HNO3 or KOH. The ionic strength of the
solutions (0.05 M) was kept constant by the addition of KNO3.
Measurements. pH measurements were performed on a Methrom 623 pH meter. UV−vis
spectra were recorded in gastight cuvettes on a Shimadzu UV-2100 spectrophotometer
equipped with a thermostated (±0.1 oC) cell compartment.
Kinetics Measurements. Stopped-Flow Studies. Stopped-flow kinetic measurements at
ambient pressure were performed on an SX 18.MV (Applied Photophysics) stopped-flow
apparatus. In a typical experiment, a deoxygenated buffer solution was mixed in varying
volume ratios with a saturated NO solution in a gastight syringe to obtain the appropriate
NO concentration (0.2−1.8 mM). The NO solution was then rapidly mixed with
deoxygenated iron(III) porphyrin in a 1:1 volume ratio in a stopped-flow apparatus.
Changes in absorbance were monitored at 440 nm for (RSP4+)FeIII(OH) at pH 8.8 and 424
nm for (RSP4+)FeIII(H2O) at pH 3.5. Rapid scan measurements were performed using a
J&M-Tidas 16-416 diode-array detector connected to a SX-18MV (Applied
Photophysics) thermostated (± 0.1 oC) stopped-flow spectrometer coupled to an online
data acquisition system. All kinetic experiments were performed under pseudo-first-order
conditions and the reported rate constants are the mean values from at least five kinetic
runs.
6.3 Results and Discussion
6.3.1 Spectrophotometric Studies
Spectrophotometric titrations in the pH range of 3.5 – 11 were performed. Figure 2
shows the UV-vis spectral changes observed at 0.05 M ionic strength adjusted with KNO3.
Spectra are characterized by the appearance of new bands at 420 and 533 nm from 422
and 538 nm on increasing the pH from 3.5 to 11, accompanied by isosbestic points at 412
and 530 nm, as shown in Figure 2(b). The corresponding plot of absorbance at 394 nm
- 158 -
P450 model complex
versus pH is shown in the inset of Figure 2(b), from which the pKa value was calculated
to be 6.2 for which the corresponding equilibrium is presented in equation (1). The
absorbance of the Soret band decreases and shifts as the pH is decreased. In addition, the
(RSP4+)FeIII complex is not stable at low pH over several hours (see below).
0,5
0,5
(a)
0,4
0,28
0,26
0,4
0,24
Absorbance
Absorbance
Absorbance
0,30
(b)
0,3
0,2
0,3
0,22
0,20
0,18
0,16
0,14
0,12
0
0,2
2
4
6
8
10
12
pH
0,1
0,1
0,0
250
300
350
400
450
500
550
600
650
700
0,0
250
300
350
400
450
500
550
600
650
700
Wavelength, nm
Wavelength, nm
Figure 2. (a) UV-vis spectral changes observed for aqueous solutions of (RSP4+)FeIII in
the pH range 1.3 − 3.5 (b) pH 3.5 − 11. Experimental conditions: [(RSP4+)FeIII] = 8 × 10-6
M, temp = 25 °C, I = 0.05 M (adjusted with KNO3).
(RSP4+)FeIII-(H2O)
Ka
(RSP4+)FeIII-(OH) + H+
pKa = 6.2
(1)
6.3.2 Reactivity of (RSP4+)FeIII toward NO at pH 8.8
The UV-vis spectrum of the (RSP4+)FeIII complex in water shows Soret- and Qband maxima at 422 and 538 nm at pH 8.8. Treatment of the degassed water solution of
(RSP4+)FeIII with an excess of NO results in spectral changes in the Soret band to 424 nm
and the Q-band to 552 nm. The formation of the nitrosyl complex (RSP4+)FeII(NO+)
seems to be irreversible. Absorbance time traces recorded at 440 nm indicate that the
observed reaction occurs within 0.5 s at 7.5 oC. Figure 3 reports the typical spectral
changes that are observed immediately following stopped-flow rapid mixing of an
aqueous solution of (RSP4+)FeIII with NO at 7.5 oC.
- 159 -
P450 model complex
0,092
0,15
0,090
440 nm
Absorbance
Absorbance
0,088
0,086
0,084
0,082
0,10
0,080
0,0
0,1
0,2
0,3
0,4
0,5
Time, sec
0,05
0,00
300
350
400
450
500
550
600
W avelength, nm
Figure 3. Representive spectral changes and typical absorbance-time plot for the binding
of NO by (RSP4+)FeIII measured with the stopped-flow instrument. Experimental
conditions: [(RSP4+)FeIII] = 3 × 10-6 M, [NO] = 0.5 mM, pH 8.8 (0.025 M Tris buffer), μ
= 0.05 M with KNO3, temp = 7.5 oC, λdet = 440nm
The kinetics of the binding of NO to (RSP4+)FeIII-(OH) at pH 8.8 was studied by
stopped-flow technique for the “on” and “off” reactions, respectively. The processes
occurring in the stopped-flow measurement can be summarized as follows.
4+
III
(RSP )Fe (OH) + NO
kon
koff
(RSP4+)FeIII(NO)
(RSP4+)FeII(NO+) + OH- (2)
The process was shown to follow pseudo-first-order kinetics for which the observed rate
constant, kobs, is expressed by eq 3.
kobs = kon[NO] + koff
(3)
According to eq 3, a plot of kobs versus [NO] appeared to be linear with a slope kon = (3.6
± 0.1) × 105 M-1 s-1 and an intercept koff = 40 ± 2 s-1 at 15 oC. In general a more accurate
value for koff can be obtained using an NO-trapping technique that involves the rapid
binding of NO by for instance [RuIII(edta)(H2O)]–. However, the studied system does not
seem to be suitable for the application of such NO-trapping methods as a result of rapid
- 160 -
P450 model complex
subsequent reactions that interfere with the release of NO. The overall equilibrium
constant obtained from the kinetic data, KNO = kon/koff = (9.2 ± 0.4) × 103 M-1 at 15 oC.
The binding (kon) and release (koff) rate constants were determined as a function of
temperature over the limited temperature range of 3 – 15 oC as a result of the limitations
of the stopped-flow technique (see Figure 4 and Table 1). The Eyring plot constructed on
the basis of these data was found to be linear (see Figure 4b) and gave ΔH‡on = 35 ± 1
kJ/mol and ΔS‡on = −16 ± 3 J/mol K, and ΔH‡off = 154 ± 7 kJ/mol and ΔS‡off = +323 ± 24
J/mol K for the “on” and “off” reactions, respectively. The large intercepts suggest that
the off reaction increases significantly with temperature, indicating that the overall
equilibrium in (2) is exothermic and the NO complex can be stabilized at low temperature.
In addition to, the equilibrium constant (KNO) decreases with increasing temperature that
clearly show the exothermic character of eq (2). It was impossible to study the pressure
dependence of the binding of NO by the (RSP4+)FeIII complex because of too small
absorbance changes observed for the reaction.
250
8
o
15 C
(a)
(b)
o
12 C
200
"on" reaction
7
o
9C
o
6C
ln(k/T)
kobs, s
-1
150
o
3C
100
6
-1
-2
50
-3
"off" reaction
-4
0
0,0000
-5
0,0001
0,0002
0,0003
0,0004
0,0005
0,0006
[NO], M
0,00348
0,00352
0,00356
1/T, K
0,00360
0,00364
-1
Figure 4. (a) kobs versus [NO] as a function of temperature for the first reaction of the
(RSP4+)FeIII complex with NO as measured by stopped-flow technique. (b) Eyring plot of
ln(kon/T) versus 1/T. Experimental conditions: [(RSP4+)FeIII] = 8 × 10-6 M, in water, λdet =
440 nm.
On a longer time scale, the band at 440 nm representing the (RSP4+)FeIII(NO)
complex disappears with the concomitant appearance of a new band at 423 nm. The
- 161 -
P450 model complex
resulting spectrum resembles the absorption spectrum of the parent (RSP4+)FeIII complex
in aqueous solution, however its Soret band intensity appears to be much higher than that
of the (RSP4+)FeIII complex. Moreover, there is a shift in the Q-band from 549 to 546 nm,
as shown in Figure 5.
The rate constant of the subsequent reaction was shown to depend on the NO
concentration. The plots of kobs versus [NO] at different temperatures (see Figure 6) are
linear with almost zero intercepts within the experimental error limits, viz. koff = 1.4 ± 1.7
s-1. The resulting second-order rate constant, kon, determined from the slope of this plot
equals (86 ± 3) × 103 M-1 s-1 at 25 oC. The linear Eyring plot enabled calculation of the
activation parameters, ΔH‡on and ΔS‡on for the “on” reaction. The rate constants and
activation parameters are summarized in Table 1.
0,25
0,100
445 nm
0,095
0,090
Absorbance
Absorbance
0,20
0,085
0,080
0,075
0,15
0,070
0,065
0,0
0,1
0,2
0,3
0,4
0,5
Time, sec
0,10
0,05
0,00
350
400
450
500
550
600
Wavelength, nm
Figure 5. Typical spectral changes and an absorbance-time plot for the subsequent
reaction of the (RSP4+)FeIII complex with NO measured with the stopped-flow instrument.
Experimental conditions; [(RSP4+)FeIII] = 4 × 10-6 M, [NO] = 1 mM, pH 8.8 (0.025 M
Tris buffer), μ = 0.05 M with KNO3, temp = 7.5 oC, λdet = 445 nm
- 162 -
P450 model complex
90
80
6,0
o
25 C
(a)
5,8
70
o
20 C
(b)
5,6
60
5,4
40
ln(kon/T)
kobs, s
-1
o
15 C
50
o
10 C
30
5,2
5,0
o
5C
20
4,8
10
4,6
0
0,0000
4,4
0,0002
0,0004
0,0006
0,0008
0,0010
0,00335
0,00340
0,00345
[NO], M
0,00350
0,00355
0,00360
1/T
Figure 6. (a) kobs versus [NO] as a function of temperature for the second reaction of the
(RSP4+)FeIII complex with NO measured with the stopped-flow technique. (b) Eyring plot
of ln(kon/T) versus 1/T. Experimental conditions: [(RSP4+)FeIII] = 8 × 106 M, in water, λdet
= 424 nm.
The second reaction step is suggested to correspond to the formation of nitrosothiol as
shown below:
NO
NO
FeIII
S-
NO
NO
FeIII
S-
FeII
NO
SNO
The UV-Vis spectra indicated the occurrence of a further reaction step which is
accompanied by a rather small decrease in absorbance at 423 nm with a concomitant
small increase at 436 nm as shown in Figure 7(a). Plots of kobs versus [NO] as a function
of temperature in Figure 7(b) gave straight lines with slopes (kon) and relatively large
intercepts (koff). For instance kon = (30 ± 1) × 102 M-1 s-1 and koff = 6.2 ± 0.1 s-1 at 25 °C.
Eyring plots of these data were found to be linear allowing for the determination of ΔH‡
and ΔS‡ for the “on” and “off” reactions (see Figure 8). The resulting kon and koff values
along with the activation parameters are summarized in Table 1.
- 163 -
P450 model complex
11
0,25
0,120
441 nm
(a)
Absorbance
0,15
(b)
o
25 C
9
0,110
0,20
0,105
8
0,100
7
0,095
6
-1
0,090
kobs, s
Absorbance
10
0,115
0,085
0,0
0,2
0,4
0,6
0,10
0,8
1,0
1,2
1,4
1,6
Time, sec
o
20 C
5
4
o
15 C
3
0,05
o
10 C
2
o
5C
1
0,00
350
400
450
500
550
600
0
0,0000
650
0,0002
0,0004
0,0006
0,0008
0,0010
[NO], M
Wavelength, nm
Figure 7. (a) Typical spectral changes and absorbance-time plot for the third step in the
reaction of the (RSP4+)FeIII complex with NO measured with the stopped-flow instrument.
[(RSP4+)FeIII] = 4 × 10-6 M, [NO] = 1 mM, pH 8.8 (0.025 M Tris buffer), Temp = 25 oC,
λdet = 441 nm (b) kobs versus [NO] as a function of temperature for the third reaction, λdet
= 440 nm.
2,8
2,6
-3,0
(a)
-3,5
2,4
2,2
(b)
-4,0
-4,5
1,8
ln(koff/T)
ln(kon/T)
2,0
1,6
1,4
-5,0
-5,5
1,2
1,0
-6,0
0,8
-6,5
0,6
0,00335
0,00340
0,00345
0,00350
0,00355
0,00335
0,00360
0,00340
0,00345
0,00350
0,00355
0,00360
1/T
1/T
Figure 8. Eyring plot for the third reaction of the (RSP4+)FeIII complex with nitric oxide
as measured with the stopped – flow technique. (a) ln(kon/T) versus 1/T. (b) ln(koff/T)
versus 1/T. Experimental conditions: [(RSP4+)FeIII] = 4 × 10-6 M, in water, λdet = 424 nm.
The third reaction step is suggested to involve the weak binding of a second NO molecule
to the five-coordinate FeII-NO center as shown in the following scheme. The unfavorable
- 164 -
P450 model complex
equilibrium position is demonstrated by the overall equilibrium constant (kon/koff) of 480
M-1 at 25 °C for this reaction step. As studied by Ford et al, the formation stability of
dinitrosyl iron(II) porphyrin complexex is relatively low (K < 3 M-1 at 25 oC).5 This
observation leads to conclusions that the attack of N2O3 on the five-coordinate (RSP4+NO)FeII-NO2 occurs to yield a nitrosyl-nitrite complex as shown in scheme. This reaction
is accelerated with increasing NO concentration in terms of the presence of the higher
nitrogen oxides present as impurities in the saturated NO solutions.5,6
NO
NO
NO
FeII
NO
FeII
FeIII
or
NO
N 2O 3
NO
SNO
NO2
SNO
SNO
The kinetics of the reductive nitrosylation of (P4+RS-NO)FeII-NO or (P4+RSNO)FeIII-NO2 to form (P4+RS-NO)FeII-NO at pH 8.8 was observed as a slow, fourth
reaction step. The solution spectra show shifts in the Soret- and Q-bands from 423 and
534 nm to 424 and 552 nm, respectively (see Figure 9). Kinetic plots for the
disappearance of (RSP4+)FeIII monitored at 440 nm could be fit to a single exponential
function, from which kobs was determined to be 2.2 × 10-3 s-1 at 25 °C (see inset in Figure
9).
0,3
0.180
0.175
0.170
Absorbance
Absorbance
0.165
0,2
0.160
0.155
0.150
0.145
0.140
0.135
0
200
400
600
800
1000
1200
1400
Time, sec
0,1
0,0
300
350
400
450
500
550
600
650
700
750
Wavelength, nm
Figure 9. Spectral changes following the binding of NO to (RSP4+)FeIII(OH) at pH 8.8.
Inset: Absorbance vs. time trace at 440 nm fitted with a single exponential function.
- 165 -
P450 model complex
Experimental conditions: [(RSP4+)FeIII] = 4 × 10-6 M , [NO] = 1 mM, temp = 25 oC, I =
0.05 M (KNO3) in water.
As already mentioned, the observed reaction is catalyzed by the addition nitrite ion,
always present as impurity in NO aqueous solution. The solution of (RSP4+)FeIII(OH) was
mixed with a buffered NO solution containing various concentrations of NaNO2 at 298 K.
A linear plot of kobs versus [NO2–] with an almost zero intercept was obtained (see Figure
10 and Table 1). The slope of this plot equals 0.21 ± 0.01 M-1 s-1 from which the knitrite
value of 0.31 M-1 s-1 was obtained with the help of the following equation.
knitKNO[NO2-][NO]
kobs =
1 + KNO[NO]
The suggested reductive nitrosylation mechanism is given in the following reaction
scheme.
NO
NO
FeIII
FeII
NO2
NO2
SNO
NO
Reductive
Nitrosylation
FeII
NO2-
SNO
SNO
0.0014
0.0012
0.0010
kobs, s
-1
0.0008
0.0006
0.0004
0.0002
0.0000
0.000
0.001
0.002
0.003
0.004
0.005
0.006
-
[NO2 ], M
Figure 10. Nitrite concentration dependence on the reductive nitrosylation for the
reaction of (P4+RS-NO)FeII(NO+). Experimental conditions: [(RSP4+)FeIII] = 8 × 10-6 M,
[NO] = 1 mM, temp = 25 oC, pH = 8.8, I = 0.05M (KNO3) in water, λdet = 424 nm.
- 166 -
Table 1. Summary of Rate and Activation Parameters for the Different Reaction Steps Observed in the Reaction of
(RSP4+)FeIII with NO at pH 8.8
temp
(oC)
3
6
9
12
15
25b
ΔH‡,
First reaction
kon/104
koff
Second reaction
temp
kon/103
koff
(M s )
(s )
(oC)
18.8 ± 0.5
22.5 ± 0.6
26.7 ± 0.4
31.7 ± 0.7
36.2 ± 0.4
60.7± 0.6
-4 ± 2
5±2
12 ± 1
22 ± 1
40 ± 1
300 ± 2
6
10
15
20
25
-1 -1
-1
temp
(M s )
-1
(s )
(oC)
26.4 ± 0.8
36.9 ± 0.8
51.1 ± 0.9
66 ± 2
86 ± 3
-0.5 ± 0.7
0.9 ± 0.5
1.8 ± 0.7
1.3 ± 1.5
1.4 ± 1.7
5
10
15
20
25
-1 -1
Third reaction
kon/102
koff
(M-1 s-1)
(s-1)
6.8 ± 0.1
9.6 ± 0.5
14.7 ± 0.1
21.8 ± 0.5
30.1 ± 0.4
0.7 ± 0.1
1.2 ± 0.2
1.8 ± 0.1
3.3 ± 0.2
6.2 ± 0.1
35 ± 1
154 ± 7
38 ± 1
48± 1
(kJ/mol)
ΔS‡,
-16 ± 3
+323 ± 24
-22 ± 3
-16 ± 3
(J/mol⋅K)
a
Temperature Dependence of the Nitrite-Catalyzed Reaction of (RSP4+)FeIII with NO.
298K on the basis of Eyring plot, Figure 4(b).
- 167 -
b
Fourth reactiona
temp
k/10-3
(oC)
(M-1 s-1)
15
20
25
30
35
1.1 ± 0.1
1.6 ± 0.2
2.8 ± 0.2
4.0 ± 0.2
6.1 ± 0.4
69 ± 5
62 ± 3
+1 ± 18
-87 ± 9
These values are calculated for
P450 model complex
6.3.3 Reactivity of (RSP4+)FeIII toward NO at pH 3.5
The reversible binding of NO to (RSP4+)FeIII(H2O) at pH 3.5 proceeds significantly
faster than that observed for (RSP4+)FeIII(OH) at pH 8.8. As is seen in Figure 11, there are
two reaction steps that occur within 50 ms and 1 s, respectively. Unfortunately, due to the
rapidity of the first reaction and limitation of the rapid-scan instrument, it was not
possible to record the gradual spectral changes that are suggested to corresponds to NO
binding to (RSP4+)FeIII(H2O). It is assumed that the second reaction step can be ascribed
to the formation of the nitrosothiol complex under these conditions.
0,134
0,132
Absorbance
0,130
Second
reaction
0,128
0,126
0,124
0,122
First
reaction
0,120
0,118
0,116
0,0
0,2
0,4
0,6
0,8
1,0
Time, sec
Figure 11. Typical absorbance-time plot for the reaction of (RSP4+)FeIII(H2O) with NO
measured with the stopped-flow instrument. Experimental conditions: [(RSP4+)FeIII] = 2 ×
10-6 M, [NO] = 0.4 mM, pH = 3.5, λdet = 424 nm.
The UV-vis spectrum observed for the (RSP4+)FeIII(H2O) complex in aqueous
medium displays Soret- and Q-band maxima at 420 and 533 nm for pH 3.5. Following
rapid binding of NO to (RSP4+)FeIII(H2O) results in changes in the Soret band to 422 nm
and Q-band to 533 nm (no change in the Q-band) with isosbestic points at 434 and 462
nm as shown in Figure 12. Shown are the spectral changes observed immediately after
stopped-flow rapid mixing of (RSP4+)FeIII with a NO solution at 6oC and pH 3.5.
- 168 -
P450 model complex
Absorbance time traces recorded at 424 nm indicate that the observed reaction occurs
with 0.5 sec at 6oC (see Figure 12 inset). The uptake of NO seems to be irreversible since
it is followed by fast subsequent reaction (see below).
0,3
0,230
0,225
Absorbance
0,220
Absorbance
0,2
0,215
0,210
0,205
0,200
0,0
0,1
0,1
0,2
0,3
0,4
0,5
Time, sec
0,0
300
400
500
600
Wavelength, nm
Figure 12. Typical spectral changes and absorbance-time plot for the second reaction step
observed for the reaction of the (RSP4+)FeIII(H2O) complex with NO measured with the
stopped-flow instrument. Inset: Absorbance-time trace recorded at 424 nm from which
the observed rate constant kobs = 18.5 s-1. Experimental conditions: [(RSP4+)FeIII] = 4 × 106
M, [NO] = 0.8 mM, temp. = 6 oC, the obtained spectra were recorded every 5 ms.
The rate of the second reaction was found to depend on the NO concentration. The
plot of kobs versus NO was found to be linear with an almost zero intercept as seen in
Figure 13. The determined second order rate constant kon = (74 ± 1) × 103 M-1 s-1 at 25 oC,
and values over the temperature range of 5 to 25 oC are summarized in Table 2. The
corresponding Eyring plot was found to be linear and resulted in the activation parameters
summarized in Table 2.
- 169 -
P450 model complex
100
5,8
o
6C
o
10 C
o
15 C
o
20 C
o
25 C
(a)
80
5,6
5,4
5,2
-1
ln(kon/T)
60
kobs, s
(b)
40
5,0
4,8
4,6
20
4,4
0
0,0000
4,2
0,0002
0,0004
0,0006
0,0008
0,0010
0,0012
0,00335
0,00340
[NO], M
0,00345
0,00350
0,00355
0,00360
1/T
Figure 13. (a) kobs versus [NO] as a function of temperature for the second step of the
reaction of (RSP4+)FeIII(H2O) with NO as measured by stopped-flow technique. (b)
Eyring plot of ln(kon/T) versus 1/T. Experimental conditions: [(RSP4+)FeIII] = 4 × 10-6 M
in water, pH = 3.5, λdet = 424 nm.
On the basis of the observed spectral changes and kinetic data, we suggest that the first
reaction corresponds to water substitution bound to the metal center by NO and the
second reaction to the formation of the nitrosothiol complex in agreement with
conclusions drawn for a closely related system in the literature.7 The overall reaction
scheme is shown below.
OH2
FeIII
NO
FeIII
NO
S-
S-
NO
FeIII
OH2
NO
FeII
NO
S-
SNO
For the second reaction, the observed kon = (74 ± 1) × 103 M-1 s-1 at pH 3.5 and 25 °C is in
good agreement with kon = (86 ± 3) × 103 M-1 s-1 found at pH 8.8 and supports our
mechanistic assignment.
- 170 -
P450 model complex
The kinetics of reductive nitrosylation of (P4+RS-NO)FeII(NO) was also
investigated at pH 3.5. The solution spectra exhibited a corresponding shift in the Soretand Q-band from 420 and 533 nm to 424 and 552 nm as shown in Figure 14, respectively.
The final spectrum is identical to that observed at pH 8.8.
0,5
0,42
0,41
0,40
Absorbance
Absorbance
0,4
0,39
0,38
0,37
0,36
0,3
0,35
0,34
0,33
0
0,2
200
400
600
800
1000 1200 1400 1600 1800 2000
Time, sec
0,1
0,0
300
350
400
450
500
550
600
650
700
750
Wavelength, nm
Figure 14. Overall spectral changes observed for the second step of the reaction of the
(RSP4+)FeIII complex with NO. Inset: Absorbance-time trace at 423 nm. Experimental
conditions: [(RSP4+)FeIII] = 8 × 10-6 M, [NO] = 1 mM, μ = 0.05 M with KNO3, pH 3.5,
temp = 25 °C. Spectra were recorded every 5 s, kobs = 2.0 × 10-2 s-1
As already mentioned, the observed reaction at pH 3.5 is also catalyzed by the
addition of nitrite ion, always present as impurity in aqueous solutions of NO. A solution
of (RSP4+)FeIII was mixed with a buffered NO solution containing various concentrations
of NaNO2 at 298 K. A linear plot of kobs versus [NO2–] with an almost zero intercept was
obtained (see Figure 15 and Table 2), from which the slope is 13.2 ± 0.2 M-1 s-1. The
slope of this plot is similar to that found for positive charged porphyrin complex,
(TMPyP)FeIII.8 The observed reductive nitrosylation at pH 3.5 is two orders of magnitude
faster than measured at pH 8.8. This is due to the presence of the NO2 ligand at high pH
that decreases the electrophilicity of coordinated NO+ and slows the reductive
nitrosylation process.
- 171 -
P450 model complex
0,08
-9,0
0,07
-9,2
-9,4
0,06
-9,6
-9,8
ln(k/T)
kobs, s
-1
0,05
0,04
0,03
-10,0
-10,2
-10,4
0,02
-10,6
0,01
-10,8
0,00
0,000
-11,0
0,001
0,002
0,003
0,004
0,005
0,006
0,00330
-
0,00333
[NO2 ], M
0,00336
0,00339
0,00342
0,00345
0,00348
-1
1/T, K
Figure 15. (a) Nitrite concentration dependence of the reductive nitrosylation of (P4+RSNO)FeII(NO+) at pH 3.5 and 25 °C. (b) Temperature dependence of the nitrite catalyzed
reductive nitrosylation reaction.
Table 2. Summary of Rate and Activation Parameters for the Different Reaction Steps
Observed in the Reaction of (RSP4+)FeIII with NO at pH 3.5
Second reactiona
temp
o
( C)
kon/103
(M-1 s-1)
koff
(s-1)
Fourth reactionb
temp
(oC)
k/10-2
(M-1 s-1)
6
15
24.0 ± 0.9
-0.9 ± 0.5
0.8 ± 0.1
10
20
33 ± 1
-0.5 ± 0.7
1.2 ± 0.1
15
25
44 ± 1
2±1
1.5 ± 0.1
20
30
59 ± 2
5±1
2.3 ± 0.2
25
74 ± 1
8±2
‡
ΔH , (kJ/mol)
38 ± 2
46 ± 4
ΔS‡, (J/mol⋅K)
-24 ± 7
-125 ± 12
a
4+
III
temperature dependence of the reaction of (RSP )Fe (H2O) with NO
b
temperature dpendence of the ntrite-dtalyzed rductive nitrosylation
raction of (P4+RS-NO)FeII(NO+)
- 172 -
P450 model complex
Based on the reported rate and activation parameters the following reaction scheme
is proposed to account for the reductive nitrosylation reaction at pH 3.5.
(P4+RS-NO)FeII(NO) + HONO
N2O3
(P4+RS-NO)FeIII(NO2)(NO) + H+
(4)
Reductive nitrosylation
(P4+RS-NO)FeII(NO) + HONO
6.3.4. Discussion of kinetic data
The axial coordination sites of Fe(III) in (RSP4+)FeIII are occupied by thiolate as
the fifth and a water molecule or hydroxide ion as the sixth ligand.9 Since the RS
coordination face of the porphyrin molecule is protected by bulky groups, it is assumed
that the first fast reaction observed immediately after stopped-flow mixing of an aqueous
solution of (RSP4+)FeIII with an excess of NO represents rapid displacement of water (or
hydroxide) by NO leading to the formation of the six coordinate (RSP4+)FeIII-NO.
The binding of NO to (RSP4+)FeIII was found to be very fast with a second-order
rate constant of kon = 6.5 × 105 M-1 s-1 at pH 8.8 and 25 oC. Since the synthetic thiolate
ligated nitrosyl complex is regarded as a model for cytochrome P450 enzymes, it is
informative to compare the kon values determined for those of ferriheme-thiolate proteins.
As seen from Table 3, the rate of NO binding to the heme proteins is affected to a large
extent by the protein structure and the accessibility of the heme binding pocket.
Comparison of the association rate constant obtained for the model SR complex with
those found for the iron(III) porphyrins, (Por)FeIII (Por = TMPS, P8-), demonstrate that
thiolate ligation of the iron(III) center appears to have only a minor influence.
- 173 -
P450 model complex
Table 3. Rate constants kon and koff for the nitrosylation of ferriheme-thiolate proteins and
synthetic complexes at 25 °C
Ferric (RSP4+)FeIIIb eNOSc nNOSd P450nore P450camf
TMPS
P8- g
Proteinsa
kon,
6.1×105 6.1×106 1.9 ×107 3.2 ×105 3.0 ×105 8.2×105
6.5 ×105
M-1s-1
217
koff, s-1
40
93
60
0.35
7.3 ×102
a
Abbreviations: eNOS = endothelial nitric oxide synthase; nNOS = neuronal nitric oxide
synthase; P450cam = Cytochrome P450, enzyme in the hydroxylation of camphor. b
Calculated for 298K from the Eyring plot reported in Figure 4. c H4B-saturated eNOS in
the absence of the substrate, measured at 10 oC. d Holoenzyme in the presence of H4B. e
pH 7.2 at 20 oC. f Substrate-free P450cam, pH 7.4, at 25 oC. g measured at 24 oC.
Due to the anionic nature of the RS- group, it is clear that the iron atom in the
thiolate-ligated heme proteins should be stabilized in its higher 3+ oxidation state.
Unfortunately, the determination of activation parameters for the release of NO from the
nitrosyl complex, (RSP4+)Fe(NO) was complicated by the occurrence of subsequent
reaction steps.
The second reaction step appeared to be nearly 10 times slower than the formation
of (RSP4+)FeII-NO+. Temperature dependent measurement were performed to obtain the
activation parameters ΔH‡ = 38.3 ± 0.8 kJ/mol and ΔS‡ = –22 ± 3 J/mol⋅K, which support
the operation of an associative mechanism. A suitable explanation for the second reaction
step is the direct attack of the second NO molecule on the sulfur atom of the thiolate ligand
in the initially produced (RSP4+)FeIII-NO complex. This is also observed in stable
thiolatecobalamin, which are also able to bind two molecules of NO.10,11 These reactions
suggest that one is bound to the central heme pocket and the other to the cystein thiolate
ligand to produce S-nitrosyl (SNO) conjugate, from which homolytic cleavage of the
Fe(III)-S-Cys bond occurred to give Fe(II)-NO.
It is supposed that the third reaction corresponds to the attack of excess NO or
NO2-, present as impurity in a large excess of NO, or hydroxide ion on (RSP4+)FeII(NO+).
However, we do not have any evidence to prove this.
- 174 -
P450 model complex
The (RSP4+)FeIII(OH) complex undergoes a similar subsequent reaction at pH 8.8
following the binding of NO, observed for synthetic ferric porphyrins. Thermal activation
parameters for the reductive nitrosylation process were determined for (RSP4+)FeIII(OH)
at pH 8.8.
6.4. References
1. (a) Ignarro, L. J.; Buga, G. M.; Wood, K. S.; Byrns, R. E. Proc. Natl. Acad. Sci. U.S.A.
1987, 84, 9265 (b) Stamler, J. S.; Jia, L.; Eu, J. P.; McMahon, T. J. ; Demchenko, I. T.;
Bonaventura, J.; Gernert, K. & Piantadosi, C. A. Science, 1997, 276, 2034
2. (a) Higuchi, T.; Shimada, K.; Maruyama, N.; Hirobe, M. J. Am. Chem. Soc. 1993, 115,
7551. (b) Urano, Y.; Higuchi, T.; Hirobe, M.; Nagano, T. J. Am. Chem. Soc. 1997, 119,
12008
3. Higuchi, T.; Hirobe, M. J. Mol. Catal. A: Chem. 1996, 113, 403.
4. Rich, A. M.; Armstron, R. S.; Eillis, P. J.; Lay, P. A. J. Am. Chem. Soc. 1998, 120,
10827
5. (a) Lim, M. D.; Lorkovic, I. M.; Wedeking, K.; Yanella, A. W.; Works, C. F.; Massick,
S. M.; Ford, P. C. J. Am. Chem. Soc. 2002, 124, 9737(b) Lorkovic, I. M.; Ford, P. C.
Inorg. Chem. 2000, 39, 632
6. Settin, M. F.; Fanning, J. C. Inorg. Chem. 1988, 27, 1431.
7. Frank, A.; Stochel, G.; Suzuki, N.; Higuchi, T.; Okuzono, K.; van Eldik, R. J. Am.
Chem. Soc. 2005, 127, 5360
8. Theodoridis, A.; van Eldik, R. J. Mol, Catal, A. 2004, 24, 197
9. Higuchi, T.; Uzu, S.; Hirobe, M. J. Am. Chem. Soc. 1990, 112, 7051
10. Zheng, D.; Birke, R. L. J. Am. Chem. Soc. 2002, 124, 9066
11. (a) Walker, F. A. J. Inorg. Biochem. 2005, 99, 216. (b) Weichsel, A.; Maes, E. M.;
Andersen, J. F.; Valenzuela, J. G.; Shokhiereva, T. Kh.; Walker, F. A.; Monfort, W. R.
Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 594
- 175 -
Summary
In Chapter 2 of this thesis, introduction of four flexible malonate substituents on
the highly negatively charged iron(III) porphyrin, (P8-)FeIII increases the pKa1 of
coordinated water in (P8-)FeIII(H2O)2. This is ascribed to through-space interactions of
negatively charged substituents with coordinated water, as well as to electronic effects.
The latter effects are reflected in partial depopulation of the iron dx2-y2 atomic orbital,
leading to ca. 26% contribution of the S = 3/2 spin state in the spin-admixed (P8)FeIII(H2O)2 system. This contribution is relatively high in comparison with those of other
(P)FeIII(H2O)2 species and correlates with the high lability of coordinated water, as is
indicated by rapid water-exchange and NO coordination rates observed for the (P8)FeIII(H2O)2 complex. The solution pH determines the nature of the axial ligands in (P8)FeIII and thereby controls the coordination number, spin state, and reactivity of the
iron(III) center toward NO. The predominantly five-coordinate, purely high-spin (P8)FeIII(OH) formed at pH > 9 binds NO according to an associative mechanism, in contrast
to a dissociatively activated process observed for six-coordinate (P8-)FeIII(H2O)2 at lower
pH. In the case of (P8-)FeIII(OH), however, the NO binding step is no longer controlled by
the lability of the metal center. Other (most probably electronic and structural) factors
involved in the rate-limiting Fe-NO bond formation and breakage (for the “on” and “off”
reactions, respectively) apparently account for the slower rate of NO binding and release
observed at high pH for (P8-)FeIII(OH). Correlations between spin/ligation states and
reactivity inferred on the basis of spectroscopic and kinetic data for other water-soluble
iron(III) porphyrins studied to date further support this conclusion. As suggested by
preliminary studies on other water soluble iron(III) porphyrins, the pH-reactivity pattern
reported here for water-exchange and reversible NO binding to (P8-)FeIII is a common
phenomenon for diaqua- and monohydroxo ligated (P)FeIII species in aqueous media.
In Chapter 3, the results of kinetic and mechanistic studies on the reversible binding
of NO to (P8+)FeIII indicated that the reactivity of model water-soluble iron(III)
porphyrins toward nitric oxide is tuned by the pH of the solution. In this context, the pH-
Summary
induced change from diaqua- to monohydroxo-ligated porphyrin slows down the binding
and release of NO and results in a mechanistic changeover in the coordination of NO to
the iron(III) center. A comparison of experimental data obtained for (P8+)FeIII with those
reported for other water-soluble (P)FeIII porphyrins clearly shows that the nature and
charge of substituents in the porphyrin periphery affect the dynamics of both the binding
and release of NO. These effects are particularly evident in the reactions of diaqua-ligated
porphyrins, where the effective electron density on the metal center influences the lability
of coordinated water (which determines the rate of the “on” reaction) and the strength of
the Fe–NO bond in the {FeNO}6 product (as evidenced by variation in koff values
determined for the complexes studied). Other (mainly steric and electrostatic) factors are
likely to influence the binding and/or release of NO in specific porphyrin systems.
Despite a rather limited set of studied porphyrins, it can be concluded on the basis of the
available data that reversible binding of NO to iron(III) porphyrins is very sensitive to the
nature of axial ligands and the porphyrin periphery. The NO+ ligand in the {FeNO}6
nitrosyl formed by (P8+)FeIII undergoes subsequent reductive nitrosylation to form
(P8+)FeIINO ({FeNO}7 nitrosyl) as the final reaction product. This reaction is addressed in
detail along with data for related complexes in a following report.
The important role of electronic, structural, and environmental factors (especially pH)
on the reactivity of the studied porphyrin complexes toward NO can be of biological
significance. Although the pH ranges selected to differentiate between the reactivity
pattern of the diaqua and hydroxo complexes are in some cases far away from biological
conditions, local effects in the catalytic “pockets” of enzymes can induce a drastic change
in the apparent acidity and result in a mechanistic changeover as reported, in this study.
Chapter 4 presents that the results of kinetic and mechanistic studies on NO2binding to (p8+)FeIII as a function of solution pH, in which pH-induced changes from
diaqua- to monohydroxo-ligated porphyrin slows down and results in the mechanistic
changeover in NO2- coordination to the iron(III) center. This observation is in line with
our results for the reactivity of model water-soluble iron(III) porphyrins toward nitric
oxide, as supported by previous studies. The rate of reductive nitrosylation consequently
- 177 -
Summary
correlates with the formation of ferric nitrosyl porphyrin complex (P)FeII(NO+) from the
binding of NO toward Fe(III) center depending upon the basicity of the porphyrin ring
linked electrophilicity of the metal center. It can be concluded on the basis of the
available data that electronic features of the Fe(III) center is primarily controlled by the
nature of substituent (i.e., electron-releasing or electron-withdrawing meso substituents)
and steric effect, overall charge of porphyrin ring and in addition electrostatic effects. In
these studies, we report that the rate of reductive nitrosylation is varied over two orders of
magnitude through appropriate modification of the porphyrin structure and investigated
the nitrite catalysis on the observed reaction. There is still controversy regarding the
separation of the inner- and outer- sphere mechanisms.
In Chapter 5, the presented results provide mechanistic information on the reaction
of NO with an extremely negatively charged porphyrin complex, (P16−)FeIII. The
speciation of (P16−)FeIII in aqueous solution depends on pH and involves the formation of
diaqua- and monohydroxo-ligated complexes. The experimental data obtained for
(P16−)FeIII(H2O)2 in comparison with the reported reactivity pattern for other water-soluble
(P)FeIII(H2O)2 porphyrins demonstrate that the number and nature of charged substituents
on the porphyrin affect the dynamics of the water exchange reaction and the reversible
binding of NO, in which both processes follow a dissociative substitution mechanism. For
the five-coordinate (P16−)FeIII(OH) complex, the addition of NO follows an associative
bond formation process which is in agreement with that found for a series of (P)FeIII(OH)
porphyrins. The nitrosyl adduct (P16−)FeII(H2O)(NO+) is subjected to slow reductive
nitrosylation that is catalyzed by nitrite to yield (P16−)FeII(NO) as product via an innersphere redox pathway. The rate-limiting step of this process shows that the reaction is
controlled by the electrophilicity of coordinated NO+. Importantly, potential catalytic
activity of hydroxide ions during reductive nitrosylation was found to be several orders of
magnitude larger than for the nitrite-catalyzed process. Because of the potential
significance of this observation for studies on NO interactions with heme proteins (in
which a variety of spin and ligation states are observed), the mechanistic conclusions
- 178 -
Summary
inferred here will be supplemented and verified by further studies that will be reported in
a subsequent paper.
Finally in Chapter 6, presents the spectrophotometric, kinetic and mechanistic
studies for the reversible binding of NO toward the thiolate ligated iron(III) porphyrin,
(SRP4+)FeIII, soluble in aqueous solution, as a function of pH and temperature. It can be
concluded on the basis of the results obtained that the stopped flow kinetic measurements
indicate that initial NO binding occurs at the ferric heme and is followed by heme
reduction with nitrosothiol formation (SNO). The second order rate constants determined
for the formation of (SRP4+)FeIII(NO) follow a dissociative mechanism, which involves
the release of bound H2O and OH– axial ligands. Subsequently, rapid formation of the
nitrosyl product, (SRP4+)FeIII(NO) undergoes subsequent reactions that involve direct
attack of a second NO molecule on the thiolate ligand, and reaction with higher nitrogen
oxides lead to the nitrosyl-nitrite complex are also involved. Lastly, reductive
nitrosylation occurs over a longer time scale. To improve the mechanistic understanding
for the sulfur ligated ferric proteins, several additional methods (i.e., FT-IR, EPR) should
be applied and the solubility of the complex should be improved.
- 179 -
Zusammenfassung
Im ersten Teil dieser Arbeit erhöht die Einführung von vier flexiblen MalonatSubstituenten an das hoch negativ geladene Eisen(III) Porphyrin- Molekül (P8-)FeIII den
pKs1 Wert des koordinierten Wasser-Moleküls von (P8-)FeIII(H2O)2. Das ist auf die
Wechselwirkungen von den negativ geladenen Substituenten mit dem koordinierten
Wasser-Molekül und auf elektronische Effekte zurückzuführen. Die letzteren Effekte
reflektieren sich in der partiellen Depopulation vom Eisen dx2-y2 Atomorbital, was zu ca.
26% Beteiligung vom S = 3/2 Spinzustand im spingemischten (P8-)FeIII(H2O)2 system führt.
Dieser Beitrag ist relativ gross und korelliert mit der grossen Labilität vom koordinierten
Wasser-Molekül, wie es von dem gemessenen schnellen Wasseraustausch und der hohen
NO Koordinierungsgeschwindigkeiten für den (P8-)FeIII(H2O)2 Komplex angezeigt wird.
Der pH-Wert der Lösung bestimmt die Natur des axialen Liganden in (P8-)FeIII und
kontrolliert dadurch die Koordinationszahl, den Spinzustand und die Reaktivität des
Eisenzentrums gegenüber NO. Der vorwiegende fünffach- koordinierte, reiner high-spin
(P8-)FeIII(OH) Komplex der bei pH > 9 gebildet wird, bindet NO über einen assoziativen
Mechanismus, im Gegensatz zu einem beobachteten dissoziativ aktivierten Prozess für
den sechsfach koordinierten (P8-)FeIII(H2O)2 bei niedrigerem pH. Dagegen ist der Schritt
der Bindung des NO Moleküls im Fall des (P8-)FeIII(OH) nicht mehr durch die Labilität
des Metallzentrums kontrolliert. Andere, involvierte (wahrscheinlich elektronische und
strukturelle) Faktoren in der geschwindigkeitsbestimmende Fe-NO Bindungsbildung und
–aufspaltung (für die „Hin“- und Rück-Reaktion entsprechend) erklären die niedrigere
Geschwindigkeit von der NO-Bindung und -Spaltung bei höherem pH für (P8-)FeIII(OH).
Korrelationen zwischen spin/ligation Zustände und Reaktivität abgeleitet von
spektroskopischen und kinetischen Daten für andere Studien von wasserlöslichen
Eisen(III)-Porphyrinen, unterstützen diese Folgerung. Wie durch vorläufigen Studien für
andere wasserlösliche Eisen(III)-Porphyrinen empfohlen, ist das pH-Reaktivitätsbild, von
dem hier berichtet wird, ein übliches Phänomen für Di-aqua und monohydroxo-(P)FeIII
Spezies im wässrigen Medium.
Zusammenfassung
In Kapitel 3, werden die Ergebnisse der kinetischen und mechanistischen Studien
von der reversiblen Bindung von NO an (P8+)FeIII präsentiert. Diese wird von dem pHWert der Lösung bestimmt. In diesem Kontext verlangsamt die vom pH-Wert induzierte
Änderung von diaqua- zum monohydroxo- porphyrin die Bindung und Abspaltung von
NO und resultiert zu einem mechanistischen Übergang der Koordination von NO zum
Eisen(III)-Zentrum. Ein Vergleich von experimentellen Daten für (P8+)FeIII mit denen für
andere wasserlösliche (P)FeIII zeigt dass die Natur und Ladung von Substituenten in der
Porphyrin-Peripherie beeinflusst die Dynamik von beiden, der Bindung und Abspaltung
von NO. Diese Effekte werden besonders klar in den Reaktionen von diaqua-Porphyrinen,
wo die effektive Elektronendichte im Metallzentrum auf die Labilität vom koordiniertem
Wasser (welches die Geschwindigkeit für die „Hin“-Reaktion bestimmt) und auf die
Stärke der Fe-NO Bindung im {FeNO}6 Produkt (wie von der Variation der koff Werte der
studierten Komplexen bewiesen) Auswirkungen hat. Andere (hauptsächlich sterische und
elektrostatische) Faktoren könnten die Bindung und/oder Abspaltung von NO in
spezifischen Porphyrinen beeinflussen. Trotz eines eher limitierten Sammlung von
studierten Porphyrinen, kann auf der Basis der vorliegenden Daten darauf geschlossen
werden, dass die reversible Bindung von NO an Eisen(III)-Porphyrinen sehr empfindlich
für die Natur der axialen Liganden und die Porphyrinperipherie ist. Der NO+ Ligand im
{FeNO}6 nitrosyl-Komplex, der von (P8+)FeIII entsteht, reagiert sofort weiter und es
entsteht (P8+)FeIINO ({FeNO}7 nitrosyl) als Produkt der Reaktion. Über dieser Reaktion
wird detailliert berichtet mit Daten für die entsprechenden Komplexen in einem folgenden
Bericht.
Die wichtige Rolle von elektronischen, strukturellen und Umweltsfaktoren
(besonders pH) auf die Reaktivität von den studierten Porphyrinkomplexen gegenüber
NO kann von biologischer Bedeutung sein. Obwohl die gewählte pH Bereiche der diaqua
und hydroxo Komplexen in vielen Fällen weit von den biologischen Bedingungen sind,
können lokale Effekte in den katalytischen „Taschen“ von Enzymen einen drastischen
Wechsel in der offensichtlichen Acidität induzieren und einen mechanistischen Wechsel,
wie in dieser Studie berichtet, hervorrufen.
- 181 -
Zusammenfassung
Kapitel 4 präsentiert die Ergebnisse der kinetischen und mechanistischen Studien
über die NO2- Bindung an dem (P8+)FeIII abhängig vom pH Wert stimmt hervorgerufenen
Änderungen von diaqua- zu monohydroxo-Porphyrin. Diese Bemerkung überein mit
unseren Ergebnissen über die Reaktivität von wasserlöslichen Modell Eisen(III)Porphyrinen gegenüber NO. Die Geschwindigkeit der reduktiven Nitrosylierung korelliert
mit der Bildung vom Eisen(II)-nitrosyl-komplex (P)FeII(NO+) über der Bindung von NO
an das Eisen(III)-Zentrum, die abhängig von deren Elektrophilie ist, die von der Basizität
des Porphyrinrings abhängig ist. Es kann darauf geschlossen werden, auf der Basis der
erhältlichen Daten, dass elektronische Eigenschaften vom Eisen(III) Zentrum an erster
Stelle von der Natur des Substituenten kontrolliert werden (Elektronen-abweisende und –
anziehende meso-Subtituenten) und sterische Effekte, Gesamtladung des Porphyrinrings
zusätzlich beeinflussen. In diesen Studien haben wir berichtet, dass die Geschwindigkeit
der reduktiven Nitrosylierung über zwei Zehnerpotenzen variiert wird durch Modifikation
der Porphyrinstruktur, und haben die Nitritkatalyse an die beobachtete Reaktion erforscht.
Es gibt noch Diskussionen über die Trennung vom „Inner“- und „Outer“-Sphere
Mechanismen. Wir erweiterten auch unsere Forschungen mit hoch positivgeladene
Eisenporphyrine die klare Eigenschaften aufweisen.
In Kapitel 5, die werden mechanistische Informationen über die Reaktion zwischen
NO und einem extrem negativ geladenen (P16-)FeIII-Porphyrinkomplex präsentiert. Die
Ausbildung des (P16-)FeIII-Porphyrinkomplexes in wässriger Lösung ist von dem pH-Wert
der
Lösung
abhängig
und
schließt
die
Entstehung
der
Diaqua-
bzw.
Monohydroxokomplexe mit ein. Die erhaltenen experimentellen Daten stehen im
Einklang mit den bereits berichteten Reaktivitätsmustern für andere wasserlösliche (P16)FeIII(H2O)2-Porphyrine und beweisen, dass die Anzahl und die Natur der geladenen
Substituenten am Porphyrin die Dynamik der Wasseraustauschreaktion sowie die
reversible Bindung des NO-Moleküls beeinflussen, wobei die beiden genannten Vorgänge
nach einem dissoziativen Reaktionsmechanismus ablaufen. An einem fünffach
koordinierten (P16-)FeIII(OH)-Komplex erfolgt die Addition des NO-Moleküls in einem
assoziativen Prozess in Übereinstimmung mit dem, was für eine Serie von (P)FeIII(OH)- 182 -
Zusammenfassung
Porphyrine herausgefunden wurde. Das Nitrosyladdukt (P16-)FeII(H2O)(NO+) unterliegt
langsamer reduktiver Nitrosylierung, die durch Nitrit katalysiert wird und über den Innersphere-redox-mechanismus
(P16-)FeII(NO)
als
Produkt
ergibt.
Der
geschwindigkeitsbestimmende Schritt dieses Prozesses zeigt, dass die Elektrophilie des
koordinierten NO+ die Reaktion kontrolliert. Wichtig ist, dass die potentielle katalytische
Aktivität des Hydroxid-ions während der reduktiven Nitrosylierung um mehrere
Größenordnungen höher als die für den nitritkatalysierten Prozess gefunden wurde.
Wegen der potentiellen Signifikanz dieser Beobachtung für Studien über die NO
Interaktion mit Häm-Proteinen (in denen eine grosse Auswahl von Spin- und
Koordinationszustände beobachtet werden) werden die mechanistische Rückschlüsse, die
hier abgeleitet werden, noch mit anderen Studien ergänzt, die in einer weiteren
Publikation berichtet werden.
Schliesslich in Kapitel 6, werden die spektrophotometrischen, kinetischen und
mechanistischen Untersuchungen für die reversible Bindung von NO an ein
wasserlösliches Eisen(III)-Porphyrin mit einem Thiolatliganden, (SRP4+)FeIII, in
Abhängigkeit vom pH und der Temperatur behandelt. Aus den Ergebnissen der stoppedflow Messungen kann geschlossen werden, daß die Bindung von NO am Eisen(III)-Häm
erfolgt, gefolgt von einer Reduktion des Eisens und Bildung von Nitrosothiol. Die
Geschwindigkeitskonstanten zweiter Ordnung für die Bildung des (SRP4+)FeIII(NO)
Komplexes lassen erkennen, daß dieser Komplex als ein gutes Modell für die
Thiolateisen(III)-proteine angesehen werden kann. Die Aktivierungsparameter zeigen,
daß die Bildung von (SRP4+)FeIII(NO) einem dissoziativen Mechanismus folgt, wobei die
Dissoziation eines axial gebundenen H2O oder OH- Liganden erfolgt. Der schnellen
Bildung des Nitrosylkomplexes, (SRP4+)FeIII(NO), folgen schließlich mehrere Reaktionen,
bei denen ein direkter Angriff eines zweiten NO-Moleküls an den Thiolatliganden und
Reaktionen höherer Stickstoffoxide eine Rolle spielen, die letztlich zu dem NitrosylNitrit-Komplex führen. Die reduktive Nitrosylierung erfolgt außerdem auch über längeren
Zeitabschnitten.
Um
die
Vorgänge
in
den
mechanistischen
Studien
der
schwefelkoordinierten Eisen(III) Proteine besser zu verstehen, sollten weitere Messungen
- 183 -
Zusammenfassung
(z.B. FT-IR, EPR) durchgeführt und die Löslichkeit des Komplexes in Wasser verbessert
werden.
- 184 -
Appendix
Appendix
Table A1. General Crystallographic Data for Zinc(II)-54,104,154,204-tetra-t-butyl52,56,152,156-tetrakis-(2,2-bis-ethoxycarbonyl-ethyl)-5,10,15,20-tetraphenylporphyrin (2).
Formula
C108 H140 N4 O20 Zn
Formula weight
1879.61
Diffractometer
Nonius KappaCCD
Temperature [K]
173(2)
0.71073
Wavelength λ(MoKα)[Å]
Crystal system
triclinic
Space group
P-1
a [Å]
13.0099(2)
b [Å]
13.5022(3)
c [Å]
16.3638(4)
α [°]
70.651(1)
β [°]
80.928(1)
γ [°]
68.970(1)
3
2529.04(9)
V [Å ]
1
Z
-3
1.234
ρcalcd [g cm ]
-1
Absorpt. coeff. [mm ]
0.313
F(OOO)
1006
3
0.35×0.35×0.20
Crystal size [mm ]
2θmax [°]
54.98
Index range (h, k, l)
-16 to 16; -17 to 17; -21 to 21
Reflections collected
21873
Independent reflections
11565 [R(int)=0.0212]
Reflections [I>2σ(I)]
8971
Data / restraint / parameters
11565 / 0 / 601
2
Goodness-of-fit on F
1.022
Final R indices [I>2σ(I)]
R1 = 0.0631; wR2 = 0.1753
R indices (all data)
R1 = 0.0821; R2 = 0.1925
-3
largest diff. peak and hole [e Å ]
0.747 and -0.573
The structure was solved by direct methods (SHELXS-97); parameters were refined with
all data by full-matrix least-squares on F2 (SHELXL-97) [G. M. Sheldrick, SHELX-97,
Program for refinement of crystal structures, University of Göttingen, 1997].
- 185 -
Curriculum Vitae
Curriculum Vitae
Personal Data
Name : Joo-Eun Jee
Date of Birth : February 10th 1979
Date of Place : Yeosu
Nationality : Republic of Korea
Education and Training
03.1985
-02.1997
Primary, middle, and high schools, Yeosu, South Korea
03.1997
-02.2001
Sunchon National University BSc. Chemistry
03.2001
-02.2003
07. 2002.
- 08. 2002
09. 2003.
- 03. 2004
03. 2003.
- 08. 2003
04. 2004.
- present
Sunchon National University MSc. Chemistry
Title of the thesis: “Preparation and Properties of Heteronuclear Multimetallic
Complexes Containing Nickel (II)”. Director: Prof. Dr. Chee-Hun Kwak
Visiting Researcher at the University of Erlangen - Nürnberg
Program name: Summer Institute Program for Korean Graduate Students
Korea Science and Engineering Foundation (KOSEF) and Deutscher
Akademischer Austausch Dienst (DAAD)
Researcher in Sunchon National University
This work was funded by Korea Science and Engineering Foundation
(KOSEF)
Special Researcher in Polymer Chemistry Plant in Sunchon National
University
PhD studies under the guidance of Prof. Dr. Dr. h.c mult. Rudi van Eldik
at the Institute for Inorganic Chemistry, University of Erlangen Nürnberg
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