The Electronic Configurations of Catalases and Peroxidases in their
High Oxidation States: A Definitive Assessment
DAVID DOLPHIN
Department of Chemistry, University of British Columbia, Vancouver, B.C.. Canada VfYI IYft
(Received 12May 1 9 8 0 )
Abstract. The highest oxidation states of catalase and horseradish peroxidase
contain two oxidizing equivalents more than the resting ferrihemoproteins. While
numerous electronic configurations for these primary complexes have been suggested,
the most widely accepted formulation is that of an he(IV) (S = 1) porphyrin p--cation
radical ( S = 1/2). The evidence for and against these electronic configurations is
reviewed and assessed.
1
2
The catalases and peroxidases (of which horseradish
(HRP) and Japanese-radish are typical examples) are
heme enzymes which contain non-covalent!y bound iron
protoporphyrin IX as their prosthetic group. As pari of
the enzymatic cycle of both catalase and peroxidase, the
resting enzymes, which contain ferric heme, undergo
revers ble
two-electron
oxidations.
These
highestoxidation states, which contain two electrons less than
the resting ferric enzymes, are green (Fig. 1A&B) and
are termed the primary compounds (Catalase I (Cat I)
and HRP I). The primary compounds both function
enzymatically as oxidants with catalase oxidizing hydrogen peroxide to dioxygen, while peroxidase oxidizes
organic substrates (primarily amines and phenols) by one
electron. When HRP I undergoes a one-electron reduction, HRP II is generated; Cat II can also be generated
via the one-electron reduction of Cat I by the use of
exogenous reductants, although Cat II is not enzymatically significant. HRP II, on the other hand, is an
enzymic oxidant which further catalyses the one-electron
oxidation of amines and phenols to regenerate the
resting enzyme.
Clearly, the primary compounds have a formal oxidation state of Fe(V). In addition, magnetic susceptibility
measurements' give a value of .S = 3/2. On the basis of
just this information, several electronic configurations
for these primary compounds can be envisaged, and
indeed, all of the possible permutations have been
suggested at one time or another.
The formal Fe(V) oxidation state has been suggested
as the actual electronic configurations for these systems.4
However, at the time this suggestion was made, no
complexes of iron in the 5 + oxidation level were known;
to our knowledge none have yet been reported and
5
substantiated. It was initially suggested that the primary
compounds were classical enzyme-substrate complexes
of the type Fe(III)OOH. Despite the fact that this
6
concept was shown to be wrong, non-peroxidatic oxidants such as hexachloroiridate(IV) can give rise to the
primary compounds, this formulation has taken a long
time to die, and one can still see it discussed at the
present time.
Instead of a two-electron oxidation of the ion, it has
7
been suggested that the porphyrin ring is the sight of the
two-electron oxidation and that HRPcould be envisaged
as an intermediate spin (3/2) ferric heme, with the two
oxidation equivalents stored in the porphyrin macrocycle. Since this formulation, intermediate spin ferric porphyrins have been reported for both natural and synthetic porphyrin systems and 3/2 spin systems are thus
8,9
achievable.
Nevertheless, we have carefully examined
both the chemical and spectral properties of porphyrin
10
p-dications
(where the porphyrin macrocycle has lost
two electrons from the highest filled molecular ( p)
orbital). Neither the optical spectra of metalloporphyrin
p-dications, which are essentially featureless over the
whole visible region, nor their high reactivity with nuc11
Ieophiles, parallels the properties of the primary compounds of catalase or peroxidase. This leads one to
conclude that the p-dication formulation for the primary
compounds is untenable.
Further candidates as electronic configurations for Cat
I and HRP I could be envisaged as resulting from the
one-electron oxidation of the porphyrin ring, coupled
with either an oxidation of the iron to Fe(IV) or with a
one-electron oxidation of the protein. Such suggestions
6,7,12,13
were considered unlikely
due to both the absence of
14
ESR signals, even at low temperature,
and to the
presumed instability of radical structures as enzymic
intermediates. However, since these objections were
raised to the possible intermediacy of porphyrin radicals,
we have shown that such species, in model systems, are
readily accessible and stable.
Numerous metalloporphyrins exhibit revers ble one10,15,16
electron oxidations
.The oxidized species exhibit
ESR signals (with g values close to that of the free
electron), and a detailed analysis of these spectra confirm
that the radicals are indeed metalloporphyrin p-cations,
where the remaining unpaired electron is delocalized
17
over the porphyrin periphery.
No matter what the
peripheral substituents on the porphyrin, nor, in general,
the nature of the coordinated metal, once oxidation to
the ir-cation radical has been achieved, only two classes
of optical spectra are observed for the green oxidation
18
products.
The two highest filled p-molecular orbitals of metalloporphyrins are essentially degenerate and have alu and
1
a2u symmetries. '' Removal of an electron from either of
these orbitals would generate a n-cation radical with a
2
2
Aiu or A2u ground state, and the two classes of optical
spectra noted above correspond to these two ground
states. Thus the p-cation radical of magnesium oc-
lsrael Journal of Chemistry Vol. 21 1981 pp. 1-71
Fig. 1. A. Electronic spectrum of ferrihorseradish peroxidase HRP ( ----------------------) and HRP I (............. ). B. Comparison of the electronic
spectra of the primary compounds of Cat I ( --------------- ) and HRP I ( .............). C. Comparison of the 2A2u and 'Alu electronic spectra of
.
[Co(III)OEP]2+ . 2Br ( ---------- ) 2CIO4 , (......... ). D. Comparison of the electronic spectra of Cat I (------------------) and deutero HRP I (...............).
E. Comparison of the electronic spectra of HRP I ( ------------------ ) and HRP II ( .............. ). F. Comparison31 of the electronic spectra of the
complexes of protoporphyrin dimethyl ester Fe(IV) T P P C = C(p-C1C6H4)2. N-methylimidazole (-------------- ) and its one electron oxidation
.
31
of the electronic spectra of Cat I ( -------------------------). [Fe(IV)TPP C = C(p-ClC6H4)2]+ Clproduct with Cl as counter ion. G. Comparison
.
( ........ ) and [Fe(IV)TPP C = C(p-ClC6H4),]+ F ( ---------------- ).
taethylporphyrin
([Mg(II)OEP]'),
which
exhibits
a
strong absorption band near 700 nm and a high energy
2
shoulder, is typical for that of the Alu ground state,
while
the
cation
radical
of
zinc
mesotetraphenylporphyrin
([Zn(II)TPP]'),
which
exhibits
several overlapping peaks in the whole visible region,
2
typifies the A2u ground state. Moreover, we observed
that the IT-cation radical of cobaltic octaethylporphyrin
2
([Co(III)OEP] ') could reversibly exhibit the spectrum
of either ground state depending upon the anion present
(Fig. IC)." All of these porphyrin radicals exhibit a high
stability which is perhaps not too surprising when one
remembers that the unpaired electron is dclocalized in an
orbital which extends over much of the porphyrin macrocycle. Of even more significance is the close similarity
2
2
between the optical spectra of the Alu and A2u ground
states of simple metalloporphyrins and those of Cat I and
HRP I (Fig. 1B&C). Indeed, this close similarity in
optical spectra led us to suggest that the electronic
configurations of catalase and peroxidase, in their highest
Israel Journal of Chemistry 21 1981
oxidation states, were those of porphyrin p--cat ion radicals."
Since one, but only one, electron can be lost from the
macrocyclic porphyrin ligand, during the two-electron
hydrogen peroxide mediated oxidation, a second site for
the other one-electron oxidation must be found. A
reasonable site for this is the iron atom, and an oxidation
state of Fe(IV) in HRP I and II is consistent with
20
Mossbauer studies, which show that the oxidation state
of the iron is the same in these complexes but different
from that of ferric iron. In addition the optical spectra of
HRP II and Cat II are typical of metalloporphyrins as are
those of simple Fe(IV) porphyrins that we have generated by the electrochemical oxidation of ferric porphy21,22
rins.
All of the above observations are consistent with the
overall electronic configurations of the primary compounds as being the TT-cation radicals of [Fe(IV) proIoporphyrin]''. The overall charge is balanced by ligands
provided by the protein or hydroxide. Furthermore, the
3
2
different ground states exhibited by HRP I ( A2u) and
2
Cat I ( A1u) would have to be controlled by the protein,
presumably via axial ligation.
The p-cation radical formulation for the electronic
configurations of Cat I and HRP I had gained wide
acceptance, but was recently questioned by Morishima et
23
1
al. on the basis of H NMR studies of HRP compounds I
and II. The experiments suggested that the iron in
compounds I and II were indeed high and low spin
Fe(IV). However, the NMR resonances of the peripheral
methyl groups of the heme in HRP I were observed at
only
5()-80
ppm
downfield
from
4,4-dimethyl-4silapentane-5-sulfonate. These chemical shifts are similar
to those observed for simple high spin Fe(IV) complexes.
It was argued, if HRP I was indeed an Fe(IV) p-cation
radical, that this might result in a dramatically paramagnetically shifted NMR spectrum coupled with a considerable broadening of the peripheral heme groups. Since
23
neither of these effects was observed, it was concluded
that the additional oxidizing equivalent of compound I
was not retained on the heme ring as a p-cation radical.
Since the magnitude and nature of the coupling of
spins in an Fe(IV) porphyrin p-cation radical system,
and the mechanisms of relaxation are unknown, we
considered the conclusion drawn by Morishima ct al. to
be premature, and indeed further experiments have
shown this to be the case.
24
1
La Mar and de Ropp have carried out similar H
NMR studies on deuterohemin reconstituted horseradish
25
peroxide (deutero-HRP), which we had earlier shown
2
exh bits a Alu ground state (like that of catalase) while
24
still exhibiting peroxidase activity. La Mar and de Ropp
found that resting HRP and deutero-HRP exhibited
heme methyl (rings A-D) shift patterns similar to other
high spin ferric heme proteins. However, the NMR
spectrum of deutero-HRP I showed the methyl shifts to
be downfield and the b-pyrrolic shifts to be upfield. It
21
was concluded that the similarity observed earlier in the
methyl shifts between HRP I and high spin Fe(IV)
models, rather than the Fe(IV) 77-cation radical, is an
unfortunate coincidence,and that the large methyl downfield and b-pyrrolic hydrogen upfield shifts in deuteroHRP I are consistent with extensive spin delocalization
primarily in the p-system. It was further pointed out
that, although the observed hyperfine shifts in deuteroHRP I are consistent with a porphyrin p-cation radical,
they do not prove the point. Nevertheless, it was
suggested the broader line width of the methyl groups in
deutero-HRP I does indirectly suggest a porphyrin cation
radical.
Calculation, using unrestricted Hartree-Fock SCF
26
techniques,
for Fe(IV) (S = 1) porphyrin p-cation
2
(S = 1/2) suggests that in the A2u configuration spin
density resides principally on the nitrogens and methine
2
carbons, while in the Alu configuration spin density
occurs mainly on the methine and C„ carbon atoms. In
neither case is there any appreciable spin density on the
23
Cp atoms. Consequently, Morishima*s results
may in
fact support the Fe(IV) p-cation radical description. It
13
can be expected that C NMR spectroscopy on HRP I,
where all the carbon atoms can be examined, will prove
useful in further examination of these electronic configurations and as a test of the above calculations.
Calculations on the relative energies, quadrupole splitting and electric field gradients for all of the other
suggested electronic configurations discussed above for
26
HRP I have also been made. Both the a1u and a2u
ground states have similar calculated quadrupole splittings and these fit best with the experimental observations. Only the Fe(V) (S = 3/2) configuration gave calculated values of DE0 in similar agreement to the experimental observations, and of all the hypotheses the 5 +
state is the least tenable.
Further support for a porphyrin free radical coupling
27
to Fe(IV) comes from the University of Illinois group,
who have presented Mossbauer and ESR studies which
suggest inter alia that in HRP I the Fe(IV) has S = I and
weakly couples to a radical of S = 1/2, and that the
28
previously observed ESR signal of HRP I is the sharp
central feature (with a g value of that of a free electron)
of a much broader spectrum (accounting for 0.7
spins/heme) which extends over 2000 G.
Clearly, developing a model Hamiltonian to account
for the ESR, NMR and Mossbauer data is an undertaking of some magnitude, and while considerable progress
is being made, one must wait until final conclusions can
be drawn from these studies. The subtle interactions
between unpaired spins on iron and porphyrin a2u-cation
radical, which make difficult the interpretation of the
resonance techniques described above, do not so dramatically effect electronic absorption spectra. Indeed, since
16
our initial assignment on the basis of optical spectra of
the p-cation formulation for the primary complex, additional evidence in support of this electronic configuration
has been obtained.
26 29,,30
Semiempirical MO calculation ,
suggests that in the
2
Alu ground state, spin density is primary on the a2
pyrrolic carbons and on the nitrogens, while the A2u
ground state places spin density largely on the mesocarbon atoms and that the two ground states are similar
in energy. These calculations are supported experimentally and suggest to us that the ground state of a p-cation
radical might be changed by changing the electron
donating or releasing power of the peripheral substituents. To this end we have reconstituted HRP with a
25
variety of different disubstituted deuterohemins.
The
majority of the reconstituted heme proteins exhibited the
2
same ( A2u) ground state as that of the natural protohemin enzyme, except for the case of deutero-HRP I which
exhibited an optical spectrum (Fig. ID) characteristic of
2
the catalase p-cation radical type ( A1u). Despite this
change in its electronic ground state to that of a Cat I,
deutero-HRP I still exhibited the enzymic activity of a
peroxidase. Nevertheless, the fact that the optical spectrum of the primary complex can be so dramatically
changed between two extremes, both of which are typical
of p-cation radicals, adds further support to the
hypothesis that the electronic ground states in these
enzymes are indeed best described as one-electron porphyrin ring oxidized systems.
Additional support for the hypothesis comes from the
31
work of Mansuy et al., who have isolated a number of
stable iron porphyrin carbene complexes (1). These
systems can be considered as the carbon analogs of the
"Fe(IV)" compounds II of catalase and peroxidase, and
indeed the carbene complexes exhibit optical spectra
similar to those of Cat II and HRP II (Fig. 1E&F).
Furthermore, the one-electron oxidation products of the
carbene complexes generate species (2) which are optically similar to Cat I and HRP I and to those of other
metalloporphyrin p-cation radicals which have already
been well characterized (Fig. 1F&G). These observations
31
of Mansuy et al. are of special importance since they
show that, in order to mimic the electronic spectra of the
Dolphin I Configurations of Catalases and Peroxidases
primary complexes, all of the oxidizing equivalents can
be stored on the iron porphyrin, such that any model
which suggests an oxidation of the protein is untenable.
Note, however, that while oxidation of the protein
does not occur with catalase or horseradish peroxide,
there is now very strong evidence that protein redox
chemistry does occur in cytochrome c peroxidase.
Cytochrome c peroxidase, like Cat and HRP, is a ferric
heme protein in its resting state which is oxidized by
32
hydrogen peroxide to a complex called ES by Yonetani.
ES is at the same level of oxidation as HRP I and Cat I,
but differs from them in several fundamental ways. All of
the appropriate physical techniques show ES to contain
Fe(IV) and a species with S = 1/2 which, unlike Cat I or
33, 34
HRP I, exhibits a strong ESR signal.
The optical
32
spectrum of ES is like that of Cat II and HRP II; this is
in further support of Fe(IV), but eliminates an oxidation
of the porphyrin. Ring oxidation is further discounted by the strong ESR signal that is observed from a
33, 34
non-metal-central radical.
Since the site of the additional oxidizing equivalent cannot be accounted for at
the metalloporphyrin, oxidation of the protein must
occur in this case. Indeed, recent work from Hoffman's
35
laboratory
has reached the interesting conclusion that
the site of protein oxidation is not, as has been earlier
suggested, an aromatic amino acid to generate the corresponding cation radical, but rather that the radical is
l
Israel Journal of Chemistry 21 l )Sl
sulfur based within a cluster of methionine residues
which share the unpaired electron.
So far all of the evidence for a p-cation radical
formulation as the electronic configuration of HRP and
CAT is based upon physical measurements. There is, in
addition, chemical evidence in support of the primary
11
36,37
complexes being ring oxidized. We and others
have
shown that ring oxidized porphyrins can react with both
radicals and nucleophiles to give meso-substituted
38
isoporphyrins (3). If the isoporphyrin is substituted by
hydrogen at the meso-position, then loss of a proton will
generate a porphyrin now meso-substituted by the original radical or nucleophile (Scheme 1). When horseradish
peroxidase is treated with an excess of oxidizing agent, a
transient species (absorbing at 940 nm) and named P-940
39
is
observed
which
decays
to
P-670,
(mesohydroxyheme. Scheme 1). Few tetrapyrrole macrocycles
show intense absorption in the near infrared. In fact
isoporphyrins (3) are the only compounds which do. This
suggests that P-940 is in fact a meso-hydroxy isoporphyrin which upon deprotonation gives the meso-substituted
heme. The significance of this chemistry is that it mimics
that now well established for porphyrin p-cation radicals
and adds further support for ring oxidation in the
primary compounds.
CONCLUSION
Chemical evidence, coupled with physical measurements on the enzymes and model systems, all support the
formulation of the primary complexes of catalase and
horseradish peroxidase as porphyrin p-cation radicals of
Fc(IV) protoporphyrin. In addition, the reinterpretation
of 'H NMR experiments, which were originally suggested
not to support the p-cation radical configuration, also
point in favour of the ring oxidized species.
Acknowledgements. Ihis work was supported
States National Institutes of Health (AM 17W)).
b\
the
United
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