Vrije Universiteit van Amsterdam
Faculty of Science
Literature study submitted for the degree
MSc in Analytical Sciences
S UPERVISOR : G ERT VAN DER Z WAN
The role of manganese in photosynthesis
Maria Marioli
Amsterdam, March 29, 2012
ABSTRACT
One transmembrane protein, photosystem II (PS II), located in the thylakoid membranes of photosynthetic organisms is responsible for the water oxidation into dioxygen, electrons, and protons during
photosynthesis. The key component of PS II is a µ-oxo bridged Mn4 Ca cluster in which water is
the substrate. Apart from manganese and calcium, other important elements for the water-splitting
activity are ligand aminoacids, chloride ions, and the electron acceptor tyrosine. The manganese
complex together with the aforementioned essential cofactors is designated as oxygen-evolving complex (OEC) or water-oxidation complex (WOC). The electrons are extracted in different time intervals, resulting to five distinct subsequent oxidation states Sn , where n = 0 - 4. The structure and
the catalytic mechanism of OEC has been investigated mostly with electron paramagnetic resonance
(EPR), X-ray spectroscopy, and computational studies. Recently, the elucidation of the structure by
X-ray crystallography with resolution 1.9 Å has provided much information, but the mechanism is yet
to be clarified. Considering that chemical and electrochemical water oxidation requires high activation energy, this unique property of biological water splitting with solar energy has drawn the attention of many scientists and inspired the search for manganese-based catalysts. Such catalysts can be
proven very useful for the production of alternative renewable, and clean energy from solar fuels (e.g.
H2 ). This literature study intends to review the studies about OEC, not to evaluate or criticize them.
i
Contents
Abstract
i
List of Figures
vi
1
Photosynthesis
1
1.1
Photosynthetic parts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1
1.2
Light absorption in PS II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2
1.3
Activity of PS II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4
2
3
Oxygen-evolving complex
7
2.1
Kok’s cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8
2.2
Experimental and theoretical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10
2.3
Oxidation states . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11
2.4
Cofactors of Mn cluster . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12
2.4.1
Calcium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12
2.4.2
TyrZ and TyrD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12
2.4.3
Ligand amino acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13
2.4.4
Chloride
14
Electron paramagnetic resonance
17
3.1
S0 and S2 EPR signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
19
3.2
Parallel and pulsed EPR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
21
3.3
Electron nuclear double resonance . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22
3.4
4
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Split
Si Y•Z
signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
23
X-ray spectroscopy
25
4.1
X-ray absorption near-edge structure . . . . . . . . . . . . . . . . . . . . . . . . . . . .
27
4.2
Extended X-ray absorption fine structure . . . . . . . . . . . . . . . . . . . . . . . . . .
28
4.3
X-ray emission spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
30
4.4
Time-resolved X-ray absorption Spectroscopy . . . . . . . . . . . . . . . . . . . . . . .
31
iii
CONTENTS
5
6
X-ray diffraction
33
5.1
35
X-ray radiation and cluster damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Manganese based catalysts
39
Appendix: Abbreviations
45
Bibliography
47
iv
List of Figures
1.1
1.2
1.3
Transmembrane enzymes responsible for the photosynthetic process. Adapted from [3] .
Absorption spectra of photopigments. Adapted from [1] . . . . . . . . . . . . . . . . . .
(a) The photon absorption by an antenna pigment molecule is transferred to a RC chloro-
2
3
phyll, or is re-emitted as fluorescence. (b) The electron ends up on the RC chlorophyll
1.4
1.5
because of the lower energy of the excited state. Adapted from [5] . . . . . . . . . . . .
Photoinduced charge separation. Adapted from [6] . . . . . . . . . . . . . . . . . . . .
Electron transfer chain in PS II. (1) Light absorption of P680 leads to electron transfer to
3
4
Pheo and formation of P680+• and Pheo− , (2) the electron is transferred from Pheo− to
PQA , (3) P680+• regain an electron from TyrZ , forming a radical Tyr•Z , (4) Tyr•Z oxidizes
1.6
the Mn4 complex and (5) PQB is reduced by PQA . Adapted from [1] . . . . . . . . . .
Redox potentials of every electron transmission. Adapted from [1] . . . . . . . . . . . .
2.1
Possible structure of Mn4 Ca complex derived from XRD studies with resolution 1.9 Å.
5
6
The atoms illustrated as spheres are Ca (yellow), Mn (purple), O (red) and H2 O (orange).
7
2.2
Adapted from [9] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Kok’s cycle: Oxygen evolution occurs after the advancement of OEC in four higher
8
2.3
oxidation states. Adapted from [18] . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Oxygen evolution as a function of the (accumulated) number of short flashes. The plot
9
2.4
has a damped oscillation pattern with a period of four flashes. Adapted from [18] . . . .
A) Classical Kok’s model and B) extended Kok’s model involving misses, double hits,
2.5
2.6
inactivations and backwards transitions [27] . . . . . . . . . . . . . . . . . . . . . . . .
Extensive hydrogen-bonding network [9] . . . . . . . . . . . . . . . . . . . . . . . . .
Ligand amino acids from X-ray diffraction studies with resolution 1.9 Å(2011). Adapted
10
14
2.7
from [9] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Location of Cl− inside a hydrogen network (XRD studies with resolution 1.9 Å) [9] . .
15
16
3.1
3.2
3.3
Hyperfine coupling and Zeeman effect. Adapted from [78] . . . . . . . . . . . . . . . .
Optional caption for list of figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
EPR signals of S0 , S1 , S2 and S2 Y•Z states. The narrow signal of tyrosine D (TyrD ) is
18
20
removed in the two last spectra. Adapted from [29] . . . . . . . . . . . . . . . . . . . .
21
v
LIST OF FIGURES
3.4
ENDOR in S0 and S2 state. Adapted from [93] . . . . . . . . . . . . . . . . . . . . . .
4.1
XANES and EXAFS analysis from the K-edge X-ray Absorption spectrum of Mn cluster.
Adapted from [35] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2
22
26
XANES spectra of every Si state expressed as fluorescence intensity divided by the incident X-ray intensity, F/Io , against the energy of the incident X-rays and their 2nd derivative spectra. Missinger et al. (2001), adapted from [32] . . . . . . . . . . . . . . . . . .
28
4.5
Extended Kok’s cycle with time-resolved XAS. Adapted from [110] . . . . . . . . . . .
31
5.1
Structures defined in 2001 with resolution 3.8 Å a) Spatial organization of PSII b) Suggested model for Mn positions. Adapted from [111] . . . . . . . . . . . . . . . . . . . .
5.2
33
OEC structure proposed in 2004, defined at 3.5 Å resolution a) Cuboidal structure of
the Mn4 CaO5 with one Mn atom outside of the cube b) Proposed mechanism for water
oxidation, c) Aminoacids associated with the complex. Adapted from [113] . . . . . . .
34
5.3
Structural analysis with 3.0 Å resolution (2005). Adapted from [115] . . . . . . . . . . .
35
5.5
Structural analysis with 1.9 Å resolution (2011). Adapted from [9] . . . . . . . . . . . .
37
6.1
First attempt to mimic the electron transfer in PS II [121] . . . . . . . . . . . . . . . . .
39
6.2
Structure of a manganese dimer linked to a Ru (II) compex which is photo-oxidized
with [Co(NH3 )5 Cl]2+ as the irreversible electron acceptor. EPR electron transfer studies
6.4
6.5
revealed oxidation state Mn(II,II) which evolve to Mn(III,IV). Adapted from [61] . . . .
40
Photoelectrochemical cell. Adapted from [118] . . . . . . . . . . . . . . . . . . . . . .
42
Mn k-edge (a) XANES and (b) pre-edge XAS data. Green line: [Mn4 O4 L6
line: the product of [Mn4 O4 L6
]+
]+ ,
blue
when it is sudpended in Nafion, red line:the reoxidized
product. Adapted from [134] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
vi
43
1
Photosynthesis
A
LL
living organisms need energy for function and growth; humans and animals receive this energy
from food, while plants, algae and many bacteria convert solar energy into chemical energy of re-
duced organic compounds. The redox equation which describes this process in oxygenic photosynthetic
organisms is,
light
2nCO2 + 2nH2 O −→ 2(CH2 O)n + nO2
and it is of paramount importance because chemical compounds abundant in the environment (water and
CO2 ), are converted into valuable O2 and carbohydrates. Conversely, plants during night, like the aerobic
heterotrophs, generate energy by respiring O2 to degrade the energy-rich carbohydrates and produce CO2
and H2 O. The CO2 returns to the atmosphere completing the cycle [1].
1.1
P HOTOSYNTHETIC PARTS
Three transmembrane enzymes, i.e. photosystem II, cytochrome b6 /f and photosystem I (fig. 1.1) are
responsible for photosynthesis. They are located in the thylakoid membranes of higher plants, algae and
cyanobacteria and convert water into molecular oxygen with a subsequent NADPH and ATP production,
by only using light as source energy [2].
Briefly, the function of every enzyme is:
1. Photosystem II (PS II)
This complex catalyzes the water cleavage into dioxygen and reduce plastoquinone (PQ) into plastoquinol (PQH2 )
hv
2H2 O + 2PQ −→ O2 + 2PQH2
2. Cytochrome b6 /f
This complex facilitates the electron transport from PS II to PS I, by oxidizing PQH2 and reducing
the protein plastocyanin (PC). Both molecules act as the mobile electron carriers between PSI and
PSII:
1
CHAPTER 1. PHOTOSYNTHESIS
Figure 1.1: Transmembrane enzymes responsible for the photosynthetic process. Adapted from [3]
PQH2 + 2PCox −→ PQ + 2PCred + 2H+
During the aforesaid stages, H+ is released on the lumen side of the membrane causing a gradient
which activates the ATP synthase.
3. Photosystem I (PS I)
This complex catalyzes the reduction of the terminal acceptor: NADPH.
hv
H+ + 2PCred + NADP+ −→ 2PCox + NADPH
NADPH oxidase is a strong reducing agent (in contrast to water) and, assisted by ATP synthase, enables photosynthetic species to reduce CO2 through a procedure known as Calvin cycle. In the following
sections the focus is shifted to the PS II and its water-splitting activity.
1.2
L IGHT ABSORPTION IN PS II
Photosystem I and II require solar energy in order to exhibit their catalytic activity. The light absorption
in these enzymes is facilitated by an efficient mechanism which involves light harvesting (LH) antennae
systems and reaction centers (RC). LH systems absorb solar energy and transfer it into the reaction
center. They are protein chains which contain photopigments and encompass the RC in an orderly way
[4]. Photopigments are molecules with an extensive network of alternating single and double bonds and
this polyenic structure offers them ability to absorb visible light with a very high extinction coefficient
(π → π ∗ absorption).
The most abundant photosynthetic photoreceptor in plants and green algae is chlorophyll. Chlorophyll is a cyclic tetrapyrrole, similar to protoporphyrin, and it contains Mg2+ for central ion [5]. It
2
CHAPTER 1. PHOTOSYNTHESIS
Figure 1.2: Absorption spectra of photopigments. Adapted from [1]
exhibits a green color because it absorbs red and purple light (fig. 1.2). Other photopigments are phycoquanin (cyanobacteria), β-carotene (carrot) and phycoerythrin (red algae). Photopigments transfer light
to the reaction center.
In PS II the RC in plants and algae is a dimer of chlorophyll a molecules, known as special pair (SP)
or primary donor P680. In cyanobacteria, it is a dimer of bacteriochlorophylls (BChls) or P870. P is due
to “pigment” and 680 nm, 870 nm are the wavelengths they absorb. The excited state of the special pair
of chlorophyll molecules has lower energy than those of the other antenna pigment molecules, therefore
it is able to “trap” the photon (fig. 1.3). The overall procedure of photon transmission and trapping is
very fast (100 ps) [4].
Figure 1.3: (a) The photon absorption by an antenna pigment molecule is transferred to a RC chlorophyll, or is re-emitted as fluorescence. (b) The electron ends up on the RC chlorophyll
because of the lower energy of the excited state. Adapted from [5]
3
CHAPTER 1. PHOTOSYNTHESIS
When the SP absorbs the photon, it goes through a photo-induced charge separation; a positive charge
is formed on the chlorophyll dimer and a negative charge on the acceptor (fig.1.4). In PS II the electron
acceptor is one pheophytin. When this charge separation takes place, the positively charged chlorophyll
dimer (P680+• ) is seeking to regain the lost electron triggering a series of redox reactions (fig. 1.5).
Figure 1.4: Photoinduced charge separation. Adapted from [6]
1.3
ACTIVITY OF PS II
PS II not only exhibits the unique water-splitting activity but also it is the part of plants that reacts with
many commercial herbicides as well as the most light-sensitive protein which limits plant growth at high
light intensities. For all these reasons, it has drawn the attention of many scientists. At the end of the
80s, two discoveries extensively broadened our knowledge over PS II: (i) the development of functional
PS II-enriched membrane preparations, free from other electron transfer proteins and (ii) the elucidation
of the three-dimensional structure of the purple bacterial PS II. The similarity between plantal PS II
and bacterial PS II was validated with site-directed mutagenesis, thus the function of plantal PS II was
explained by extrapolating the information obtained by the purple bacterial activity [7]. For the second
discovery Deisenhofer, Michel and Huber were honored with the Nobel Prize in Chemistry in 1988 [3].
The detailed structure of PS II has been elucidated and it can be found in a 2007 review [8] and in a
2011 XRD study with resolution 1.9 Å [9]. Photosystem II is a homo-dimer with a molecular weight of
350 kDa. Each monomer comprises of 20 polypeptide subunits and many cofactors. Cofactors such as
chlorophylls and carotenoids are responsible for the capturing and conveying of photonic energy to the
reaction center P680. The polypeptide subunits with known functions are the D1, D2, CP43, CP47 and
PsbO. The SP chlorophyll dimer (P680) and a Mn4 cluster, which is responsible for the water oxidation,
are located on D1 subunit (fig. 1.5). The Mn cluster is further stabilized by the subunit PsbO. The
photopigments are concentrated in two peripheral antenna proteins i.e. CP47 and CP43 [10].
The water-splitting activity of PS II is induced by a series of redox reactions that lead to the oxidation
of water into molecular dioxygen by the Mn cluster: Charge separation of the P680 leads to P680+• , and
the released electron follows a transmembrane electron transfer chain (ETC) involving one pheophytin
(Pheo) and two plastoquinones (QA and QB ). The positively charged P680+• regains its lost electron
4
CHAPTER 1. PHOTOSYNTHESIS
Figure 1.5: Electron transfer chain in PS II. (1) Light absorption of P680 leads to electron transfer to
Pheo and formation of P680+• and Pheo− , (2) the electron is transferred from Pheo− to
PQA , (3) P680+• regain an electron from TyrZ , forming a radical Tyr•Z , (4) Tyr•Z oxidizes
the Mn4 complex and (5) PQB is reduced by PQA . Adapted from [1]
from the Mn cluster via a redox-active tyrosine, the TyrZ or D1-Tyr161 (fig. 1.5). Thus solar energy
is converted into electron transfer energy with subsequent redox reactions (fig. 1.6). After two cycles,
QB is released as plastoquinol QB H2 to be re-oxidized by cytochrome b6 f complex and the electrons are
conveyed to photosystem I [11]. After four cycles, the Mn cluster having stored four charges, it is able
to oxidize water. Recently, the studies about the function of PS II have been extensively reviewed [12].
5
CHAPTER 1. PHOTOSYNTHESIS
Figure 1.6: Z-scheme of photosynthesis: Redox potentials of every electron transmission. Adapted from
[1]
6
2
Oxygen-evolving complex
T
HE
key component of PS II, where water-splitting and dioxygen formation take place, is called
oxygen-evolving complex (OEC) or water-oxidation complex (WOC). It is a tetramanganese - cal-
cium complex (Mn4 Ca) bound with µ-oxo bridges. The exact structure of the complex is not established
yet. A possible structure which stems from X-ray diffraction studies with resolution 1.9 Å, is illustrated
on fig. 2.1. In this structure three manganese atoms and a calcium form a (non-ideal) cube while the
fourth manganese lay aside. In addition, the cluster contain chloride ions, ligand aminoacids and involves
a nearby tyrosine (TyrZ or YZ ). All the last cofactors, although absent on fig. 2.1, are indispensable for
the catalytic function and they are also considered part of OEC. The theoretical and experimental studies
about OEC have been well reviewed in 1991 [2], 1996 [13], 2001 [14], 2006 [10], 2007 [15, 16], 2008
[17] and 2011 [3].
Figure 2.1: Possible structure of Mn4 Ca complex derived from XRD studies with resolution 1.9 Å.
The atoms illustrated as spheres are Ca (yellow), Mn (purple), O (red) and H2 O (orange).
Adapted from [9]
The OEC has an efficient mechanism to convert water molecules into molecular dioxygen. The
exact catalytic mechanism is yet to be established but the basic principles are known: (1) The Special
Pair of chlorophylls (P680) absorbs one photon from solar light, goes through charge photoseparation
7
CHAPTER 2. OXYGEN-EVOLVING COMPLEX
forming P680+• and, subsequently, attracts an electron from a nearby tyrosine YZ (2) Tyrosine forms
a radical (Y•Z ) and attracts an electron from the OEC (3) The procedure is repeated four times and the
OEC accumulates four charges (4) The lost electrons of the cluster are regained via oxidation of two
water molecules which are bound to the cluster and when the reaction
2H2 O → 4H+ + 4e− + O2
occurs, other two water molecules bind to the cluster to continue the catalytic cycle.
The ejection of every electron happens in different time intervals resulting to five distinct subsequent
oxidation states of the manganese complex S0 → S1 → S2 → S3 → S4 (→ S0 ). In every state the OEC
accumulates one positive charge apart from the oxidation state S4 which is unstable and is converted
rapidly into S0 . The transition S4 → S0 is light independent and very fast. This oxidation model of four
states was introduced by Kok et al and is designated as ”Kok’s cycle”.
Figure 2.2: Kok’s cycle: Oxygen evolution occurs after the advancement of OEC in four higher oxidation states. Adapted from [18]
The S1 state is the most dark-stable state; when no illumination occurs, transitions S0 → S1 ← S2 ←
S3 take place until approximately 25% of the population of PS II is in S0 state, while the remaining 75%
is in the S1 state.
2.1
KOK ’ S CYCLE
The complementary work of Kok et al. and Joliot et al. revealed the fundamental characteristics of the
oxidation procedure and proposed the Kok’s cycle (fig. 2.2) which is the oxidation model still in use
today. First, Joliot et al in 1969 [18] discovered a periodical fluctuation of the concentration of oxygen.
The experiment was illumination by a series of single-turnover flashes with duration of 2 µs and 0.3 s
duration of the dark period. By plotting oxygen concentration as a function of the (accumulated) flash
number, they observed a period-four oscillation (fig. 2.3). Moreover, the first maximum was observed
after 3 flashes, the second maximum after 7 flashes, and so on [18, 19].
After one year (1970) Kok et al [20] interpreted this result by a four-step oxidation mechanism in the
oxygen-evolving system. In this model the interaction between P680+ and water oxidation occurs via
the accumulation of four oxidizing redox equivalents, the Si states, where i is the accumulated oxidizing
equivalent. The first maximum in the 3rd flash number was explained with the dark-stable S1 state
(dark incubation leads to 75% population in S1 state, therefore most of the sample starts from this state).
8
CHAPTER 2. OXYGEN-EVOLVING COMPLEX
Figure 2.3: Oxygen evolution as a function of the (accumulated) number of short flashes. The plot
has a damped oscillation pattern with a period of four flashes. Adapted from [18]
The damping of the oscillation with time was attributed to misses (lose of a state) and double hits (fig.
2.4A) [2]. Double-hits are mostly a result of a long flash and can be reduced significantly by the use
of laser activation. These flashed-induced oxidation states have a lasting impact to our understanding in
photosynthesis and all the experiments involving OEC are based to this model.
Several solutions have been proposed to measure the misses and the double hits. The first solution
proposed by Forbush et al (1971) [21] assumed equal number of misses and double hits for every state
and the best fit with experimental data was found for probability α = 0.1 of misses and probability
β = 0.05 of double hits. Since then, traditionally, α refers to the probability of misses and β to that of
double hits. Delrieu introduced a new model assuming very unequal misses between the states which
gave better quantitative agreement [22, 23]. In this model misses occur nearly exclusively on S2 state
(one-miss model).
In 1976 Lavorel noticed that the Kok model of oxygen evolution can be described as a Markov chain
i.e., a stochastic process with discrete states and discrete time [24]. Mathematical problems involving
Markov chain can be solved with matrix analysis by describing the model with the row vector of probabilities pi (n) and a transition matrix Q, which in Kok’s case are [25],

0


 0 α γ β
pi (n) = (p0 (n), p1 (n), p2 (n), p3 (n)) and Q = 
 β 0 α γ

γ β 0 a





α γ
β
where α, β, γ are the probability of misses, double hits and of the (successful) hits, respectively. Therefore, α + β + γ = 1. The parameter n is the (accumulated) flash number e.g the initial vector is
pi (0) = (p0 (0), p1 (0), p2 (0), p3 (0)). Computational mathematics software is able to solve this matrix
analysis for many cases and find the best fit between simulations and experimental data. As a result the
probabilities of misses, double hits and initial conditions are known. In 1996, Meunier et al. added into
the model one more state, the S−1 and also backward transitions (probability δ) and inactivations (probability ) and presented slightly different results than the classical Kok’s model [26]. Shinkarev presented
simple analytical solutions in 2003 for the simple Kok’s model [25] and in 2005 for the extended model
9
CHAPTER 2. OXYGEN-EVOLVING COMPLEX
[27]. Figure 2.4 displays the classical and the extended Kok’s models.
Figure 2.4: A) Classical Kok’s model and B) extended Kok’s model involving misses, double hits, inactivations and backwards transitions [27]
In order to solve the Markov chain, it is necessary to assume that the probability of misses and double
hits is equal among states. Otherwise, the matrix would be underdetermined. However, direct estimation
of the miss probability of each S-state transition with electron paramagnetic resonance spectroscopy
(EPR) has given evidence that probabilities of misses are unequal for every state. Recently, Tyystjarvi
et al. published a paper assuming unequal probabilities [28]. They eliminated the double hits by using
too short laser flashes (4 ns) and they preconditioned the thylakoid membranes so as all the population
to start from S1 state. These practices made the model simpler and the best fit was found for one miss
model, namely for the transition S3 → S0 with probability α = 0.346. Moreover, they studied the
decay kinetics after one- or two-flashes illumination concluding that there are slow and fast transitions.
Fast transitions happen with recombination of S2 or S3 states and Q−
B and slow transitions are one step
deactivations (i.e. S2 → S1 ) and two-step deactivations (i.e. S3 → S1 and S2 → S0 ).
2.2
E XPERIMENTAL AND THEORETICAL STUDIES
A plethora of analytical techniques has been applied to characterize the structure and the function of
the manganese complex. Among them the most suitable methods are X-ray spectroscopy studies and
electron paramagnetic resonance (EPR) because they are atom-specific. Most of our knowledge of the
OEC structure and the changes on its intermediates S-states stems from these studies. EPR studies have
been well reviewed in 2001 [29] and 2007 [30] and X-ray spectroscopy studies were discussed in several
papers in 2001 [31], 2005 [32, 33], 2006 [34], and 2008 [35, 36]. Also mutagenesis studies have offered
a lot of information about the aminoacid ligands of OEC [37, 38, 39].
Last decades computational chemistry has been proven very beneficial for the investigation of biochemical systems and OEC is no exception. Considering the experimental difficulties to synthesize and
10
CHAPTER 2. OXYGEN-EVOLVING COMPLEX
define Mn cluster models in order to compare them with OEC, the complementary research with computational chemistry is a powerful tool. In addition, the contradicting results of XRD, XAS and EPR, about
the structure or about the oxidation during S2 →S3 transition, emerge further the use of simulations.
Early computational studies are using XANES and EPR data to find possible structures of OEC. Recent
studies (after 2003) have more interest since they include the XRD data [40, 41, 42, 43, 44]. In 2011
three reviews over computational studies have been published [45, 46, 47].
2.3
OXIDATION STATES
To investigate the oxidation and structure of OEC in every Si state in order to understand its catalytic
mechanism, we need first to advance the PS II population in the state of interest. However, this cannot
be achieved directly by using short saturated flashes, because the dark-stable PS II has a distribution of
75% S1 and 25% S0 and every S-state transition has only a 85 to 95% quantum yield. Early studies
used to pretreat the PS II with sufficient reagents to increase the population of a Si state. As an example
Messinger et al. have developed an efficient pretreatment to enhance the population of S0 -state by the
use of reducing agents such as hydroxylamine and hydrazine to convert S1 -state PS II centers into the
0
S0 -state, which otherwise would require three flashes and the quantum yield would be around 50% [48].
0
0
However, these pretreatments are artificial and the obtained states typically are expressed as S0 , S1 ,
0
0
S2 and S3 . Natural methods have been developed to advance the 100% of PS II population to the S1
and S2 states and then “trap” them in helium nitrogen temperatures. The advancement to S1 state is
achieved by one-flash illumination and subsequent dark incubation several minutes and to the S2 state
with continuous illumination in low temperatures (200K) [30]. In addition, there are methods to quantify
the S-state distribution in samples after n number of flash(es) by measuring their EPR signals [49, 50].
In order to understand the catalytic mechanism of OEC it is necessary to clarify whether the oxidation
in every state is Mn-center or another cofactor of the cluster is oxidized. If Mn is not oxidized, the
electron ejection may happen from a redox-active organic residue such as a protein side chain, a bound
water, or bridging oxygen. Many techniques have been applied to explore manganese redox changes e.g.
electron paramagnetic resonance (EPR), Fourier transform infrared (FTIR), near-edge X-ray absorption
(XANES). Among these techniques there is a consensus that Mn oxidation takes place during transitions
S0 → S1 , S1 → S2 whereas the transition S2 → S3 remains elusive. Two different mechanisms for
O2 evolution have been suggested, one assuming Mn oxidation in S3 state and the other one assuming
ligand or substrate oxidation. The charges of Mn4 in S1 and S2 states have been suggested from the
majority of the studies as Mn4 (III,III,IV,IV) and Mn4 (III,IV,IV,IV), respectively and less groups proposed
Mn4 (III,III,III,III) and Mn4 (III,III,III,IV) whereas for S0 state the presence or not of Mn(II) is in question
[51].
Another crucial point for our understanding in OEC activity is to detect when the formation of the
O-O bond takes place. Theoretical studies have proposed two pathways of O-O formation: (1) the O-O
bond is formed in the S4 oxidation state or (2) the O-O bond has already be formed from the S3 state and
occurs as a ligand peroxide. For the first case the reaction that leads to O-O formation may stem from
11
CHAPTER 2. OXYGEN-EVOLVING COMPLEX
a nucleophilic attack of a free or loosely bound substrate water molecule onto a highly valent Mn ion
Mn(V) = O or reaction of µ-oxo-bridge radical with another oxo- radical [16].
2.4
2.4.1
C OFACTORS OF M N CLUSTER
Calcium
Calcium is a metal commonly encountered in biological systems with a coordination number 6 or 7
[52]. The first indication that calcium plays a significant role in photosynthesis was in 1984 when it
was reported by Babcock et al. that PS II which had lost its catalytic activity by PsbP/PsbQ-depletion,
could regain it with the addition of Ca2+ [53]. The requirement of this cation was confirmed in 1988 when
extraction of Ca2+ with simple pH pretreatment resulted in inactivation of O2 evolution while subsequent
incubation with Ca2+ was able to restore the catalytic activity [54]. Shortly after, EPR studies of Mn in
native and in calcium-depleted PS II manifested a strong dependency between Ca2+ and Mn structure
and oxidation states [55].
Since then, various studies with EPR, XAS and FTIR have been conducted to discover calcium
functionality by analyzing native PS II, calcium-depleted PS II and also PS II in which Ca2+ has been
replaced by Sr2+ . With Sr2+ replacement, PS II maintain its function but the catalytic rate is reduced by
a factor of 2. All these studies have been recently reviewed [52, 56]. Some studies concluded that PS II
contained two or more populations of Ca2+ but this conception was abandoned with experiments using
45 Ca2+
which showed that only one extractable Ca2+ atom is associated with the OEC and that other
calcium-binding sites reside outside the complex [57].
In 1995 EXAFS studies manifested the proximity of calcium to manganese atoms [58, 59] and EXAFS studies in all the Si states indicated that significant changes into the distances Mn-Ca take place in
the transitions S2 →S3 and S3 →S0 [56]. The Ca2+ -depletion prevents the advancement in states higher
than S2 . However, it was demonstrated that Ca2+ removal does not lead to fundamental distortion or
rearrangement of the Mn cluster. That means that the Ca2+ in the OEC is not essential for structural
maintenance of the cluster, but it has an important function during the catalytic cycle [56].
2.4.2
TyrZ and TyrD
The most important aminoacid of PS II is tyrosine YZ because its neutral tyrosyl radical (Y•Z ) is the
direct oxidant of the manganese cluster. It is located in D1 polypeptide (D1-Tyr161) and forms a radical
after electron transfer to the special pair of Chlorophylls, which it rapidly regains from the Mn cluster.
Apart from a charge-carrier interface between the Mn cluster and P680 reaction center, it serves another
purpose, to release the protons from the PS II [60]. A tyrosine in water changes its pKa-value from 10
to -2 when it is oxidized, therefore the oxidation of the tyrosine is often coupled with hydrogen transfer
and this is possible the implication for proton release during transition states. In addition, oxidation of
a tyrosine without deprotonation, generating TyrOH•+ , requires potential +1.46 V (vs SHE), but for the
proton coupled potential at pH=7 a more moderate value of +0.93 V is needed [61].
12
CHAPTER 2. OXYGEN-EVOLVING COMPLEX
The mirror image of TyrZ in the homologous D2 polypeptide is the TyrD (D2-Tyr160) and although
it forms a neutral radical, it is dark stable because it is decaying very slow. The role of TyrD is not
clear yet but mutation studies manifested its importance for O2 evolution [60]. It has been proposed that
YD can oxidise the Mn cluster when it is in low valence states [62]. Before identification, they were
known as cofactors D and Z giving rise to identical EPR spectra with a g-factor of 2.0046. In 1987 they
were identified as tyrosyl unprotonated radicals [63] and in 1988 studies with site-directed mutagenesis
assigned TyrD and TyrZ to the aminoacids D2-Tyr160 and D1-Tyr161 respectively [64, 65]. A method
to obtain stable YZ was discovered in 1990 by Ono and Inoue. The procedure was Ca2+ depletion of
PS II and then illumination with subsequent liquid nitrogen freezing [66]. Pulsed EPR studies in 1994
disclosed that the distance of the two tyrosines is approximately 30 Å [67].
EPR characterization of Y•Z has been limited to inactive PS II until the discovery of preparations to
trap Y•Z in intact PS II by NIR illumination at helium liquid temperatures [68]. Reviews of the studies
of the two proteins have been published on 2001 [60], 2004 [62] and 2011 [69]. As is widely known
H+ cannot exist alone in nature and it has to be bound to water or other molecules. Thus, an extensive
hydrogen bond network is expected from tyrosine to the lumenal side of PS II. Indeed, X-ray diffraction
studies revealed an extensive hydrogen-bonding network between YZ and the Mn4 CaO5 cluster and from
YZ to the lumenal bulk phase (fig. 2.5). It was found YZ has hydrogen bonds with the nitrogens of D1His190 and the two water molecules bound with calcium. D1-His 190 was further hydrogen-bonded to
D1-Asn 298 and to several waters.
2.4.3
Ligand amino acids
The manganese cluster is bound to amino acid residues of two polypeptides of PS II, i.e. D1 and CP43.
Mn and Ca ions are connected with these amino acids via carboxylate bonds (with aspartic acid, Asp or
glutamic acid, Glu) or via nitrogen bonds (with arginine, Arg or histidine, His). These ligand amino acids
modulate the properties of the Mn4 Ca cluster. In order to elucidate the structure and make theoretical
models that explain the O2 evolution is crucial to include in the model the ligand aminoacids, not only of
the first coordination sphere (direct Mn or Ca binding) but also of the second (indirect binding through
bound water or oxygen of the µ-oxo bridges).
In 1992 the first site-directed mutations [70, 71] revealed that replacement of certain carboxylate
amino acids leads to partly or total inhibition of the catalytic activity. Namely, mutations of D1-Asp170
and D1-Ala344 resulted in a reduction of O2 evolution measured with EPR and fluorescence studies.
Therefore, these aminoacids were identified as possible direct ligands to OEC. Many other mutation
studies followed using X-ray spectrometry, EPR and FTIR to identified possible ligands. The ligands
of the D1 subunit, that their mutations were found to affect CaMn4 structure and activity, were: Asp59,
Asp61, Glu65, Asp170, Glu189, Glu333, Asp342, His92, His190, His332, His337 and Ala344. All these
studies involving site-directed mutations or depletion of amino acids are summarized on a review in 2001
[37]. Mutations of D1-Asp170 had the biggest impact on structure and catalytic activity so many studies
have been focused on this aminoacid [38, 39].
13
CHAPTER 2. OXYGEN-EVOLVING COMPLEX
Figure 2.5: Extensive hydrogen-bonding network [9]
X-ray crystallography studies revealed all the amino acids bound to the OEC (fig. 2.6): D1-His332
and D1-Glu189 are monodentate ligands bound to a manganese ion and D1-Asp170, D1-Glu333, D1Asp342, D1-Ala344 and CP43-Glu354 serve as bidentate ligands bound to two Mn ions or a Mn ion and
Ca. In addition, three more amino acids (D1-Asp61, D1-His337 and CP43-Arg357) are located in the
second coordination sphere stabilizing further the OEC [9].
2.4.4
Chloride
The significant role of Cl− in photosynthesis was denoted by Ono et al in 1986 [72] who presented
EPR measurements of Cl− -depleted photosystem II. The Cl− depletion resulted to inhibition of the
advancement in higher oxidation states; the EPR spectra of Cl− -depleted PS II did not show the EPR
multiline signal of S2 oxidation state neither with flash excitation nor with continuous illumination.
Moreover, after addition of Cl− ions, the multiline EPR signal of S2 was restored, which confirmed the
role of chloride [72]. However, another EPR signal of the S2 was observed which indicated that Cl− depleted PS II can be oxidized to form a modified S2 state. Early studies have showed that Cl− is not
required for the S0 →S1 and S1 →S2 , but it is for the transitions S2 →S3 and S3 →S0 [73]. Furthermore,
the exchange rate of Cl− in the S1 state was measured very low indicating a kind of strong bonding.
14
CHAPTER 2. OXYGEN-EVOLVING COMPLEX
Figure 2.6: Ligand amino acids from X-ray diffraction studies with resolution 1.9 Å(2011). Adapted
from [9]
In 2006 mutations of two basic residues, R151 and R161 resulted to inefficient binding of chloride to
the OEC. Thus, this aminoacids were identified as potential Cl-ligands [74]. In 2008 bromide anomalous
X-ray diffraction analysis have been employed to locate Cl− binding sites. Anomalous XRD analysis in
crystals of PS II whose Cl− was replaced by Br− revealed two Br− binding sites in the vicinity of the
OEC, which were not in the first coordination sphere but in a distance 6-7 Å. One Br− ion was located
close to the nitrogens of D2-K317 and D1-Glu333 whereas the other Br− was near the nitrogens of CP43Glu354 and D1-Asn338 [75]. The following year XRD studies were carried out in PS II crystals where
Cl− was replaced by I− . The Br− replacement did not inhibit O2 evolution while I− replacement does,
indicating that Cl− has more similar function with I− [76]. The results were similar with the previous
study but also it was found that the I− ions were located inside proton channels (hydrogen-bonding
network), therefore they may facilitate H+ and electron transfer.
With X-ray crystallography and resolution 1.9 Å, the electron density of the two chloride ions have
been identified in 2011. Each of Cl− ions is surrounded by four species, two water molecules and two
amino-acids. One Cl− is in proximity to the amino group of D2-Lys 317 and the nitrogen of D1-Glu 333
and the other Cl− with the nitrogens of D1-Asn 338 and CP43-Glu 354. The aminoacids D1-Glu 333
and CP43-Glu 354 are direct ligands of the Mn4 CaO5 cluster, therefore one function of the ions may be
to stabilize structure of the Mn4 CaO5 cluster [9]. Like tyrosine YZ , the Cl− was also located inside an
extensive hydrogen-bonding network (fig. 2.7).
15
CHAPTER 2. OXYGEN-EVOLVING COMPLEX
Figure 2.7: Location of Cl− inside a hydrogen network (XRD studies with resolution 1.9 Å) [9]
16
Electron paramagnetic resonance
E
LECTRON
3
paramagnetic resonance (EPR), or at other times designated as electron spin resonance
(ESR), is a technique involving the absorption of electromagnetic energy by the magnetic moments
of molecular systems, arising from one or more unpaired electrons (total spin state S>1/2) [29]. In an
EPR experiment a continuous wave (CW) of electromagnetic energy is applied and the magnetic field
is swept. The majority of commercial instruments provide microwave electromagnetic energy of 9.5
GHz (X-band) and occasionally energies with higher frequencies. They are calibrated to display spectra
in Gauss (G) or Tesla (T, 1T = 10−4 G) and the varying magnetic field is in the range of 1 - 10,000 G
[77]. EPR spectra usually are presented with the first derivative of the absorption, hence, the resonance
magnetic field corresponds to the zero crossing point. Nuclear magnetic moments are 1000 times weaker
than electron magnetic moments and, typically, unobservable in the EPR spectra. Nevertheless, there
is one technique, i.e. electron nuclear double resonance (ENDOR) where the transitions arising from
nuclear magnetic moments are enhanced and detectable. The theory of EPR and ENDOR is briefly
explained below.
When an external magnetic field is applied, every unpaired electron lines up along the direction of
the field and this lifts the degeneracy of the ±1/2 quantized spin states (Zeeman effect). Resonance
occurs when the applied field induces a splitting whose energy is equal to that of an applied microwave
field. Apart from Zeeman effect, hyperfine coupling is splitting further the energy levels of a molecular
system. Each nuclear spin split the EPR signal into 2I + 1 lines, where I is the spin of the nucleus that
is interacting with the unpaired electron (fig. 3.1(a)). The hyperfine coupling produce spectra rich in
information and the most important structural information is obtain by the interpretation of this property
[77].
The relationship between Zeeman energy splitting and magnetic field is
hν = gµB B
(3.1)
where g is the proportionality factor, µB is the Borh magneton constant (9.27401*10−24 JT−1 ) and
B is the strength of the magnetic field. If the electron spin is the only source of magnetism then ge =
2.0023 (this value has been calculated by the Dirac equation and the theory of quantum electrodynamics).
For metals and radicals, the g-value deviates from ge because orbital angular momentum is present (fig.
3.1(b)). The g-value or g-factor is a dimensionless quantity, independent from hyperfine electron-nuclear
17
CHAPTER 3. ELECTRON PARAMAGNETIC RESONANCE
(a) Energy level splitting for a molecular system of an electron (b) The g-value can be smaller or greater that ge .
(ms =±1/2) and a nucleus (mI =±1/2). Allowed transitions have ms =±1
and mI =0.
Figure 3.1: Hyperfine coupling and Zeeman effect. Adapted from [78]
coupling and it helps to characterize species [78]. The strength of g-anisotropy depends from the spinorbit coupling which is small for radicals and greater for transition metals. Hence for radicals we expect
small deviations from ge and significant deviations for transition metals [77]. In addition, molecular
systems with more than one unpaired electron (S > 1/2) may show zero field splitting, i.e. splitting of
the energy levels even in zero field strength.
The general spin Hamiltonian for a system with n electron and n nuclear magnetic moments, like in
the case of Mn cluster, is [29]
Ĥuncoupled =
n
X
~ i Ŝi + Ŝi Ãi Iˆi + Ŝi D̃i Ŝi + Iˆi P̃i Iˆi − γi B
~ i Iˆi ] +
[Bg̃
i
n
X
Jjk Sˆj Sˆk
(3.2)
j,k,j6=k
The terms of the hamiltonian in order are: (1) electron Zeeman term, (2) hyperfine (electron-nuclear)
coupling term, (3) electronic zero-field splitting term, (4) nuclear quadrupole zero-field splitting term,
(5) nuclear Zeeman term and (6) magnetic exchange couplings between the paramagnetic ions. The
exchange couplings between the n ions correlate their spins and the equation 3.2 can be rewritten in a
term of the total coupled spin ST ,
~ 0 ST +
Ĥcoupled = Bg̃
n
X
~ i Iˆi ] + ŜT D̃0 ŜT
[ŜT Ã0i Iˆi + Iˆi P̃i Iˆi − γi B
(3.3)
i
The coupled g-tensor g̃ 0 , the zero-field splitting tensor D̃0 , and the hyperfine tensors Ã0i in eq. 3.3 are
not identical to the tensors of eq. 3.2 and they are called ’effective tensors’. The correlation between
effective Ã0i and intrinsic Ãi takes place through a projection matrix ρ which depends from the total spin,
the intrinsic zero field splitting term and the exchange magnetic couplings, Ã0i = Ãi ρ(ST , Jjk , D̃i ).
18
CHAPTER 3. ELECTRON PARAMAGNETIC RESONANCE
All manganese ions are paramagnetic (have unpaired electrons) with electron spin 3/2, 2 and 5/2
for the ions Mn(IV), Mn(III) and Mn(II) respectively, thus all four Mn of OEC contribute to the EPR
spectra. In addition, 55 Mn has a nuclear spin of I = 5/2, resulting to a multiline splitting because of the
electron-nuclear hyperfine coupling. EPR is a powerful tool to determine the oxidation of Mn4 in every
state Si and it is a complementary method to X-ray absorption spectrometry for OEC characterization
[29]. The first EPR measurements were reported in 1981 when pretreatments to enrich membranes in PS
II were discovered (the signal of PS II was obscured by PS I before with untreated membranes) [79].
EPR was the technique that in the early 80s correlated the Mn4 cluster with the periodic fluctuation of
O2 evolution [80, 81, 82, 83] and since then it has been used to characterize Mn, Ca, TyrZ and TyrD and
to confirm inhibition of the catalytic mechanism when inhibitors are used. Considering the complexity of
the OEC (four high valence Mn ions), traditional EPR studies (with continuous wave and perpendicular
polarization) are not sufficient to characterize OEC in every state. Therefore, advanced EPR techniques
(parallel polarization EPR, pulsed EPR) [84, 50] and associated techniques (electron nuclear double
resonance ENDOR) [30] have also been applied.
3.1
S0
AND
S2 EPR SIGNALS
The spin state of the Mn4 Ca cluster is the result of the coupling of the four Mn ions electron spins. The
S0 and S2 states present as half integer spin states (odd number of electrons, ground spin state S = 1/2)
and the S1 and S3 as integer spin states (even number of electrons, diamagnetic ground spin state S = 0).
Half integer spin states are more well described by EPR because of the paramagnetic ground state S =
1/2, while integer species require parallel-polarization mode. States with spin S > 1/2 show zero field
splitting (separation of the MS energy levels in the absence of a magnetic field) which results to g values
very different from the free electro value of 2.0023 [30].
The signal of S2 state was first observed because it is a half integer state and requires only a single
flash. Dismukes and Siderer reported in 1981 [81] a multiline EPR signal centered at g = 2 (with 1920 hyperfine lines spaced by 80-90 G) following excitation with a single flash and subsequent rapid
freezing to “trap” the state. After a year, the same signal was reported by Hansson and Andreasson
with illumination in frozen PS II [82] and by Brudvig et al. under continuous illumination at 200 K
and subsequent freezing [85]. All these studies correlated the signal with the S2 state and was identified
as an EPR signal arising from antiferromagnetically coupled mixed high valence Mn ions. Different
pretreatments to advance PS II populations to S2 state by using inhibitors or by Ca2+ depletion resulted
in slightly different multiline g = 2 signals indicated different structure possibly because of ligation of
the inhibitors (fig. 3.2(a)).
In 1984 Casey and Kenneth discovered another EPR signal, a broad 320G-wide signal centered at g
= 4.1, that appear under illumination at T = 140K and disappear when it was warming at T = 190K [86]
and they identified it as a S = 5/2 state of a non-heme Fe in a rhombic structure. In 1986 Zimmermann
and Rutherford recognized it as a second signal of the S2 state because it showed the same four-period
oscillation pattern with the S2 state [87]. They concluded that since both signals are functional (they
19
CHAPTER 3. ELECTRON PARAMAGNETIC RESONANCE
(a) EPR spectra of the g = 2 signal of the S2 state obtained by (b) X-band (9.65 GHz), P-band (15.5 GHz), Q-band (34
different pretreatments
GHz) EPR spectra of the g = 4.1 signal
Figure 3.2: EPR signals of the S2 state [30]
exhibit four-period oscillation pattern with maxima on the 1st and the 5th flashes), then minor differences
should occur in the structure and the difference stems from the spin state. They proposed a spin state of
S = 3/2 but in 1992 Haddy et al. established it as a S = 5/2 signal. Haddy et al. performed multifrequence
studies at 4 GHz (S-band), 9 GHz (X-band) and 16 GHz (P-band) demonstrated the anisotropy of the g
= 4.1 signal (fig. 3.2(b)) [88].
EPR experiments often are conducted at X-band or at lower frequences mainly due to the ready availability of the necessary microwave components. However, measurements in increased frequencies can
improve resolution and split the signal into more than one g-factors. These g-factors contain information
about the Mn properties (anisotropy, Jahn-Teller effect). Studies of the g = 2 signal at Q-band frequency
(34 GHz) showed that the signal is generally isotropic [30], with a central g-factor of 1.98 but a W-band
(94 GHz) study revealed three principal g-factors of 1.988, 1.981, and 1.965 [89].
Table 3.1: Proposed valences in every oxidation state Si : EPR cannot discriminate between these different valent S-cycles, whereas ENDOR favors the high-valent S-cycle
Oxidation state
S0
S1
S2
20
High valent S-cycle
(II,III,IV2 ) or (III3 ,IV)
(III2 ,IV2 )
(III,IV3 )
Low valent S-cycle
(II,III3 )
(III4 )
(III3 ,IV)
CHAPTER 3. ELECTRON PARAMAGNETIC RESONANCE
The sample pretreatments developed by Missinger et al. in 1997 to enhance the population of S0 -state
enabled the characterization of S0 state with EPR. Messinger et al. demonstrated that a g = 2 CW-EPR
signal is present in the S0 -state [48]. The S0 state shows a multiline signal with 24–26 hyperfine lines
with spacings of 80–90 G (fig. 3.3). Three groups attempted to extract the information of the hyperfine
couplings of S0 and S2 states and, taking into account the X ray absorption data, they suggested two
S-cycles, high valent or low valent (tab. 3.1) [29].
Figure 3.3: EPR signals of S0 , S1 , S2 and S2 Y•Z states. The narrow signal of tyrosine D (TyrD ) is
removed in the two last spectra. Adapted from [29]
3.2
PARALLEL AND PULSED EPR
In 1992 Dexheimer and Klein applied parallel polarization EPR which is sensitive to integer spin states,
to the S1 state, and observed a broad peak at g = 4.9 [91]. The temperature variation of the signal intensity
between 1.9 and 10 K indicated that the signal originates from an excited state with a spin S = 1 [90].
21
CHAPTER 3. ELECTRON PARAMAGNETIC RESONANCE
Moreover, pulsed EPR spectra with multipulse electro spin echo (ESE) sequences of PS II have been
recorded. Pulsed-EPR is more sufficient method than convetional CW-EPR for multinuclear clusters
because it reduces inhomogeneous broadening [84].
In addition, the EPR spectra of the reduced S1 and S2 oxidation states have been reported with EPR
microwave power studies [92]. The many signals discovered by the conventional/ parallel/ pulsed EPR
studies are not totally interpreted (e.g. the multiline and g = 4.1 signals in the S2 state, the low field
multiline and g = 4.9 signals in the S1 state) but they may be useful in future studies when the knowledge
will be broader [30].
3.3
E LECTRON NUCLEAR DOUBLE RESONANCE
All the hyperfine interaction tensors of the individual Mn ions were not defined by the EPR multiline
signals of the S0 and S2 , and that emerged the use of
55 Mn
electron nuclear double resonance (EN-
DOR) spectroscopy to probe directly the Mn nuclei.[93]. For a tetranuclear Mn cluster with a S = 1/2
electron spin ground state and four I = 5/2 55 Mn nuclei, there are 1296 allowed EPR transitions. However, the multiline signals exhibit only 20 hyperfine interactions, therefore the EPR spectral analysis is
significantly underdetermined [29].
In an ENDOR experiment the nuclear magnetic moments are enhanced and it can measure electronnuclear hyperfine couplings between the unpaired electrons and neighboring nuclei with much higher
precision than EPR spectroscopy alone. It can provide information that conventional NMR misses, due
to the short relaxation times of these nuclei. In an ENDOR experiment electron spin transitions are
saturated by a CW-microwave radiation, while an electromagnetic wave of radio frequency is swept.
ENDOR experiments and comparison with simulated models (fig. 3.4), favored the high valent S-cycle.
For the S0 state, it favored the Mn4 (III,III,III,IV) assignment [93, 94, 95].
Figure 3.4: ENDOR in S0 and S2 state. Adapted from [93]
22
CHAPTER 3. ELECTRON PARAMAGNETIC RESONANCE
3.4
S PLIT Si Y•Z
SIGNALS
The direct observation of the tyrosyl radical TyrZ in every Si state by EPR spectroscopy is rather difficult
because of its rapid reduction by Mn cluster. However, with pretreatment of PS II to advance in every
state and then NIR illumination in very low temperature (5K), the split EPR signals of Si Y•Z states have
been reported [96, 97]. These split signals may provide significant information as they are proposed to
reflect the magnetic interaction between Y•Z and the Mn4 Ca cluster. The split signal of S2 Y•Z is illustrated
on fig. 3.3.
23
4
X-ray spectroscopy
X
- RAY absorption or emission spectroscopy (XAS, XES) is the method of choice when a metal of a
complex cluster (e.g. metalloenzyme) needs to be analyzed, because it is an element - specific an-
alytical technique. In addition, synchrotron sources provide a range of X-ray energies that can selectively
excite most of the metals present in biomolecules. The full X-ray absorption spectra provide valuable
information for the oxidation state and the structure for the cluster. By comparison with model Mn complexes, the charge and structure of Mn4 cluster can be estimated in all the oxidation states without being
interfered with aminoacids or other present metals (Ca, Mg, Fe) [35].
Typically, X-ray spectrometry studies of Mn involve excitation of a K-shell (1S) electron (k-edge
XAS). The study of the absorption spectra with energies beyond the absorption edge energy (when the
electron is extracted from the atom) is called XANES (X-ray Absorption Near-Edge Spectroscopy) (fig.
4.1). This part gives information about the charge and coordination environment of the metal ions.
In higher oxidation states, the nucleus carries higher positive charge which attract more the electrons.
Therefore more energy is required to excite an electron from an orbital and XAS spectrum is shifted to
higher energies. The transition 1s → 3d has ∆l = 2 and is quantum mechanically forbidden, therefore it
has very weak intensity. An intense peak stems from the 1s → 4p transition. The transition to 4p orbital,
which is the lower unoccupied, has ∆l = 1 and is quantum mechanically allowed. Transitions to higher
np and nd unoccupied orbitals occur in higher energies, with the 1s → np transitions to absorb stronger
than the 1s → nd transitions. Thus, with XANES studies we can use model compounds to estimate the
oxidation state of CaMn4 O5 cluster and determine the oxidation changes among the Si states.
The study of the K edge XAS spectrum at energies above binding energy is called EXAFS (Extended
X-ray Absorption Fine-structure Spectroscopy)(fig. 4.1). In this region, part of the incident energy (equal
to the binding energy Eo ) is used to remove the electron from the ion and the rest is converted into kinetic
energy of the electron. The energy of the extracted electron due to its wave nature is scattered by the
nearby atoms, resulting in a modulation pattern in the X-ray absorption spectrum above the binding
energy. The EXAFS oscillations from heavy backscatterers (Mn and Ca) are different and stronger
from the EXAFS oscillation of light backscatterers (O, N, and C). The photoelectron wavevector k of
the kinetic energy is estimated, typically in Å−1 , by subtracting the binding energy from the excitation
energy
k=
2π(2me (E − Eo ))1/2
h
25
(4.1)
CHAPTER 4. X-RAY SPECTROSCOPY
Figure 4.1: XANES and EXAFS analysis from the K-edge X-ray Absorption spectrum of Mn cluster.
Adapted from [35]
and the energy axis of the spectrum is converted to the wavevector k. Then the absorption of the spectrum
is converted to the absorption coefficient µ,
µ=
ln(Io /I)
t
(4.2)
where t is the thickness and µ has units cm−1 . The data are then expressed as a relative EXAFS modulation χ(k),
χ(k) =
µ(k) − µBG (k)
µo (k)
(4.3)
where µo (k) is the photoelectric absorption coefficient of the free atom (normalization to one atom absorption) and the µBG (k) is the pre-edge background absorption (experimental baseline). The theoretical
EXAFS equation for χ(k) is [98, 99]
ns
X
Ni σ2 k2
i
χ(k) =
|fi (k, π)| sin(2kRi + αi )
2e
kR
i
i=1
(4.4)
where ns is the number of scattering shells, Ni is number of atoms in the ith shell, Ri is the distance,
fi (k, π) the backscattering amplitude, σi2 is the variation of the distance (disorder parameter) and ai (k)
26
CHAPTER 4. X-RAY SPECTROSCOPY
the scattering phase shift. The distance between Mn and nearby atoms within a distance of 5 Å can
be estimated by Fourier transform (FT) of the wave vector k by applying suitable models and find the
best curve fitting in the plot χ(k) = f(k). The amplitude of the plot diminishes with increasing energy,
therefore it needs to be weighted to a power of k, often to the 3rd power, i.e. χ(k)·k3 = f(k), to compensate
for the χ(k) dependence from 1/k3 . If there is a big increase in noise with k3 -weight, then lower power
numbers are preferred. As a result, with Mn EXAFS studies on the CaMn4 O5 cluster, we can estimate
the type and number of the Mn neighbor atoms and the distances from Mn atoms.
4.1
X- RAY ABSORPTION NEAR - EDGE STRUCTURE
Based on Mn k-edge XANES (X-ray Absorption Near-Edge Structure) spectroscopy, there is a consensus
that Mn oxidation occurs during transitions S0 →S1 and S1 →S2 whereas there is an on-going debate
about Mn oxidation during transition S2 →S3 . Traditionally, the transition 1s→4p, corresponding to the
inflection point of the main edge of the spectra, is used as an indicator of the oxidation states and the
inflection point energy (IPE) is given by the zero-crossing of the 2nd derivative [51]. In 1981 Klein et
al. presented XANES studies in active and inactive (tris treated) membranes and they discovered Mn
oxidation state higher than +2 in active membranes [100]. In 1992 Ono et al. carried out Mn XANES
experiments in different oxidation states induced by short laser flashes. Similar edge energy shifts were
observed between flashes and they concluded that Mn-centered oxidation occurs during all the transitions
S0 →S1 , S1 →S2 , S2 →S3 [101]. However, they did not mention measurements of the real S-state
distributions after every flash and therefore these spectra cannot be attributed to pure states.
In 1996 Roelofs et al. [49] reported Mn k-edge XANES data from PSII samples of the S0 through
S3 “pure” states which they obtained by short flashes and the relative state distributions after every flash.
To estimate the relative state distribution they followed this procedure: First they concentrated all PS
II population in the S1 state (S1 synchronization) by two preflashes and 60 min dark incubation. Then,
they monitored the EPR signal of the S2 state after every flash and they calculated the relative S-state
populations in samples treated with 0, 1, 2, 3, 4, or 5 flashes from fitting the flash-induced EPR multiline
signal oscillation pattern to the Kok’s model. The edge positions were defined as the zero-crossing of
the second derivatives and they were estimated 6550.1, 6551.7, 6553.5, and 6553.8 eV for the oxidation
states So , S1 , S2 , and S3 , respectively. The authors concluded that Mn-centered oxidation changes take
place during the transitions S0 →S1 (1.6 eV) and S1 →S2 (1.8 eV). The small edge position shift (0.3 eV)
for S2 → S3 transition was an indication that no Mn oxidation take place and they proposed oxidation
states S0 (II, III, IV, IV) or (III, III, III, IV), S1 (III, III, IV, IV), S2 (III, IV, IV, IV), S3 (III, IV, IV, IV).
In 1998 Dau et al. presented also XANES data with similar procedure with Roelofs et al. Again the
Mn K-edge spectrum of each pure S-state of spinach PS II stemmed from the deconvolution of the spectra obtained from consecutive flash illumination and EPR measurements. Conversely with Roelofs et al.,
they found that transition occurs during S2 →S3 transition [102]. Namely, the shift of the edge position
was estimated 0.8-1.5, 0.5-0.9, and 0.6-1.3 eV for the transitions S0 →S1 , S1 →S2 , and S2 →S3 respectively. In 2001 Missinger et al. repeated the experiment by applying very short flashes to prevent double
27
CHAPTER 4. X-RAY SPECTROSCOPY
hits for more precise determination of the state distribution. The results are similar to Roelofs et al.
(small energy shift during the S2 →S3 transition) and they are illustrated in figure 4.2 [32]. In 2005 Dau
et al. did XANES experiments [33] following with three different approaches: (1) illumination-freeze
approach (XAS at 20 K), (2) flash-and-rapid-scan approach (RT), and (3) a novel time scan/samplingXAS method (RT). The methods were in very good agreement and the shifts in energy were 0.6, 0.8, 0.7
eV for the three transitions.
Figure 4.2: XANES spectra of every Si state expressed as fluorescence intensity divided by the incident X-ray intensity, F/Io , against the energy of the incident X-rays and their 2nd derivative
spectra. Missinger et al. (2001), adapted from [32]
However, apart from the ongoing contradicting results of the aforementioned studies, it is important
to mention that Mn k edge energies are not affected only from the oxidation state but also from the
ligands, therefore it is erroneous to conclude whether oxidation changes occur or not only with XANES
energy shifts [33].
4.2
E XTENDED X- RAY ABSORPTION FINE STRUCTURE
Extended X-ray absorption fine structure spectroscopy (EXAFS) was the first technique that revealed the
µ-oxo bridged nature of the multinuclear Mn cluster of OEC. EXAFS studies of PS II samples shows
three peaks, designated as peaks I, II and III (fig. 4.3(a)). The Fourier peak at 2.7 Å is characteristic of
di-µ- oxo bridged models, therefore it was proposed that the Mn atoms form µ-oxo bridges before it was
reported with other techniques. This conclusion was reinforced by a presence of a short Mn-O distance
at about 1.8 Å, also characteristic of bridging Mn-oxo. A peak at 3.3 Åthat corresponds to one Mn-Mn
28
CHAPTER 4. X-RAY SPECTROSCOPY
imply a mono-µ-oxo-bridged Mn complexes which later will be verified with XRD.
In 1981 Klein et al. presented the first X-ray study on whole chloroplasts washed free of unbound
or loosely bound Mn2+ [98]. Before that date, only indirect methods were introduced for monitoring
Mn activity such as NMR water proton relaxation experiments and Mn release experiments. In this study
EXAFS spectra of active PS II, inactive PS II (Mn-depleted) and synthetic models were compared, over a
wavevector in the range of 4 - 11 Å. Fourier transformation revealed good curve fit and three peaks. The
first peak with average distance 1.81 Å was associated with 2-3 Mn-C/N/O bonds, the second at distance
2.15 Å associated with 2-4 Mn-C/N/O and a peak at 2.72 Å related to a Mn-Mn/Fe bond. From the small
Mn-Mn distances of OEC and its similarity with complexes Mn2 (III,IV)bpy and Mn2 (IV,IV)phen, it was
concluded that Mn atoms in OEC are bridged with di-µ-oxo bridges. In 1986 with PS II sub-chloroplast
preparations [99] they revised the results to distances 1.76 Å, 1.98 Å for Mn-O/N distances and 2.69
Å for Mn-Mn distance. The experiment was performed at temperature T = 170K to assure minimal
radiation damage and that there is no advancement from S1 state to hgher oxidation states. These early
studies established that at least one dinuclear Mn cluster exist, possibly multinuclear, connected with
µ-oxo bridges.
(a) Exafs studies revealed the µ-oxo bridges of the Mn cluster. (b) Extended-range EXAFS studies.
Adapted from [35]
[107]
Adapted from
Figure 4.3: Traditional and extended EXAFS
Penner-Hahn et al. (1990) performed EXAFS in isolated, highly purified and highly concentrated PS
II samples and demonstrated at least two Mn-Mn distances at 2.7 Å and at least one distance Mn-Mn/Ca
at 3.3 Å [104]. After the early 90s, with the introduce of FEFF software to simulate EXAFS spectra of
different models to find the best fit, the interpretation of EXAFS spectra became more easy and accurate.
In 2000, Liang et al. assigned the two 2.8 Å Mn-Mn distances to di-µ-oxo Mn bridges and the 3.3 Å MnMn distance to a mono-µ-oxo Mn bridge [105]. In addition, a comparison between the EXAFS spectra
of S1 to the higher oxidation states, disclosed negligible structural changes during S1 →S2 transition
and significant structural changes during S2 →S3 transition. Namely, the two short Mn-Mn distances
increased from 2.72 Å in the S2 state to 2.82 and 2.95 Å to the S3 state.
The previous EXAFS studies were conducted in the wavevector range 4.1 Å - 11.8 Å because it was
limited by the presence of 2- 3 Fe (Fe edge at 7120 eV), which are obligatory for the activity of PS II.
29
CHAPTER 4. X-RAY SPECTROSCOPY
Traditional EXAFS spectra of PS II are collected as an excitation spectrum by electronically windowing
the Kα fluorescence of Mn. In 2005, a high-resolution EXAFS study, involving a crystal monochromator
was able to selectively separate the Mn and the Fe fluorescence, allowing higher excitation energies with
an extended wavevector range to 15.5 Å [106]. This technique revealed three short distances in the range
of 2.7-2.8 Å. Furthermore, in 2008, another extended range EXAFS study clearly showed that the III
peak (3.3-3.4 Å) was split into two peaks, 3.2 Å and 3.7 Å (fig. 4.3(b)) [107].
4.3
X- RAY EMISSION SPECTROSCOPY
In contrast to XANES that measures the absorption of 1s electron to a higher unoccupied orbital (1s→4p),
kβ X-ray emission spectrometry detects the X-ray emission from the relaxation of an 3p electron in the
1s hole that has been created after X-ray absorption. In metals, the 3p orbitals are much less extended
than the 4p orbitals and therefore the emission energy 3p→1s depends mainly to the oxidation state and
not to the ligands. Kβ1 and kβ3 emission is the emission from 3p1/2 and 3p3/2 states respectively. Two
final spin states exist with either a constructive Kβ1,3 or destructive kβ ‘ spin (fig. 4.4(a)).
(a) kβ XES studies on Mn oxides
(b) Difference in XES spectra between the Sstates. The difference between S2 and S3 spectra is negligible
Figure 4.4: X-ray emission spectroscopy to Mn oxides and PS II. Adapted from [108]
The shape and energy of the kβ1,3 emission is mainly influenced by the number of unpaired 3d electrons and less by to the symmetry and ligand-bonding. In 2001 Visser et al. measured and compared
the XANES and kβ XES spectra of two structurally homologous dinuclear Mn compounds in different
oxidation states, i.e. Mn2 (III,III), Mn2 (III,IV), Mn2 (IV,IV), and concluded that kβ X-ray emission energies are more sufficient to examine only oxidation changes. Namely the XANES spectra showed energy
shifts of 0.7-2.2 eV for oxidation changes and 0.5-2.0 eV for ligand-environment changes while kβ XES
spectra showed an energy shift of ~0.21 eV for oxidation state and only ~0.04 eV for ligand-environment
30
CHAPTER 4. X-RAY SPECTROSCOPY
changes [109]. Thus, this technique could be used for determination of oxidation changes in OEC during
S-cycle.
High-resolution Mn Kβ X-ray emission spectroscopy was performed on samples treated by 0, 1, 2,
or 3 high-power laser flashes. The energy of the Mn Kβ emission was that of the transmission 3p→1s.
The shape and energy of the kβ1,3 emission was selected to reflect the oxidation state(s). Between states
S2 → S3 no change was observed (fig. 4.4(b)) [108].
4.4
T IME - RESOLVED X- RAY ABSORPTION S PECTROSCOPY
Figure 4.5: Extended Kok’s cycle with time-resolved XAS. Adapted from [110]
Haumann et al (2005) [110] employed time-resolved X-ray Absorption Spectroscopy to monitor the
redox processes. They detected the changes in the Mn X-ray fluorescence after laser-flash illumination
with a time resolution of 10 µs. They ascertained, at an excitation energy of 6552 eV, an absorption
decrease (Mn oxidation) between 1st , 2nd , 4th flashes which came from the transitions S1 → S2 , S2 →
S3 and S0 → S1 , respectively with halftimes of 70 µ and 190 µ and ≤ 30 µ respectively. Conversely, an
absorption increase (Mn reduction) was recorded between the transition S4 → S0 with halftime 1.1 ms
preceded by a lag phase of about 250 µ which was attributed to deprotonation between S3 → S4 . The
deprotonation must be followed by electron transfer to Y•Z , thus implying an S4 ’ state. All these novel
findings are summarized on the extended Kok’s cycle fig. 4.5.
31
5
X-ray diffraction
In the previous decade, the X-ray diffraction (XRD) measurements of PS II resulted to rough data as the
resolution used to be very low (8 Å). The first high-resolution structure of PS II was reported in 2001
when the PS II crystal structure of the cyanobacterium Synechococcus elongatus was analyzed with
resolution 3.8 Å by Zouni et al. [111]. In the lumenal site they defined at least 14 subunits, including
the reaction center, the subunits D1 and D2, the chlorophyll-containing inner-antenna subunits CP43
and CP47, the cytochrome b-559, the membrane-extrinsic cytochrome c-550 (PsbV) and the manganesestabilizing 33K protein (PsbO). This structural analysis revealed also unprecedented information about
the localization of OEC in PS II and the Mn positions (fig. 5.1).
Figure 5.1: Structures defined in 2001 with resolution 3.8 Å a) Spatial organization of PSII b) Suggested
model for Mn positions. Adapted from [111]
The long axis had orientation 23o against the membrane plane and from the electron density, the
inter-atomic distances were estimated about 3 Å. The dimensions of the Mn Complex were estimated 6.8
Å x 4.9 Å x 3.3 Å. A possible structure of the Mn4 complex presented with three Mn atoms in the corners
33
CHAPTER 5. X-RAY DIFFRACTION
of a isosceles triangle and one in the center. However, the resolution was insufficient of estimating the
oxygen bridges between Mn-Mn bonds, the protein ligands associated with Mn and the position of the
Ca2+ , thus the model proposed in this study in figure 5.1(b) was tentative. In 2003 Kamiya and Shen
[112] provided an analysis with resolution 3.7 Å and they researched more intensively the interactions
between OEC and protein subunits. The density of the Mn cluster was similar in shape to that proposed
by Zouni et al. In addition they suggested that the Mn cluster is coordinated by the D1 protein (consistent
with mutational studies) and they proposed specific aminoacid residues.
Figure 5.2: OEC structure proposed in 2004, defined at 3.5 Å resolution a) Cuboidal structure of the
Mn4 CaO5 with one Mn atom outside of the cube b) Proposed mechanism for water oxidation, c) Aminoacids associated with the complex. Adapted from [113]
Ferriera, Iwata and Barber published two articles [113, 114] in 2004 with resolution 3.5 Å, clarifying
further the OEC structure as the oxo-bridges appear. Remarkably, it was reported a different shape,
in which 3Mn and the Ca atom form a cuboidal structure, with the 4th Mn situated outside the cube
(fig. 5.2(a)). Binding aminoacids were reported (fig. 5.2(c)) and a mechanism for water oxidation was
provided (fig. 5.2(b)).
Two subsequent studies discussed the results obtained with XRD at 3.2 Å resolution in 2004 [11] and
34
CHAPTER 5. X-RAY DIFFRACTION
Figure 5.3: Structural analysis with 3.0 Å resolution (2005). Adapted from [115]
3.0 Å in 2005 [115]. In the latter study, the structure of the Mn4 Ca cluster suggested differs considerably
from the previous cubane-like model because the Mn-Mn distances in the pyramid formed by three Mn
and Ca2+ are not equal. Moreover, although the structure is more clear, a controversy arise from the fact
that the distances calculated are not in agreement with EXAFS studies. Namely, two Mn-Mn distances
were 2.7 Å and two were 3.3 Å fig. 5.3.
5.1
X- RAY RADIATION AND CLUSTER DAMAGE
There are discrepancies among the aforementioned models of the structure of the Mn4 Ca complex that
stem from X-ray crystallography studies with 3.0 Å resolution (two long and two short Mn-Mn distances)
and the ones that stem from EXAFS studies (three short and one long Mn-Mn distance). Moreover,
polarized EXAFS experiments precluded both the models proposed with 3.5 Å resolution and 3.0 Å
resolution [35]. This disagreement is predominantly a function of X-ray-induced damage to the catalytic
metal site during XRD measurements [116]. In 2005, a XANES study on intact PS II and on X-ray
exposed PS II, revealed significant Mn reduction to Mn(II) during X-ray radiation (fig. 5.4(a)). It was
shown that at a typically X-ray dose of an XRD experiment (3.5*1010 photons/µm2 ), more than 80% of
the Mn was reduced (fig. 5.4(b)) [116, 117].
Recently (2011), the atomic crystal structure of PS II from Thermosynechococcus vulcanus was published at 1.9 Å resolution and small X-ray dose (0.28*1010 photons/µm2 ) [9]. This range of resolution
is sufficient for a reliable information over the water splitting center. The results are more reliable not
only because of the better resolution but also because the radiation energy was minimized with a slideoscillation technique. Between data collection the crystal was rotating 0.2o relative to the last position.
After collection of 100 images (20o ) the crystal was shifted by 30 mm to an adjacent point along the
oscillation axis, therefore nine different points of the crystal were used for an 180o rotation and 900
images were collected. The photon flux of the beamline used was 0.7*1011 photons/s, the X-ray beam
had a size of 50x50 µm2 , and the exposure time for each diffraction image was 1s. These conditions
35
CHAPTER 5. X-RAY DIFFRACTION
resulted to a total radiation of 2.5*1010 photons/µm2 for the 900 images and considering that the total
radiation was divided in nine spots, then each spot received a slightly lower (because of rotation) total
dose of 0.28*1010 photons/µm2 . This radiation dose is the lower that it was ever reported for XRD
measurements of OEC and it causes small damage.
(a) X-ray radiation leads to Mn reduction
(b) Cluster damage as a function of X-ray dose
Figure 5.4: Damage of the Mn cluster by X-ray radiation. Adapted from [117]
The distances among the Mn atoms are illustrated on figure 5.5 and they are significantly different
compared to previous XRD studies. The Mn-Ca distances were 3.5, 3.3, 3.4, 3.8 Å for Mn1 , Mn2 ,
Mn3 , Mn4 respectively and the interatomic Mn distances were 2.8, 2.9, 3.0 and 3.3 Å. The results bear
similarity with EXAFS data. Four water molecules were identified to bound to the Mn4 CaO5 cluster,
of which W1 and W2 are associated with Mn4 with distances of 2.1 and 2.2 Å, and W3 and W4 are
coordinated to the calcium with a distance of 2.4 Å (fig. 5.5(b)). Some of them may be oxidized to form
dioxygen. More than 1,300 water molecules were reported in each photosystem II monomer that may
serve as channels for protons, water or oxygen molecules. Moreover, every two adjacent manganeses
are linked by di-µ-oxo bridges and the calcium is linked to all four manganeses by oxo bridges. All of
the five carboxylate residues served as bidentate ligands and combined with the oxo bridges and waters,
each of the four manganeses has six ligands whereas the calcium has seven ligands.
36
CHAPTER 5. X-RAY DIFFRACTION
Figure 5.5: Structural analysis with 1.9 Å resolution (2011). Adapted from [9]
37
6
Manganese based catalysts
C
HEMICAL
and electrochemical water-splitting need high activation energy, while in nature, PS II
is able to catalyze water-splitting with moderate activation energy (solar energy). This unique
property has motivated many scientists to search for synthetic manganese systems which mimic this
natural efficient catalytic activity in order to (1) understand the catalytic activity of PS II and (2) to
produce solar biofuels. Hydrogen (H2 ) from water is broadly recognized as the ideal source of abundant,
renewable, and clean fuel. Typically TiO2 is used as a photocatalyst to oxidize water, producing O2 gas,
protons, and electrons. However, it has a large band gap (3-3.2 eV), therefore is active only in the UV
region of the solar spectrum which is a limitation of many photocatalysts, and also oxidize water only in
the vapor phase [118]. In recent years a lot of research has been conducted over techniques to modify
semiconductor photoanode surfaces of photoelectrochemical cells (e.g with light-harvesting molecules
or water oxidizing active metal-oxides) [119]. Part of this research was the discovery of cheap, abundant
Mn complexes with water-splitting activity that can operate at the low PS II potentials.
The idea of artificial photosynthesis is attractive and synthetic light-harvesting antennas, molecular light-induced charge separation systems and
synthetic manganese-based catalysts are the new
photosynthesis-inspired fields of interest [120]. In
order to design an artificial PS II model, the Special Pair of chlorophylls can be replaced with more
stable and well understood photosensitizers, like
ruthenium(II) polypyridine complexes. This replacement is justified as the redox potential of a
Ru(II)(bpy)3 /Ru(III)(bpy)3 pair is typically +1.26
V vs NHE, very close to that of a P680/P680+•
pair (1.18 V vs NHE). However, it is difficult to
establish a PS II model which can oxidize water
by accepting and storing four electrons with one
Figure 6.1: First attempt to mimic the electron
transfer in PS II [121]
electron excitation at the photosensitizer.
39
CHAPTER 6. MANGANESE BASED CATALYSTS
The first attempt to mimic PS II was in 1997 by Sun et al. [121] who reported experiments with
Ru(II) tris(bipyridyl) complex, covalently linked to a Mn(II) cluster, illustrated on fig. 6.1. The Ru(II)
played the role of the P680 reaction center: after illumination it was oxidized when an electron acceptor
was present (methylviologen MV2+ ) and subsequently, Mn(II) acted as an electron donor. Furthermore,
they concluded that the distance between the photosensitizer and the manganese is crucial: short distance
may inhibit the Ru(II) oxidation whereas long distance may not result to subsequent Mn(II) oxidation. In
the same year, Magnuson et al. used as model compound the same Ru(II) complex attached to a tyrosyl
residue and observed electron transfer from the tyrosyl moiety to the photogenerated Ru(III), when an
external sacrificial electron acceptor (Co(NH3 )5 Cl2+ ) was present, in the same mode that tyrosine TyrZ
and P680 react in PS II [122].
Figure 6.2: Structure of a manganese dimer linked to a Ru (II) compex which is photo-oxidized with
[Co(NH3 )5 Cl]2+ as the irreversible electron acceptor. EPR electron transfer studies revealed
oxidation state Mn(II,II) which evolve to Mn(III,IV). Adapted from [61]
Shortly after, the first “triad” was introduced, evolving a Ru(II) complex, a tyrosyl moiety and a
dinuclear Mn(III,III) complex. The Mn complex oxidized to Mn(III,IV) and the tyrosine intermediate
enhanced the electron transfer [123]. Moreover, in an attempt to mimic D1- His190, a dipicolylaminel
arm was linked to the tyrosyl moiety and this resulted to a increase in the electron transfer rate constant
by two orders of magnitude [124]. Shortly after, Burdinski et al. introduced various mono-, di-, and trinuclear manganese complexes with phenolate ligands covalently linked to tris(bipyridine)ruthenium(II) as
potential models [125]. All these early studies did not manage to establish a model to oxidize water but
a lot of information was gained on the mechanism of PS II.
Huang et al. in 2002 presented a dinuclear Ru-Mn(II,II) model which had two µ-OAc bridges and one
µ-oxo bridge connecting the Mn ions, namely the two Mn ions were bridged via the two bidentate acetate
ligands and the 4-methylphenolate group of the ligand (fig. 6.2). It was able to be oxidized in three
subsequent steps to give the Ru–Mn(III,IV) complex, by repeated laser flashing in the presence of an
40
CHAPTER 6. MANGANESE BASED CATALYSTS
irreversible electron acceptor (Co(III)) and the electron transfer was confirmed with EPR [126]. Although
this is close to the four electrons needed for water oxidation, no water oxidation was detected in the
experiments. Electrochemical and kinetic data indicated that ligand exchange occurred in the Mn(III,III)
state, i.e. substitution of the acetate bridges by water molecules to form di-µ-oxo bridges, which possible
added more negative charges to the complex and allowed the thermodynamically unfavorable oxidation
to the Mn(III,IV) state. The same year Sun, Hammarström, Styring et al., because of the later study
and the fact that a proposed OEC model involved a Mn(V)=O bond, synthesized a high-valent oxo-Mn
complex by replacing the two pyridines of the previous complex with phenolates [127]. This resulted in
an O/N ligand ratio closer to that in PSII and more stable higher oxidation states. All this earlier studies
are reviewed in 2003 [128] and in 2006 [61].
In another approach multinuclear Mn models are synthesized and their ability to O2 evolving is
examined with the use of strong oxidants. The first attempt was in 1999, when Limburg and Brudvig reported that O2 was formed by the reaction of [Mn(dpa)2 ]− (dpa = dipicolinate) with potassium
peroxymonosulfate [129, 130]. This reaction produced a dinuclear Mn(III/IV) dimer as an intermediate and they concluded di-µ-oxo Mn(III/IV) dinuclear complexes that provide open coordination sphere
for water are sufficient for O2 evolution. Later they examined catalytic activity of [(terpy)(H2 O)Mn(µO)2 Mn(terpy)(H2 O)]3+ (terpy = 2,2’:6’,2’-terpyridine) (fig. 6.3(a)) and they demonstrated that O2 evolution occurs when NaClO is used as oxidant with a turnover of 4. The catalytic turnover was small
18
because the sample decomposed into MnO−
4 . They labeled O from water with O isotope to prove that
O comes from the water and not the oxidant. This conclusion is under dispute since they did not take
into account the isotope 18 O exchange rate inside the solution between water and oxidant [130].
(a) Molecular structure. Adapted from [129] (b) Compound 1 exhibits O2 -evolving catalytic activity with Ce(IV) as an oxidant, only when it is adsorbed to mineral clays (mica). Adapted from [131]
Figure 6.3: Structure and activity of [(terpy)(H2 O)Mn(µ-O)2 Mn(terpy)(H2 O)]3+ (compound 1 in these
images)
However, although the latter Mn complex in a solution exhibits water-splitting activity only with
O-carrier oxidant which could indicate that O2 stems from the oxidant, when it is attached in mineral
41
CHAPTER 6. MANGANESE BASED CATALYSTS
clays like mica or kaolin it can oxidize water with cerium(IV) as an oxidant with a catalytic turnover of
15-17 (fig. 6.3(b)) [131]. That was explain with suppression of the decomposition into MnO−
4 when the
compound is adsorb on clay. Many others di-, tri-, and tetra-nuclear complexes were synthesized and
−
tested for water oxidation activity with different oxidants (e.g., Ce(IV), HSO−
5 , Ruphot , ClO ). Most
of the Mn complexes were proved inactive [132, 133] or with a very small catalytic activity (10-20
turnovers) [133]. Some of this synthetic models had cubane-like or butterfly structure to bare similarity
with PS II proposed models.
Figure 6.4: Photoelectrochemical cell. Adapted from [118]
Recently (2010), Swiegers, Spiccia et al. presented a photoelectrochemical cell consisting of a
Ruthenium photosensitizer, Ru(II)(bipy)2 -(bipy(COO)2 )) and a manganese complex (a “cubium”, a tetranuclear Mn-oxo cluster [Mn4 O4 L6 ]+ , with L=(MeOPh)2 PO2 -). It was able to exhibit photosynthetic-like
activity for > 1000 catalytic turnovers when it was illuminated with visible light [118]. The photoelectric
cell is illustrated on fig. 6.4. The manganese complex was suspended within a Nafion membrane, coated
on the electrode. The photosensitizer Ru(II) was supported on a TiO2 layer. This Mn complex did not
exhibit catalytic activity when it was not suspended in Nafion.
This remarkable enhance in catalytic activity when the Mn complex is loaded inside the Nafion
was examined by Spiccia et al. [134]. They employed X-ray absorption spectroscopy and transmission
electron microscopy studies that revealed that this cluster dissociates into Mn(II) compounds when it is
suspended in Nafion, which are then reoxidized forming nanoparticles of a disordered Mn(III/IV)-oxide.
Therefore, the initial cubane [Mn4 O4 L6 ]+ serves only as a precursor. On figure 6.5 the Mn k-edge
XANES and pre-edge XAS data are illustrated of the precursor Mn complex, of the Mn complex after
it is suspended into Nafion and of the reoxidized Mn(III,IV) complex. A significant energy reduction
occurs when the Mn complex penetrate the Nafion (green→blue), but also the XAS spectrum of the
reoxidized product differs from the initial (green and red). This redox cycling which is responsible for
the catalytic activity bare similarity with natural biogeochemistry, which also involves cycles of oxidation
into solid Mn(III/IV) oxides followed by photoreduction to Mn(II).
Amorphous solid Mn-oxides (α-Mn2 O3 ) and Mn/Ca-oxides (CaMn2 O4 ·H2 O) were also reported in
2010 to evolve O2 from water [135]. In contrast to multinuclear Mn complexes that they were not able
to oxidize water with the one-electron oxidants Ce(IV) or Ru(II), calcium-manganese oxides, created by
42
CHAPTER 6. MANGANESE BASED CATALYSTS
Figure 6.5: Mn k-edge (a) XANES and (b) pre-edge XAS data. Green line: [Mn4 O4 L6 ]+ , blue line:
the product of [Mn4 O4 L6 ]+ when it is sudpended in Nafion, red line:the reoxidized product.
Adapted from [134]
cheap and abundant materials (commercial Mn2 O3 ), were able to oxidize water at a high catalytic rate
(fig. 6.6(a)). Kurz et al. synthesized these solid compounds as they were originally suspected that the
very slow water-splitting catalysis of commercial Mn2 O3 might have been due to the small surface area
(1 mm2 g−1 ). The synthetic procedure was rather simple by oxidizing Mn(II) ions, with or without Ca2+
in aqueous solution and then heating the materials to 600o C for α-Mn2 O3 or 400o C for CaMn2 O4 ·H2 O
to dehydrate.
The structure of these amorphous solid compounds that were able to evolve O2 remained elusive until
recently (2011) that they were investigated with Mn and Ca K-edge extended-range X-ray absorption
spectroscopy (XAS) [136]. The XAS results revealed similarities in the interatomic distances of the
synthetic CaMn2 O4 ·H2 O and the Mn cluster of the PS II (fig. 6.6(b)). The oxidation state of manganese
in the active oxides was found to be 80% Mn(IV), and 20% Mn(III) and the nearby Mn ions are connected
by di-µ-oxo bridges. Two different Ca-containing motifs were identified, one has formed Mn3 CaO4
cubes, and the other one has connecting the oxide-layer fragments. The authors concluded that these
manganese–calcium oxides are the closest analogs to PS II.
The similarity in the photosynthetic activity of amorphous manganese-calcium oxides with OEC may
be an evidence of a possible evolutionary origin. It has been proposed that calcium and manganese ions
that precipitated together in the archean oceans could have formed an origin for the OEC in cyanobacteria. The aqueous Mn(II) oxidation is a thermodynamically favorable but kinetically slow reaction, which
43
CHAPTER 6. MANGANESE BASED CATALYSTS
(a) Catalytic rate as a function of time. Adapted from [137]
(b) Structure of Mn2 O3 and CaMn2 O4
determined from EXAFS studies. Green:
calcium, purple: manganese, red: oxygen. Adapted from [136]
Figure 6.6: Structure and O2 -evolving catalytic activity of amorphous solid Mn2 O3 and CaMn2 O4
can be catalyzed by a class of microorganisms referred to as manganese oxidizers. Attachment of the
oxides to protein subunits along with modification from environmental factors may have lead to OEC in
photosystem II [137].
44
Appendix: Abbreviations
ENDOR
Electron-nuclear double resonance
EPR
Electron paramagnetic resonance
EXAFS
Extended X-ray absorption fine structure
OEC
Oxygen-evolving complex
PS I/II
Photosystem I/II
WOC
Water-oxidation complex
XANES
X-ray absorption near edge structure
XAS
X-ray absorption spectroscopy
XES
X-ray emission spectroscopy
XRD
X-ray diffraction
45
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