[Frontiers in Bioscience 16, 3072-3132, June 1, 2011]
Light-induced water oxidation in photosystem II
Frank Muh1,2, Athina Zouni2
1
TABLE OF CONTENTS
1. ABSTRACT
The photosystem II core complex (PSIIcc) is the key enzyme of oxygenic photosynthesis, as it catalyzes the light-induced oxidation of water to form dioxgyen and protons. It is located in the thylakoid membrane of cyanobacteria, algae, and plants and consists of 20 protein subunits binding about 100 cofactors. In this review, we discuss what is presently known about the "donor side" of PSIIcc, covering the photosynthetic reaction center and the water oxidase part. The focus is on the catalytic Mn4Ca cluster and its protein environment. An attempt is made to connect recent crystallographic data (up to 2.9 Å resolution) with the wealth of information about Nature�s water oxidation device from spectroscopic, biochemical and theoretical work.
2. INTRODUCTION
In times of global warming and forseeable shortage of fossil fuels, the need for an efficient usage of solar energy becomes more and more evident (1). Learning from nature in this respect means to understand the molecular mechanisms of photosynthesis (2). During the last decade, the spatial structures of the relevant photosynthetic membrane proteins have been elucidated (3-17). Yet, unraveling the many functional implications of the structural information is both a fascinating and challenging enterprise. This is particularly true for the site of water cleavage in oxygenic photosynthesis, photosystem II, where many molecular details could be reliably determined only very recently (14, 15, 18).
Light-induced water oxidation is catalyzed by the photosystem II core complex (PSIIcc), a multi-subunit water:plastoquinone oxidoreductase spanning the thylakoid membrane of cyanobacteria, algae and higher plants (19-21). The light energy is harvested by specialized chlorophyll (Chl) cofactors and transferred to the reaction center (RC), where a charge separation process creates a strong oxidant that is able to extract electrons (under release of protons) from water. These electrons serve to reduce plastoquinone (PQ) to plastoquinol (PQH2) in the QB-site. PQH2 diffuses into the thylakoid membrane and is replaced by fresh PQ (Figure 1A). A byproduct of the water oxidation reactions is molecular oxygen (O2), which became essential for life on earth. The actual catalytic site of water oxidation is a metal center containing four manganese and one calcium ion (18, 22-26), the water oxidizing complex (WOC). Making this catalyst work requires the whole machinery of PSIIcc including mechanisms for efficient excitation energy transfer, charge separation, proton-coupled electron transfer and the protection against unwanted side reactions.
Cyanobacterial PSIIcc occurs in both, a dimeric and a monomeric form, which both could be crystallized (10-15, 27, 28) and the X-ray structures refined to 2.9 Å (15) and 3.6 Å resolution (28), respectively. As reviewed recently (18), each PSIIcc-monomer consists of 20 different polypeptide chains that could be identified in the crystal structures (Figures 1B, C). The central part of the core complex is build up by the two large membrane-intrinsic, symmetry-related subunits PsbA (D1) and PsbD (D2), which bind the RC cofactors and the WOC. They are flanked by the other two large membrane-intrinsic subunits PsbB (CP47) and PsbC (CP43) containing light-harvesting pigments (for reviews, see (29, 30)) and contributing significantly to the large membrane-extrinsic part of PSIIcc protruding into the lumen. The remaining membrane-intrinsic subunits of PSIIcc are small and have only one or two (PsbZ) transmembrane helices (TMH). Their function still needs clarification (18, 30-32). The lumenal extension of PSIIcc further consists of three extrinsic subunits PsbO, PsbU and PsbV visible in the crystal structure (Figures 1B, C). All three extrinsic subunits are in contact with intrinsic polypeptides and influence water oxidation with PsbO being indispensable. There is a strong variation between organism types with respect to the nature of the other two (or more) extrinsic subunits (33-35). Altogether, the peptides of PSIIcc bind 96 cofactors including 35 Chl a, two pheophytin (Pheo) a, three PQ (QA, QB, QC), a non-heme iron, bicarbonate, two heme groups (cyt b559 shown in Figure 2 as well as cyt c550 not shown and absent in green algae and plants), 12 carotenoids (b-carotene), 25 integral lipids, seven detergent molecules (n-dodecyl b-D-maltoside) probably occupying lipid binding niches, one calcium (Ca2+), four manganese and (at least) one chloride ion of the WOC as well as two additional Ca2+ ions (Ca2+-PsbK and Ca2+-PsbO, see Figures 1B, C). This abundance of non-proteinogenous molecules is suggestive of the diversity of functions that PSIIcc has to fulfil. The present review describes aspects of our current knowledge of PSIIcc taking the crystal structure at 2.9 Å resolution as a starting point (15) and focussing on the water oxidase part and its structure elucidation with different methods.
3. EXCITATION ENERGY TRANSFER, CHARGE SEPARATION, AND QUINONE REDUCTION
3.1. Excitation energy transfer and charge separation
Our current knowledge of the core antenna system of PSIIcc as well as the charge separation (CS) in the RC have been reviewed recently (21, 36-38), so that we can limit the following description to a short summary. To enhance the aborption cross section of the photochemical processes, the RC is coupled to a large number of pigments that serve to collect the light energy and to transfer it to the crucial pigments in the RC that are able to initiate CS. Besides separate antenna proteins that vary in size, shape, pigment composition and location between different organism types (39, 40), PSIIcc is equipped with a core antenna system provided by the Chl a bound to CP43 and CP47 (29, 30). One of the key questions presently debated is, whether excitation energy transfer (EET) from the core antennae to the RC pigments is significantly slower than the initial CS step as suggested by structure-based calculations (41) or significantly faster than or on a similar time scale as CS (42-44).
The RC of PSIIcc (PSII-RC) is structurally homologous to the photosynthetic RC of purple bacteria (bRC (45-48)) with two branches related by a pseudo C2-symmetry as shown in Figure 2. The excitated state formed in the RC is traditionally denoted as P
because of a bleaching observed at 680 nm in optical spectra upon formation of a radical pair (49). This led to the identification of a "pigment P680" with the "primary electron donor" in the RC (50). However, the excited state structure of PSII-RC is complex and can not be related in a simple manner to just one pigment (21, 37, 51). According to the simulations of Raszewski et al. (51), the lowest excited state of the RC is essentially localized on ChlD1. At physiological temperatures, higher excited states are populated as well, so that the excitation energy is found to only about 30 % on ChlD1 and to 10 - 20 % on each of the other five pigments. This situation is quite different from that in bRC, where the lowest excited state is localized on the "special pair" PL-PM (52), which is the counterpart of the PD1-PD2-dimer (Figure 3). The two bacteriochlorophylls PL and PM in bRC have a strong overlap of their electronic wavefunctions allowing for a significant delocalization of electrons (53, 54). As a consequence, the excited states of the PL-PM-dimer are coupled to charge-transfer states with a concomitant red-shift of the lowest excited state, so that an energy sink is formed at the "special pair" (55, 56), where the excitation energy is trapped also at ambient temperatures. In contrast, the two Chl of the PD1-PD2-dimer have a larger separation of their tetrapyrrole ring systems (Figure 3), so that the coupling of excited states to charge-transfer states is small. This decoupling avoids formation of a deep energy sink at the PD1-PD2-pair (which is therefore not so "special") and allows for a wider excitation energy distribution in equilibrium. The underlying (local) excited state energies are probably determined primarily by electrostatic pigment-protein interactions as found in other systems (57, 58), but the detailed mechanisms of excited-state tuning in PSII-RC remain to be uncovered.
In accordance with the preferential localization of the excited state of the RC on ChlD1, CS is assumed to start from this cofactor with the primary radical pair, Chl
Pheo
, being created in 0.6 - 3.0 ps. The second radical pair, P
Pheo
, is formed in 6 - 11 ps. P
is the strong oxidant needed for water splitting. The first real stabilization of CS occurs in the third step yielding P
Q
in about 300 ps (59) (Figure 2A). This primary CS is formally a one-electron process that has to take place four times to allow for the formation of one O2 (Figure 1A). As with the excited state structure, there are fundamental differences between PSII-RC and bRC concerning the mechanism of primary CS. In bRC, the PL-PM-dimer is the primary electron donor and BA, the counterpart of ChlD1, is the primary electron acceptor (52). However, also in bRC, CS can be started at BA in certain site-directed mutants that raise the energy level of the (PL-PM)
B
state (60). This finding indicates that the protein environment can alter the photochemical properties of the RC profoundly without changing the overall pigment arrangement, a fact that should be kept in mind when comparing structures. Furthermore, the crystal structures only show a kind of averaged structure, but structural variations due to protein motions cause disorder. As a result, the electronic states of protein-bound chromophores show a distribution of energy levels, so that multiple pathways of EET or CS can be operative (61, 62). Indeed, it has been proposed that there are actually two different CS mechanisms possible in PSII-RC (63, 64): One follows the path ChlPheo ®
PPheo and the other the path PChl ®
PPheo, so that PD1 and PheoD1 are always electron donor and acceptor, respectively, but ChlD1 can act as both.
A further complication arises from the observation that CS and ET leading to water oxidation can be induced with photons of wavelengths as long as 780 - 800 nm (65-71). This finding has been explained with a low-lying excited state of PSIIcc similar to the so-called long-wavelength Chl of photosystem I (62, 72). A possible candidate for such a state is a charge-transfer state of the PD1-PD2-dimer coupled to exciton states (37). It could be formed in a sub-population of PSII-RC as a consequence of structural variations resulting in an increased coupling between PD1 and PD2, so that these two Chl would after all form a "special pair". This state could then allow for CS initiated by far-red light via the path PChl ®
PPheo. We note that also in bRC, there are different conformations of the PL-PM-dimer that presumably differ in the coupling between the dimer halves (73). Another possible "red state" is suggested by the CS mechanism involving ChlPheo as the first charge-separated state: Clearly, this state has to be coupled to excited states (e.g., Chl
) to allow for light-induced CS. If this coupling is strong enough, the state ChlPheo could borrow some oscillator strength. This idea is supported by electric field effects on the optical spectra (Stark effect) of isolated PSII-RC (74). However, this study suggests the state ChlPheo to be close in energy to the lowest excited state of the RC implying a small driving force for the first ET step. Also, the spectrum of such a state should be significantly broadened by coupling to vibrations. At present, the nature of the "red Chl" in PSIIcc is unclear, and there is more to be learned about primary CS in the future.
3.2. Non-heme iron and quinones
The formation of PQH2 from PQ requires two electrons and two protons and thus needs two turnovers of the RC (Figure 1A). The first step is the creation in 200 - 400 �s of a semiquinone anion radical Q
, which is likely stabilized by protonation of a nearby amino acid side chain (21). It could be shown for the analogous bRC of purple bacteria that a glutamate residue (Glu L212) has to take up a proton from the cytoplasm prior to ET (75-77). This residue is part of a network of electrostatically coupled titratable groups that influence the proton uptake of the RC after quinone reduction (78, 79), but it is not conserved in PSIIcc. Presently, it is unknown, which group(s) take(s) over the role of the glutamate or are responsible for proton uptake from the cytoplasm. Studies on mutant bRC suggest that water molecules may functionally replace protonatable amino acid side chains (78, 80). Thus, water molecules near the QB-site in PSIIcc may also play an important role in quinone reduction, but could not yet be assigned in the crystal structure. The second ET step in PSIIcc occurs in 500 - 800 �s and is followed by protonation of the formed Q
(21). In bRC, the Glu L212 is likely one proton donor, while the second proton probably takes the route via a serine (Ser L223), which is in hydrogen bonding distance to QB and takes up a proton from Asp L213 (75-77, 81, 82). The latter serine is present in PSIIcc as well (Ser A264), and the role of the aspartate is taken over by His A252 as suggested by the crystal stucture and electrostatic calculations (83).
In recent years, the non-heme iron located equidistantly from both QA and QB has been in the spotlight again because of renewed interest in its possible participation in the ET reactions between the quinones in bRC (84-87). A mechanism is discussed, in which arrival of an electron on QA triggers ET from the non-heme iron complex to QB followed by rereduction of the non-heme iron complex by Q
. Remarkably, a similar reaction sequence has been considered two decades ago for PSII (88). An important difference is that the non-heme iron is ligated besides the four histidines by bicarbonate in PSIIcc instead of a glutamate residue (Glu M234) in bRC. Bidentate ligation of bicarbonate to the non-heme iron in PSIIcc was detected by Fourier transform infrared (FTIR) difference spectroscopy using 13C-labeled bicarbonate (89) and later confirmed by X-ray crystallography. Since bicarbonate emanates from the substrate of the dark reactions, CO2, it is tempting to assign to it a role as activator of PSIIcc and hence the light reactions. Indeed, it is well known that bicarbonate is required for efficient reoxidation of Q and protonation of Q (90, 91). The analogous Glu M234 in bRC has been shown very recently to have a direct influence on proton uptake (79). A recent study on PSIIcc applied electron paramagnetic resonance (EPR) spectroscopy and quantum chemical modeling to the Q-Fe2+ complex and concluded that the ligand is actually carbonate, i.e., deprotonated (92). Indeed, the three putative hydrogen bond donors to this carbonate identified in the crystal structure together with the Fe2+ ion could significantly lower the pKa value of HCO
from ~
10.3 in aqueous solution to below 6.0, which would be necessary for such an observation (92). In this situation, the carbonate ligand could act as a protonatable group relevant for proton transfer to Q in agreement with a proposal based on FTIR studies of a hydrogen bond network from the non-heme iron toward the QB pocket involving (bi)carbonate and His A215 (93). These findings point to a possible role of the non-heme iron in the regulation of the quinone reductase activity of PSIIcc by CO2. However, the precise function of this important metal center remains to be elucidated.
3.3. Creation of a strong oxidant
A key issue in PSII research is the question of how the oxidative power for water splitting is created. In the pH interval of 5.7 - 7.8 (the likely pH of the lumen under physiological conditions (94, 95)), the midpoint potential for the formal conversion of two water molecules into one O2 molecule, four protons and four electrons is 770 - 890 mV (verus normal hydrogen electrode (1)). Notably, the potential for oxidation of Chl a is in a similar range depending on the solvent (96). The midpoint potential of the P
/P
couple, Em(
/
), has been estimated from experimental data to be about 1.25 V (36), so that there is just enough driving force for water splitting in PSIIcc. Theoretical calculations based on the 3.0 Å structure (14) yield Em(PD1) = 1.21 V (97) and suggest that the potential increase of PD1 compared to Chl a in solution is caused by the charge distribution of the protein environment with important contributions from the polypeptide backbone (i.e., helix dipoles of TMH d of PsbA and PsbD), the Mn4Ca cluster and the peripheral protein subunits. It should be noted that the "decoupling" of PD1 and PD2 discussed in subsection 3.1 also contributes to the oxidative power of P. A strong orbital overlap between PD1 and PD2 as in the "special pair" of bRC (Figure 3) would probably decrease the potential by up to 150 mV (53).
4. REDOX STATES OF THE Mn4Ca CLUSTER
4.1. Kok-cycle and S-states
The oxidation of two water molecules to one molecule of O2 is a four-electron process that has to be linked to the one-electron process of oxidizing PD1 in the RC (Figure 1A). This linkage is provided by the ability of the WOC to store four oxidation equivalents during its reaction cycle, for which there is direct experimental evidence: If dark-adapted PSII is excited by saturating single-turnover light flashes, the oxygen evolution exhibits a damped period-four oscillation as a function of flash number (98-102) with maxima after the third, seventh, eleventh flash etc. (Figure 4A). This pattern has been interpreted by Kok et al. (99) in terms of five states of the WOC, Si (i = 0, 1, 2, 3, 4), where i is the number of stored oxidation equivalents. A one-step advancement in this so-called "Kok-cycle", i.e., Si ®
Si+1 for i = 0, 1, 2, 3, corresponds to the one-electron oxidation of the WOC by P triggered by light-induced CS in the RC (Figure 4B). The ET from the WOC to P actually involves an intermediate redox active tyrosine, YZ (see section 8), which was discovered later (103). The state S4 is not stable, but circumstantial evidence for an intermediate between S3 and S0 has been reported (to be discussed in subsection 10.3). Dioxygen is believed to be formed in the (S4) ®
S0 transition (Figure 4C), so that it is the S3 ®
(S4) ®
S0 sequence, in which O2 is released and the WOC is reset to its original redox state. This scheme implies that the stable state in the dark is S1.
Flash excitation can cause a double turnover of the RC, resulting in a "double-hit", i.e, a two-step advance Si ® Si+2 of the WOC. In fact, double-hits are necessary to explain, why the oxygen evolution after the second flash is not strictly zero. In addition, some photosystems may not be able to turn over, e.g., due to competing charge recombination processes, which results in what is called a "miss" ("zero-step advance" Si ® Si after flash excitation). Double-hits and misses have been invoked by Kok et al. (99, 100) to explain the observed oxygen evolution pattern. More recent analyses suggest that additional factors such as inactivation or backward-transitions may have to be considered (104). Processes of this kind cause a dephasing of the S-state cycling in a sample and hence a damping of the period-four oscillation until the steady-state oxygen evolution rate is reached (dashed line in Figure 4A). We note that Kok et al. (99) had to assume that besides S1 a certain amount of S0 is present in dark-adapted samples to satisfactorily fit the observed O2 evolution pattern. They proposed to illuminate samples with one saturating flash prior to the dark adaptation period to reduce the amount of S0. The initial presence of S0 in the dark was explained by a redox equilibrium between S0 and S1 (105, 106). In an alternative explanation, which is preferred nowadays (104), there is no real S0 population in the dark, but merely an apparent population being an artifact of the modeling (107, 108). Electron donation to S2 and S3 by an unknown donor has been invoked as explanation for these complications (108). For further details concerning the analysis of O2 evolution patterns, see (104, 109).
Since the publication of the original model of Kok et al. (99), several extensions have been proposed that introduced additional states. Models with additional redox states, i.e., overreduced states of the WOC, are the topic of subsection 4.5. In contrast, Dau and Haumann (25, 110, 111) supplemented the cycle with protonation states. Initially, a second "S4" state was introduced to account for time-resolved X-ray absorption data (110). However, if we take the index "i" of "Si" as representing the redox state of the WOC in the spirit of Kok et al. (99), this new "S4" is actually a deprotonated S3 state (see further discussion in subsection 10.3). Later, a nine-state model (i.e., eight states + (S4)) was proposed, in which there is a strict alternation of oxidation and deprotonation of the WOC (25, 111). Albeit intriguing, this model remains largely hypothetical; see (25) for an in-depth discussion. In the following, we shall base the description of the WOC on the traditional five-state model.
Flash-excitation of PSIIcc causes oxidation of the WOC at the "donor side" and reduction of the iron-quinone complex at the "acceptor side" (Figure 1A). While the WOC requires four turnovers of the RC to complete one cycle (period-four oscillation), the acceptor side needs two turnovers (binary oscillation). Thus, one may ask how the two reaction cycles, that are driven by the same trigger, are synchronized and whether the state of the acceptor side has any influence on the donor side. This problem was tackled by Shinkarev and Wraight (112). They distinguished between two reaction cycles of the WOC referred to as V- and W-cycle representing two different states of the iron-quinone complex and demonstrated that the details of the O2 evolution pattern depend on these states. For example, a RC with QA reduced at the instant of light-excitation will not be able to perform stable charge separation allowing for a one-step advance of the WOC. Consequently, it will produce a miss. In this way, it can be rationalized that the miss probability depends on the redox state of the acceptor side and consequently also on the flash number. We refer the reader to the work of Shinkarev (104, 113, 114) for further details.
Before discussing the structure of the Mn4Ca-cluster and the identification of its proteinogeneous ligands (see subsection 5.5), it is reasonable to look at the available information about its electronic structure, i.e., the redox states of the Mn ions in the different S-states (the Ca2+ ion is supposed not to change its redox state). Suitable methods for probing the redox states of metal centers are X-ray absorption (XAS) and emission (XES) (115-117) spectrosopy as well as electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) spectroscopy (118-120).
4.2. X-ray spectroscopy
In XAS, the energy from an X-ray beam is absorbed by the core electrons of an atom, and electrons are either promoted to a higher vacant orbital or ejected as a photoelectron. X-ray absorption spectra feature sharp increases in absorption at specific energies, which are characteristic for the absorbing element and referred to as "edges". The edge is due to an allowed transition according to the selection rule for dipole transitions, which states that the transition probability is high for Dl = 1, where l is the orbital momentum quantum number. For example, electrons from the 1s orbital (K-shell, l = 0) of Mn have a high probability for a transition into a p-orbital (l = 1). Since the lowest vacant p-orbital is 4p, it is the 1s ® 4p transition that gives rise to the "K-edge" (Figure 5A). Similarly, the lowest, not doubly occupied orbital to be reached from 2s or 2p (L-shell) is one of the 3d orbitals (l = 2) and, consequently, the L-edge is dominated by a 2p ® 3d transition. Note that the L-edge is at significantly lower X-ray energies (soft X-rays) than the K-edge (hard X-rays). In addition, there are "weakly allowed" transitions that do not obey the selection rule (e.g., quadrupole transitions) and result in weak absorption bands in the pre-edge region (121). If the incident energy is high enough to eject the electron into the continuum, it is back-scattered from the surrounding atoms, which creates an extended X-ray absorption fine structure (EXAFS) containing important information about atom-atom distances (to be discussed in subsection 5.2).
As L-edge spectroscopy on biological samples is associated with some experimental difficulties (116), the focus in applying XAS to PSII has been on the K-edge. Goodin et al. (122), in their pioneering work, observed a shift of the K-edge to higher energies upon going from the S1 to the S2 state. Later experiments with improved technology confirmed this shift and furthermore revealed a smaller shift to higher energies for the S2 ® S3 transition together with a back-shift to lower energies upon going to S0 (Figure 5B) and an oscillatory behavior as a function of flash number (123, 124). These data were interpreted to indicate a Mn-centered oxidation during the S0 ® S1 and S1 ® S2 transitions, but the absence of Mn oxidation during S2 ® S3. However, other authors came to a different conclusion and assigned a Mn oxidation to each of the three S-state transitions (125-127). As discussed in detail in the literature (124, 127-130), there are some difficulties associated with the interpretation of the K-edge data including the precise quantitation of S-states as a function of flash number, the different methods to extract edge energies from the raw data and the explanation of the K-edge shift, making the determination of the metal oxidation state on the basis of the K-edge position difficult.
In XES, a core hole is created by excitation of the metal ion with X-rays of sufficient energy. The hole has a lifetime of only about 10-15 s, because it is rapidly filled by an electron from a higher shell with the excess energy being emitted as an X-ray photon (116, 117). For example, K fluorescence originates from electrons falling into the 1s orbital (red arrows in Figure 5A). In the context of redox states, the Kb1,3 fluorescence, which is due to a 3p ® 1s transition, was found to be a good probe for the oxidation state of Mn, since its energy is sensitive to the number of 3d electrons presumably because of exchange interaction between 3p and 3d electrons (131). In contrast to the K-edge, the Kb1,3 fluorescence shifts to lower energies upon removal of 3d electrons and, on the basis of XES studies of model complexes, was suggested to be less sensitive to the ligand environment (132). The Kb1,3 fluorescence energy of the Mn4Ca cluster of PSII shows the same flash-dependent pattern as the K-edge position, i.e., a strong change after the first, a weaker shift after the second and a strong back-shift after the third flash (Figure 5B). Accordingly, these data were interpreted to confirm Mn-centered oxidation during the S1 ® S2 transition and its absence during S2 ® S3 (124). However, this interpretation is still not generally accepted, and the question was raised as to whether significant rearrangments in the ligand sphere of Mn could affect the Kb1,3 fluorescence (127).
Another complication arises from studies using resonant XES (RXES), also called resonant inelastic X-ray scattering (RIXS). In this type of experiment, the metal center is excited in the pre-edge region and X-ray emission originating from, e.g., electron relaxation from 2p orbitals into the 1s hole is monitored (116, 117). The data were analysed in terms of an effective number of 3d electrons, and it was conlcuded that the electron that is removed from the WOC during the S1 ® S2 transition occupies a strongly delocalized orbital in the Mn4Ca-cluster (133). Under these circumstances, any assignment of oxidation states to individual Mn ions within the WOC appears to be merely a formality. Nonetheless, the formal oxidation states remain a useful framework for the analysis of the redox chemistry of the WOC.
4.3. Electron paramagnetic resonance spectroscopy
So far, we have only discussed redox state changes. But what about absolute oxidation numbers? To get this information, we have to resort to EPR and ENDOR spectroscopy. EPR spectra arise from the absorption of microwave radiation by a system carrying one or more unpaired electrons (S ³ 1/2) causing spin-state transitions (134). The transition energies depend on the strength of the applied magnetic field, which is usually expressed in terms of the microwave frequency and characterized by waveguide frequency bands. The more important bands for EPR of biological samples are S (2.60 - 3.95 GHz), X (8.2 - 12.4 GHz), Ku (12.4 - 18.0 GHz, also referred to as P-band), Ka (26.5 - 40.0 GHz), Q (33 - 50 GHz) and W (75 - 110 GHz). For a single unpaired electron with S = 1/2, the energy splitting between the two spin levels (electron Zeeman interaction) is proportional to the applied field with proportionality constant gbe, where the Bohr magneton be is a universal constant and the "g-factor" g is characteristic of the spin system. Since in a molecule or cluster, the unpaired electron experiences local magnetic fields (e.g, due to orbital angular momentum), its g-factor can differ markedly from the value ge = 2.0023 of the free electron, which is particularly true for transition metal compounds. Atomic nuclei may also carry a spin (e.g., I = 5/2 for 55Mn with the nuclear g-factor gn = 1.3819), but the nuclear Zeeman splitting characterized by gnbn (with bn the nuclear magneton) is much smaller (gnbn/gebe = 37.587 ´ 10-5 for 55Mn). Of importance for EPR is rather the magnetic interaction between electron and nucleus, the hyperfine interaction characterized by the hyperfine coupling (hfc) constant a, giving rise to signal splitting. Both, g-factor and hfc constant, can show anisotropy that is characteristic of the symmetry of the molecular environment. In contrast to the Zeeman interaction, the hfc is independent of the applied field, so that studies of a system in different frequency bands can help to disentangle spectral features (135-137). If there is more than one unpaired electron (S > 1/2), additional line splittings occur due to electron-exchange and magnetic dipole-dipole interaction (134). Thus, in a system such as the WOC with four coupled Mn ions cycling through different oxidation states with possibly up to five unpaired 3d electrons per ion, a rich variety of EPR signals can be expected; and this is indeed found.
To date, EPR signals have been obtained from all (meta)-stable S-states (i.e., S0, S1, S2, S3). A recent overview is given by Haddy (119). Here, we focus on those states, in which YZ is not oxidized. So-called metalloradical signals with an additional S = 1/2 radical on YZ are discussed in subsection 8.2. The EPR signals known for the longest time are those associated with the S2 state, comprising a multiline signal centered at g = 2.0 and a broad signal at g = 4.1 in X-band (119). The amplitude of the multiline signal exhibits a damped period-four oscillation with maxima after the first and fifth flash indicating its connection to S2 (Figure 6A) (138, 139). The g-factor of the multiline signal was determined to g = 1.98 in Q-band (140). To reveal its g-anisotropy, W-band experiments were conducted on single crystals of PSII from T. vulcanus (141) and T. elongatus (142). There is consensus that the S2 multiline signal arises from the S = 1/2 ground state of the Mn4Ca cluster. The many lines giving the signal its name (about 40 - 50 in S-band (136, 143)) are due to hfc between this spin and the 55Mn nuclei. A number of different sets of hfc constants have been derived from EPR studies in various frequency bands (136, 144-146). The hyperfine data are consistent with MnIII and MnIV oxidation states, but not MnII (138). The S = 1/2 ground state arises from antiferromagnetic exchange coupling between Mn ions and leaves only two possibilities for the formal oxidation states: Either there are one MnIII and three MnIV (abbreviated in the following with (III, IV3) as indicated in Figure 4C) or three MnIII and one MnIV, i.e., (III3, IV) (144). To decide on these possibilities, EPR experiments were conducted (145) on oxygen-evolving, PSII-enriched membranes from spinach (147) that were oriented by a partial-dehydration procedure (148, 149). Both, g- and hfc-anisotropy were exploited in simulations of the S2-multiline signal (145) to conclude that the formal oxidation state is (III, IV3) and to propose an arrangement of the Mn4 moiety relative to the membrane normal (see subsection 5.4). The (III, IV3) oxidation state was later confirmed by further simulations (146) and is also in agreement with interpretations of the K-edge in XAS (129, 130).
The broad g = 4.1 signal exhibits the same damped period-four oscillation as the g = 2 multiline signal (Figure 6B) and is thus also attributed to the S2 state (150). Resolution of the hyperfine structure of the g = 4.1 signal, also exhibiting some "multiline" character, confirmed its tetranuclear Mn origin and suggested an S = 3/2 or 5/2 spin state associated with the same redox state of the Mn4Ca-cluster as the g = 2.0 signal (151, 152). Later studies favored the S = 5/2 variant (135, 146, 153-155). The relative intensity of the two S2-state signals depends on experimental conditions such as the temperature of illumination or the presence of small alcohols and cryoprotectants (119) as well as the binding of extrinsic protein subunits (156). Of particular interest is that both S2-state signals are influenced by the presence of the chloride ion (119), which is required for oxygen evolution activity (157). We return to this aspect in section 7.
By applying three flashes to a dark-adapted PSII-sample, another multiline signal at g = 2 can be produced (Figure 6C), which is associated with the S0-state (158-160). On the basis of EPR, this signal is interpreted as an S = 1/2 ground state of the WOC (161) being likely in the formal oxidation state (III3, IV) or (II, III, IV2). Assigning a spin ground state of S = 1/2 to S0 and S2 and a Mn-centered oxidation to the S0 ® S1 and S1 ® S2 transitions implies that the S1 state should have an integral spin. Indeed, an EPR signal at g = 4.8 in the S1-state consistent with S = 1 was detected (162). In coventional EPR, the microwave field is polarized perpendicular to the applied magnetic field. However, states with integer spin are better studied with parallel polarization (163), since in this case other selection rules apply. In this way, the existence of an EPR signal of dark-adapted PSII-samples from plants was demonstrated that vanishes upon illumination as expected for the S1 ® S2 transition (162, 164). However, the signal is weak, difficult to detect and absent after chloride or calcium depletion (164). Later attempts to find an EPR signal of the S1-state in parallel mode revealed yet another multiline signal at g = 12 (165). This signal only appears in cyanobacterial PSIIcc or in plant PSIIcc with extrinsic subunits other than PsbO removed (166). The hyperfine structure of the g = 12 signal confirmed its manganese origin and suggests that all four Mn ions are exchange-coupled. On the basis of a comparison with a tetranuclear Mn model complex, this state was proposed to be compatible with a (III2, IV2) redox state (167).
The S3-state can be generated by two flashes or by continuous illumination at 235 K. Matsukawa et al. (168) were the first to detect EPR signals of this state in both parallel and perpendicular mode. In particular, the parallel polarization EPR signal at g = 12 shows the expected flash number dependence (Figure 6D). Later, Ioannidis and Petrouleas (169) optimized the trapping protocol of the S3-state and confirmed the results. Very recently, Boussac et al. (170) detected additional signals and found a satisfying fit for an S = 3 spin state. So far, the EPR spectra do not allow to decide, whether a Mn-centered oxidation occurs in the S2 ® S3 transition. They suggest, however, that if a ligand is oxidized in S3, it is strongly magnetically coupled to Mn and thus from the first ligand sphere (170).
4.4. Electron nuclear double resonance spectroscopy
The multiline signals discussed in subsection 4.3 are suitable to demonstrate the limits of EPR spectroscopy as applied to the WOC: The many possible transitions arising from the hfc with 55Mn can not be resolved. For example, the up to 50 lines of the g = 2 multiline signal of the S2-state seen in S-band EPR (136, 143) represent only a small fraction of the possible 64 = 1296 EPR transitions (171). Moreover, the spectra are dominated by 55Mn hfc and weaker hfc from ligand nuclei are suppressed. Therefore, alternative methods have been applied to gain complementary information about hfc. The pulsed EPR method known as electron spin-echo envelope modulation (ESEEM) spectroscopy is sensitive to the weaker couplings arising from nuclei of putative ligands to the Mn4Ca-cluster (172, 173). Results from this type of experiment are discussed in subsection 5.5. Here, we focus on the 55Mn hfc studied with ENDOR.
In an ENDOR experiment, a radiofrequency (rf) field or pulse as used in nuclear magnetic resonance (NMR) is applied to drive nuclear spin transitions, which by virtue of the hfc act back on the electron spin system to produce a change in a specific EPR signal or the electron spin echo (ESE) intensity (for details, see (120) and references therein). The number of nuclear spin transitions observed in this way increases additively with the number of inequivalent classes of coupled nuclei (= 4 in the Mn4Ca-cluster), so that there are "only" 40 distinct allowed 55Mn nuclear spin transitions in the S2-state with S = 1/2 and I = 5/2 (174). Due to the dependence of the ENDOR spectrum on the probed EPR transition (field position), a further selection is possible. The advantage of a simultaneous simulation of EPR and ENDOR spectra to obtain reliable hfc parameters was clearly demonstrated by Peloquin et al. (174), who investigated the S2-state in X-band. Later, Kulik et al. (175, 176) extended these studies to Q-band and also examined the S0-state. They performed a simultaneous simulation of EPR and ENDOR spectra in X- and Q-band and also considered the g-anisotropy of the g = 2 multiline signal of the S2-state apparent from W-band EPR studies on single crystals (142) to arrive at a final assignment of redox states (III3, IV), (III2, IV2) and (III, IV3) to S0, S1 and S2, respectively. However, the experiments were performed in the presence of 3% methanol to enhance the multline signals, so that the variant (II, III, IV2) for S0, albeit unlikely, cannot be completely ruled out for methanol-free samples. We thus have at present the assignment of redox states to S-states as depicted in Figure 4C, with the two variants for the S3-state arising from the uncertainty concerning Mn-centered oxidation in the S2 ® S3 transition. Further implications of EPR/ENDOR data for the structure of the inorganic core of the WOC (including more recent data on single crystals (177)) will be discussed in subsections 5.4 and 5.5.
4.5. Chemical reduction of the Mn4Ca-cluster
It has been noticed quite early that treatment of PSII with chemical reductants such as hydroxylamine (NH2OH) results in a delay in oxygen evolution from the third to the fifth flash (178). These results were interpreted in terms of the formation of an S-1-state, i.e., a state of the WOC with one electron in excess of the S0-state (179-181). Further studies, in which hydrazine (N2H4) was used besides NH2OH, revealed the possibility to reversibly reduce the WOC to states S-2 and S-3 with concomitant shifts of the oxygen evolution pattern (101, 182-184). It was even possible to create states S-4 and S-5, but these states appear to be less stable (185). Nonetheless, these studies support the redox state assigment given in Figure 4C. In addition, hydroquinone was used to create the S-1-state (186, 187), and chemical reduction was applied to create an analog of the S0-state from S1 in the dark (188). This analog was shown to have the same EPR properties as the S0-state accumulated by "flashing through the Kok-cycle" (159).
Interaction of nitric oxide (NO) with dark-adapted PSIIcc was demonstrated to produce, among other products (189-191), an S1-derived state exhibiting an EPR multiline signal centered at g = 2 (192). This signal was later attributed to a MnII-MnIII interaction (193, 194) and, on the basis of the retarded oxygen evolution pattern, assigned to S-2 (195). NO also reacts with the S2 and S3-states produced by pre-flashes. The exceptionally fast reaction of NO with S3 was interpreted as the possible presence of an organic radical in the S3-state, which would be in line with the absence of a Mn-centered oxidation in the S2 ® S3 transition (196).
4.6. Net charge changes of the Mn4Ca-cluster
The fact that the Mn4Ca-cluster is oxidized at least in each of the steps leading from S0 to S3 raises the question of whether positive charges are accumulated in the WOC-site. In their seminal work, Saygin and Witt used electrochromic band shifts of carotenoids (197) as well as chlorophylls (198) to demonstrate that a positive surplus charge (i.e., the net charge change relative to S1) is accumulated in S2 compared to S1, but not in S3 compared to S2 or in S1 compared to S0. Since one electron is released in each step of the sequence S0 ®
S1 ®
S2 ®
S3, the found surplus charge pattern of 0:0:1:1 in S0: S1:S2:S3 (Figure 4C) can only be explained by processes leading to a compensation of one positive charge accompanying the formation of S1 and S3. These processes are likely related to proton release as discussed in section 9. The interpretation of the electrochromic shifts is supported by the S-state dependence of the re-reduction kinetics of P
(199). Electrochromic shifts were also studied in PSIIcc-samples treated with NH2OH indicating the presence of a negative surplus charge in the S-1-state (180).
5. STRUCTURE OF THE Mn4Ca CLUSTER
5.1. X-ray crystallography
The progress in structure elucidation of PSIIcc by X-ray crystallography can be assessed from two quantities. The indicated resolution of, e.g., 2.9 Å, implies that the crystallographic model takes into account diffraction from sets of equivalent, parallel planes of atoms with a minimum lattice plane distance of 2.9 Å. As a rule of thumb, two objects in the electron density map at 2.9 Å resolution can be resolved, if they are 2.9 Å apart. Notwithstanding, the precision of atom positions is higher, since structural constraints such as known bond lengths and angles of amino acid residues are considered in the modeling. The estimated coordinate error of PSIIcc at 2.9 Å resolution is 0.26 Å (200) as calculated by using the programm SFCHECK (201). Another measure of data quality is the number of unique reflections, which counts the number of measured reflections in a crystallographic data set after neglect of all repeated measurements of the same reflection or symmetry-related reflections. This number increased considerably from the first to the most recent 3D-structure of dimeric PSIIcc, and even the last step, albeit seemingly small as regards the resolution enhancement, implies a significant increase in the reliability of the model. We note that this latter progress is due to an improved data processing (15), whereas the earlier steps imply an improvement of crystal quality (14).
The quality of the structure of the Mn4Ca-cluster as derived from X-ray diffraction is a more complicated issue (24). Here, we face four main problems: (i) unavoidable radiation damage, (ii) high electron density in the Mn4Ca-cluster, (iii) structural heterogeneity, and (iv) lack of stringent structural constraints. As discussed in subsection 4.2., X-rays are not only scattered by atoms, but also absorbed depending on the electronic energy levels and the X-ray wavelength. Exposure of any material to X-rays thus will lead to some production of photoelectrons (202) that eventually are trapped by redox-active centers (203-207). As shown by applying XAS and EXAFS, the X-radiation indeed causes a reduction of the Mn-ions in the WOC under conditions of X-ray diffraction studies (208-210). This reduction may result in structural alterations, thereby causing a larger distribution of metal-metal distances, or even result in a destruction of the active center. The concomitant loss of phase relationship ultimately limits the quality of the electron density in this region. Even without this damage, the high electron density originating from the Ca and the four Mn ions makes it impossible even at 2.9 Å resolution to clearly resolve individual metal sites and also overshadows the electron density of proteinogeneous ligands. In addition, about 10 % of the catalytic centers can be inactive or damaged per se or only partly assembled (24). Even fully active centers in the S1-state can principally occur in different conformers and/or tautomers as suggested by quantum chemical model calculations (211, 212). Finally, there are no stringent constraints concerning bond lengths, binding topology etc. as in the case of amino acids or other cofactors except for some information from EXAFS (see subsection 5.2; in fact, some constraints have been applied in the modeling, see below). All these factors increase the coordinate error of the WOC-region considerably compared to other parts of PSIIcc.
In normal X-ray crystallography, most X-ray photons can be considered to a good approximation as being elastically scattered without any phase delay (Thomson scattering). In general, the ability of the atoms to absorb X-ray photons at specific wavelengths (see subsection 4.2) influences the scattering behavior. If the X-ray energy is close to an absorption edge, anomalous dispersion occurs, resulting in a change of amplitude and phase of the scattered radiation (213, 214). Since like the absorption edges, the anomalous scattering is element-specific, tuning of the X-ray wavelength can be used as a means of contrast variation to distinguish certain atom types and to locate their positions in the unit cell (215). In this way, Zouni et al. (10), by using a wavelength close to the Mn-edge, were able, for the first time, to locate the Mn4-cluster (at that time without the Ca). The four metal ions showed up as a pear-shaped electron density, which was interpreted tentatively in terms of an "Y"-like arrangement (Figure 7). This interpretation was essentially confirmed by Kamiya and Shen (11) at a slightly better resolution. Ferreira et al. (12), still in a similar resolution range, extended the anomalous diffraction studies to a wavelength that allowed, for the first time, to locate the Ca-ion. It is located on top of the triangle formed by three of the Mn-ions and points towards the redox-active tyrosine A161 (Figure 7A). The fourth Mn-ion appears to be somewhat segregated and has been baptized the "dangling" manganese (174). This basic arrangement was confirmed by Loll et al. (14) with some changes of ion positions within the error limits of X-ray crystallography at this site (for a comparison, see (18, 24, 26)), and no further changes were identified in the refinement made by Guskov et al. (15).
It is important to understand that structural details such as �-oxo bridges can not be inferred from the X-ray diffraction data at the presently available resolutions for reasons described above. To have at least some constraints at hand to cope with the unresolved electron density at 3.0 Å resolution, Loll et al. (14) used information from EXFAS about metal-metal distances in the Mn4Ca-cluster (Figure 7B), but the positioning of metal ions and their ligands necessarily remains tentative. Ferreira et al. (12) used a rather detailed working model for the Mn4Ca-cluster to interpret the electron density at 3.5 Å resolution, which is, in our view, an overinterpretation of the crystallographic data. Not surprisingly, some of these details will have to be revised, while other aspects are worth a further consideration (see below). In the following, we descibe the less detailed (that is, "�-oxo-free") model by Guskov et al. (15).
In Figures 7A, C we show the (tentative) arrangement of metal ions in the Mn4Ca-cluster according to the 2.9 Å resolution structure in relation to a-helical segments of the peptide backbone and some amino acid residues of interest including putative metal ligands (Figure 7B). Also shown in Figures 7C, D is the position of the chloride ion as inferred from the electron density at 2.9 Å resolution and work on bromide-substituted PSIIcc to be further discussed in section 7. Despite the uncertainties alluded to above, some points can be made: (i) The redox active Tyr A161 (YZ) is in hydrogen bonding distance to His A190, but both residues do not belong to the first ligand sphere of the Mn4Ca-cluster (the Tyr-Ca distance is about 5 Å, see Figure 2B). (ii) The Mn4Ca-cluster is located together with the Cl--ion close to three a-helices of PsbA (A-cd, A-et) and PsbD (D-et; Figure 7C), which are rather stiff structural elements. (iii) Mn1 is likely coordinated by two residues (Glu A189 and His A332), which are located at the ends of different a-helices from PsbA. In this position, Mn1 seems to form a bridge between the a-helices (Figure 7A). (iv) Likewise, the Cl--ion is coordinated by residues Asn A181, Glu A333 and Lys D317 from three different helices (Figure 7C, D) and thus may serve to stabilize the mutual arrangement of these helices, which likely contributes to the overall stability of the WOC. Also, the orientation of the putative ligand Glu A333, which is located next to His A332, but already in a loop-region, might be stabilized by the interaction of its backbone NH-group with the Cl--ion. (v) All other putative ligands from PsbA (shown with yellow carbon atoms in Figures 7A, C) are located in loop regions and thus are likely more flexible and prone to structural perturbations, which is particularly true for Asp A342 and Ala A344 in the C-terminal loop of PsbA. (vi) All carboxylate ligands are modeled as bidentate ligands bridging two metal ions (Figure 7B). Albeit tentative at the present resolution, such a bidentate ligation could stabilize the inorganic core of the WOC and also assist in structural rearrangements that might occur during the catalytic cycle. (vii) The position of the "dangling" Mn4 appears to be the least certain among the metal ion positions. This is further substantiated by anomalous scattering studies at different temperatures, suggesting that Mn4 is most prone to radiation damage (14, 24). (viii) The Mn4-Cl- distance is 6.6 Å (Figure 2B). Taking into account the uncertainty of the Mn4-position (point vii), this result is in accordance with EXAFS-studies on bromide-substituted PSIIcc, suggesting a metal-bromide distance of about 5 Å (216). (ix) Electron density between Mn4 and Cl- could be due to a water molecule, which at this position would be ligated by the side chains of Asn A181 and Glu A333 (Figure 7D). (x) There is only one putative ligand from PsbC (Glu C354) located in a small helical loop segment (not shown, but see (30) for a topological map of PsbC). A protonatable residue of potential interest, Arg C357 (not shown, but see section 9), is located close to Glu C354 on the same helical segment with its side chain pointing towards the Mn4Ca-cluster (Ca-Nh1 distance ~ 5 Å).
5.2. Extended X-ray absorption fine structure (EXAFS)
A suitable method to study the structure of multinuclear metal complexes is EXAFS (115). As discussed in subsection 4.2, an X-ray photon with sufficient energy (i.e., above an element�s absorption edge) can create a photoelectron. In a simple picture, this quasi-free electron can be envisaged as a wave that goes out from one center (the absorber, e.g., a Mn-ion) and is scattered by another center (the backscatterer, e.g., a Mn or Ca ion or a �-oxo bridge). The interference of outgoing and backscattered waves characterizes the final state of the electron after absorption of the X-ray photon and hence influences the absorption coefficient. As the interference pattern depends not only on the distance between absorber and backscatterers, but also on the wavelength of the electron, which in turn depends on the excess energy (that is, X-ray photon energy minus ionization energy), an oscillatory component of the absorption coefficient ("fine structure") results, extending to about 1 keV above the edge and containing information about the backscatterer positions. Extracting this information requires subtraction of the pre-edge background and normalization. A Fourier-transformation (FT) of the obtained oscillatory pattern then yields an FT-amplitude spectrum with peaks at the apparent absorber-backscatterer distances (115, 208). Advantages of this method are that it is element-specific as are the absorption edges, that principally each metal in any state (e.g., spin state) can be probed and that radiation damage is reduced due to the use of lower X-ray doses compared to X-ray crystallography. Problems arise from the facts that backscatterers with similar atomic numbers (e.g., C, N, O) can not be distinguished, that there is some structural disorder in the absorber-backscatterer arrangement and that a quantification of the spectra, e.g., in terms of coordination numbers is not easy. Therefore, spectra are interpreted with reference to experiments on model complexes of known structure.
As is well documented in the literature (25, 117, 217), FT-amplitude spectra derived from EXAFS at the Mn K-edge of isotropic PSII-samples exhibit three major peaks that are interpreted as Mn-O/N distances at 1.8 - 2.2 Å (peak I), Mn-Mn-distances at 2.7 - 2.8 Å (peak II) and Mn-Mn/Ca distances at 3.3 - 3.4 Å (peak III). In particular, the discovery of peak II was a significant breakthrough, as it stimulated the interpretation of the EXAFS-data of the WOC in terms of a �-oxo bridged Mn complex (218). A bridge is called �2, �3 or �4 depending on whether it connects two, three or four metal ions (examples of each type can be seen in Figure 8). Numerous studies on synthetic multinuclear Mn complexes revealed that bridging Mn-O distances in these cases are typically about 1.8 Å, and that of Mn-O/N distances pertaining to terminal ligands are between 1.9 and 2.2 Å. The Mn-Mn distances depend on the number and type of bridges: They range between 2.6 and 2.8 Å for complexes with at least two �2-oxo or two �3-oxo bridges, but complexes containing �3-oxo bridges also feature Mn-Mn distance > 3 Å, and the distance can be as short as 2.3 Å for tri-�2-oxo bridging; for reviews, see (217, 219-222). Another interesting aspect is that protonation of one bridging oxygen in a di-�2-oxo-bridged unit causes a lengthening of the Mn-Mn distance by ~ 0.1 Å (223, 224). There seems to be a consensus that peak II of the WOC is heterogeneous with contributions from ~ 2.7 Å and ~ 2.8 Å Mn-Mn-distances (25, 117, 225). Small, but distinct changes of Mn-Mn distances occur during all S-state transitions, but these are least pronounced in S1 ® S2 (117). Haumann et al. (127) interpreted their data in terms of a shortening of a Mn-Mn distance during S0 ® S1 as well as S2 ® S3 and proposed inter alia deprotonation events (e.g., deprotonation of a �2-hydroxo bridge in S0 ® S1) as a possible explanation.
A plethora of topology models of the inorganic core of the WOC is compatible with the EXAFS data of isotropic samples (217, 226). A possibility to reduce the ambiguity is the use of oriented samples, as the EXAFS FT-amplitude is orientation dependent and proportional to ~ cos2q, where q is the angle between the electric field vector of the polarized X-ray beam and the absorber-backscatterer vector. There are two types of oriented samples, on which EXAFS-experiments have been performed: (i) oriented membranes from spinach chloroplasts (227-230) and (ii) single crystals of dimeric PSIIcc from T. elongatus (22).
Already the pioneering work of George et al. (227) on oriented chloroplast membranes revealed a pronounced dichroism of peaks II and III, the latter suggesting the orientation of a 3.3 Å absorber-backscatterer vector (interpreted as Mn-Mn distance at that time) close to the membrane normal in the S1-state. Later work confirmed the dichroism of peak II with average angles of 2.7 Å vectors relative to the membrane normal of ~ 60�, but arrived at a different conclusion about peak III with a 3.38 Å vector at an angle of ~ 43� relative to the membrane normal (228). Another study reported 80 � 10� for the 2.7 Å vectors (229). More recently, improved technology (115, 117) allowed for a better resolution of absorber-backscatterer vectors, and its was concluded that there are three di-�2-oxo-bridged Mn-Mn distances of ~ 2.7 and ~ 2.8 Å in a 2:1 ratio aligned at an average orientation of ~ 60� relative to the membrane normal (230). Furthermore, the puzzle about peak III could be solved by separating it into a ~ 3.2 Å Mn-Mn vector lying nearly in the membrane plane and ~ 3.4 Å Mn-Ca vectors with their average aligned along the membrane normal (230). A problem arises from the fact that the membranes in these experiments are only partially oriented, i.e., the various membrane sheets in the sample have a different orientation with respect to the substrate plane as illustrated in (115, 231). This mosaicity causes uncertainties in the angle q of up to ~ 20�.
Polarized EXAFS was applied to single crystals of dimeric PSIIcc to obtain further constraints for the modeling of the WOC-structure (22). Although the mosaicity is significantly smaller in this type of sample, the arrangement of PSIIcc-units is more complicated as each PSIIcc-dimer contains two Mn4Ca-clusters related by a non-crystallographic C2 symmetry, and four dimers are arranged in different orientations in the unit cell. By measuring EXAFS polarized along the three different crystal axes and controlling the crystal orientation in situ by X-ray diffraction, Yano et al. (22) were able to select essentially three out of the many proposed topology models of the inorganic core of the WOC that are consistent with the EXAFS dichroism. In agreement with the later modeling based on oriented membranes (230), the remaining models obtained from the crystal study contain Mn-Mn distances of ~ 2.7 and ~ 2.8 Å in a 2:1 ratio, a ~ 3.2 Å Mn-Mn vector and two ~ 3.4 Å Mn-Ca vectors. The models were placed into the crystal structure by using information concerning the relative position of Ca and Mn ions from anomalous X-ray diffraction and the overall shape of the electron density. As an example, we show in Figure 8A the model IIa of Yano et al. (22) and a possible ligation pattern that follows from placing this model into the more recent crystal structure of Guskov et al. (15). A problem of the model is the mismatch of the inorganic core with some of the proteinogeneous ligands (e.g., Ala A344 experiences a steric clash with Mn1 with an unrealistic Mn-O distance of 0.9 Å). This type of discrepancy could result from the uncertainty of placing the Mn4CaO5-core correctly into the crystal structure, but likewise could be due to the larger coordinate error of the crystal structure in the WOC region (see subsection 5.1). Therefore, the ligand topology shown in Figure 8A remains necessarily tentative.
The filtering of models performed by Yano et al. (22) based on polarized EXAFS does not exclude the possibility that further models may be constructed that satisfy the EXAFS constraints. Sproviero et al. found such a model (232-234). They performed hybrid quantum mechanics/molecular mechanics (QM/MM) computations using density functional theory (DFT) in the QM part (for reviews, see (234-236)) based on the 3.5 Å resolution model of Ferreira et al. (12). The resulting QM/MM model of the WOC was subjected to a fitting routine to make it compatible with the polarized EXAFS data of Yano et al. (22), resulting in a so-called R-QM/MM model (233). This model features a heterocubane Mn4CaO4 core with three �3-oxo bridges and one �4-oxo bridge linking the "dangling" Mn4 (Figure 8B). We note that calculations of this kind require assumptions to be made concerning the positions of water molecules, counter ions etc. due to the lack of experimental information about these details. Also, the original QM/MM model contained a Cl- ion ligating Ca (232) in contrast to more recent experimental information (see subsection 5.1 and section 7). Nonetheless, Sproviero et al. could demonstrate that a heterocubane structure is principally in agreement with the polarized EXAFS data from single crystals. An advantage of this model is that there is no mismatch between the inorganic core and the proteinogeneous ligands. However, there is no ligation of a Mn ion by Ala A344, but ligation of the Ca2+ ion instead, which is in disagreement with spectroscopic data (see subsection 5.5). So, there remain discrepancies that require further research. A promising route to more information about the structure of the WOC is polarized EXAFS on the recently obtained crystals of monomeric PSIIcc (28). In this new crystal form, there are only four different orientations of PSIIcc monomers in the unit cell, and for all of these orientations, the membrane normal makes an angle of ~ 90� with the crystallographic b-axis. This particular arrangement could facilitate the discrimination between absorber-backscatterer vectors oriented parallel and perpendicular to the membrane normal. Work along these lines is in progress.
5.3. Ca site and Ca/Sr exchange
The binding of Ca2+ to PSIIcc and its role in water oxidation has been the subject of numerous studies; see (157) and references therein. Biochemical studies revealed several Ca2+ binding sites in PSIIcc in accordance with the most recent crystallographic model (15), but Ädelroth et al. (237) could demonstrate by using radioactive 45Ca2+ that a single exchangeable Ca2+ ion per RC is sufficient to restore oxygen-evolution activity in Ca-depleted PSIIcc. The close interaction of this Ca2+ ion with Mn in the WOC was actually established prior to the anomalous X-ray scattering experiments on single crystals of PSIIcc. One line of evidence originates from the requirement of Ca2+ besides Mn2+ for the photoassembly of the WOC; for reviews, see (238-241). Another set of experiments showed that the inactivation of the WOC by chemical reductants is retarded by Ca (186, 242-244). These data were tentatively interpreted inter alia in terms of a binding site, in which Ca2+ blocks access of bulky reductants to Mn (157). We shall return to this interesting aspect in section 6. A more direct evidence for Mn-Ca interaction is provided by EXAFS at the Ca K-edge showing backscattering from Mn at a distance of 3.4 Å (245) and complementing the data obtained at the Mn K-edge (see subsection 5.2).
A number of metals have been tested for their ability to functionally replace Ca2+ in the WOC. Apparently, the WOC has a preference for di- and trivalent metals whose ionic radii are not too different from that of Ca2+, which is 0.99 Å (157). Among these ions, only Sr2+ (1.13 Å) is an activator of oxygen evolution activity (~ 40 % according to (246)), whereas Cd2+ (1.03 Å) and La3+ (1.04 Å) are competitive inhibitors. Notably, Mn2+ (0.80 Å) is a weak inhibitor in this type of experiment (~ 20 % activity (246)). We note that reconstitution with Ca2+ itself only restores about 80 % of the oxygen evolution activity. The decrease of activity in Sr2+-reconstituted material can be traced back to a slowdown of the S-state transitions, but a significant retardation is also observed for Ca2+-reconstituted samples compared to untreated PSII membranes (247) pointing to a disturbance of the WOC by the depletion/reconstitution procedure. Notwithstanding, the possibility to perform experiments on an essentially active WOC containing a different metal ion triggered spectroscopic studies on Sr2+-reconstituted plant PSII. EXAFS at the Sr K-edge confirmed that Sr2+ is incorporated in the vicinity of Mn at a distance of ~ 3.5 Å with the slightly larger distance compared to Ca2+ (see above) likely originating from the larger ionic radius of Sr2+ (248). Corresponding experiments on oriented membranes revealed an orientation of the Mn-Sr vector along the membrane normal with an error of 23� (249) in accordance with the results on native, Ca2+-containing PSIIcc discussed in subsection 5.2. The proximity of Sr to Mn was also explored with 87Sr ESEEM spectrosocopy (250). The weak magnetic coupling between the I = 9/2 87Sr nucleus and Mn was interpreted in terms of an upper limit for the Mn-Sr (and by analogy Mn-Ca) distance of 3.8 - 5.0 Å in reasonable agreement with the EXAFS-data.
A breakthrough concerning Ca/Sr-exchange was the development of a biosynthetic procedure by Boussac et al. (251). Cells of the thermophylic cyanobacterium T. elongatus were grown under continous illumination in a medium containing Sr2+-salt instead of the normally used Ca2+-salt. Surprisingly, the cells grew at the same rate in either case, demonstrating impressively the ability of Sr2+ to replace Ca2+ in the photosynthetic light reactions of these bacteria. The isolated PSIIcc showed the same reduction in oxygen-evolution as for biochemical Ca/Sr exchange when measured under continuous illumination, which was again traced back to a retardation of the S-state transitions. However, the oxygen evolution per flash was similar to Ca-containing samples indicating full activity. Moreover, the isolation of PSIIcc had to be performed in the presence of normal amounts of Ca2+-salt to avoid loss of activity. The finally obtained core complexes contained 1 Sr/4 Mn. This result has two interesting implications: (i) Apparently, the Sr2+-ion responsible for the restoration of oxygen-evolution activity is essentially nonexchangeable under the preparation conditions (i.e., in the presence of betaine protecting against the loss of extrinsic protein subunits) in contrast to other Sr2+-ions that might have been biosynthetically incoporated into PSIIcc. This result is principally in agreement with earlier biochemical work indicating that one slowly exchangeing Ca2+ per RC is necessary for water oxidation (157). Ädelroth et al. (237) found a dissociation half-time for this Ca2+ of 80 h in intact plant PSIIcc. Furthermore, Sr2+ might be bound more tightly to the WOC than Ca2+ (for reasons to be discussed in section 6). (ii) The loss of activity in media lacking Ca2+ then points to a structure-stabilizing role of other Ca2+-ions bound to PSIIcc such as Ca2+-PsbK and/or Ca2+-PsbO (see Figure 1 B, C). In the following, we shall refer to preparations from biosynthetically exchanged bacteria as Sr-PSIIcc.
An attempt was made to crystallize Sr-PSIIcc, but crystals diffracting to 3.9 Å were only obtained in the absence of betaine, resulting in a back-exchange of about 50 % of the Sr2+ to Ca2+ (252). Anomalous X-ray scattering around the Sr K-edge was applied to locate the Sr2+ ion in the structure, but radiation damage limited the datasets to a resolution of 6.5 Å. Nonetheless, an anomalous difference electron density map was reported (252) showing a peak attributed to Sr2+ at a distance of 1.88 Å and 1.65 Å, respectively, from the Ca2+ positions indicated in the crystal structures at 3.5 Å (12) and 3.0 Å (14) resolution.
Isotropic samples of Sr-PSIIcc were investigated with EXAFS in all amenable S-states at both the Mn and Sr K-edge (253). The Mn K-edge data confirmed that there are no major structural changes concerning Mn-Mn vectors in the S1 ® S2 transition. However, the data suggested a lengthening of one Mn-Mn distance in S3 from ~ 2.75 Å to ~ 2.85 Å that persisted until S0, but was shortened again in the S0 ® S1 transition. Deprotonation of a hydroxo- to an oxo-bridge was discussed as a possible origin of the latter effect. More significant were the observed changes in Sr K-edge EXAFS. There are two major peaks in the FT-amplitude spectrum assigned to Sr-O (peak I) and Sr-Mn (peak II) vectors. Peak II is relatively narrow in S1, broadens in S2, is split into two peaks in S3, and these two peaks change their relative amplitude upon going to S0. These data were interpreted as a movement of the Sr2+ ion (and by analogy also of the Ca2+ ion in native PSIIcc) relative to the Mn-ions during the catalytic cycle of water oxidation. On the basis of model II (or IIa, see Figure 8A) of Yano et al. (22), the oxidation of the central �4-oxo bridge (also called "�3-oxo" considering only the Mn ions (253)) during S2 ® S3 was proposed as a possible cause for the structural changes.
5.4. EPR/ENDOR spectroscopy
In order to simulate the EPR and ENDOR spectra of the WOC, one has to specify the spin-Hamiltonian, which contains among others contributions the isotropic exchange interaction J between unpaired electrons on different Mn ions; for details, see (134, 146, 176). The exchange interaction causes delocalization of spin density between Mn ions and, hence, influences the effective g- and hfc-matrices determining the spectra. Since J can be expected to depend on the interaction between Mn ions, i.e., the mode of �-oxo bridging, the EPR/ENDOR spectra contain, in principle, information about the structure of the WOC. However, this information is not easy to extract. Rather, independent structural information is used to constrain the parameters used in the spectral simulation. Not surprisingly, a number of J-coupling schemes for the WOC have been discussed in the literature (see (176) and references therein).
The coupling schemes can be divided into "dimer-of-dimers" models, in which the Mn4-part of the WOC is separated into two dimers of strongly antiferromagnetically coupled Mn ions, "trimer-monomer" or "3 + 1" models, in which one Mn ion (e.g., the "dangling manganese") is segregated and "tetramer" (T) models, in which there is no obvious separation of the Mn-cluster. Based on a tetramer model, Kulik et al. (176) attempted an assignment of oxidation states to individual Mn ions. They found suitable solutions only for the single MnIII-ion in the S2-state (cf. Figure 4C) being either Mn4 or Mn2 (also called MnC). Their analysis of the S0-state suggests that there is only a medium antiferromagnetic coupling between Mn4 and Mn3, which can be explained by the protonation of a �2-oxo bridge in accordance with interpretations of EXAFS-data (see subsections 5.2 and 5.3).
EPR/ENDOR spectroscopy has been performed also on oriented samples. Hasegawa et al. (145) studied the S2-state multiline signal with EPR on oriented PSII-membranes from spinach. They attributed the observed hfc-anisotropy to the single MnIII-ion in the S2-state and arrived at an estimate for the orientation of the 2.7 Å Mn-Mn vector relative to the membrane normal of q ³ 40�. This result is roughly in agreement with the EXAFS-models (Figure 8A) and the preferred redox-state assignment and coupling scheme of Kulik et al. (176), if we identify the MnIII-ion with Mn4. Teutloff et al. (177) investigated the S2-state with 55Mn-ENDOR on single crystals of dimeric PSIIcc from T. elongatus and suggested that the MnIII-ion is Mn2. Apparently, there is at present no unique interpretation of EPR/ENDOR spectra as regards the structure of the WOC, and further work needs to be done. Measurements on single crystals of monomeric PSIIcc might be helpful in the future, since the peculiar arrangement of Mn4Ca-clusters in this new crystal form (28) could facilitate the interpretation of spectra.
5.5. Ligand sphere of the Mn4Ca-cluster
5.5.1. Asp A170
The crystal structures clearly show that Asp A170 is in the vicinity of the Mn4Ca-cluster, but it remains unclear, whether its side chain ligates a metal ion and if so, which metal ion. The modeling at 2.9 Å resolution (15, 18) implies that Asp A170 could be a bridging ligand between the Ca2+ ion and Mn4 (Figures 7A, B), but likewise it could be a unidentate ligand of Mn4 only as suggested on the basis of earlier crystallographic studies (12, 14), the R-QM/MM-model (Figure 8B) of Sproviero et al. (233), DFT-based models of Siegbahn (211, 254-256), or EXAFS model III of Yano et al. (22). Another possibility is that it solely ligates the Ca2+ ion as suggested by model IIa (Figure 8A).
Asp A170 was among the first residues related to the WOC that were extensively studied by site-directed mutagenesis in the mesophilic cyanobacterium Synechocystis sp. PCC 6803 (257-260) or the unicellular green alga Chlamydomonas reinhardtii (261). A first critical test for the role of a residue in water oxidation is the ability of the mutated strain to grow photoautotrophically. Among the many mutations made at position A170, apparently, only the exchange to Glu, His and Val did not abolish the photoautotrophic growth (258, 259) with normal growth in the Glu case, but slow growth in the other two cases. The O2 evolution activity was 40 - 60% of wild type in these mutants. Other mutants could be cultured heterotrophically and then still biosynthesized PSIIcc, so that O2-evolution activities of 10 - 30% compared to wild type could be detected for Arg, Leu, Ile, Cys but none for Ala, Gly, Ser, Thr, Tyr, Pro and almost none (~ 5%) for Asn and Trp. In the O2-evolving mutants, a significant fraction of PSIIcc (20 - 60%) lacks the Mn4Ca cluster in vivo. These results demonstrate that Asp A170 is important for assembly and proper function of the WOC. However, the fact that Asp A170 can be replaced by non-ligating residues such as Val, Ile or Leu and oxygen is still evolved at 20 - 40 % of the wild type level is actually an argument against Asp A170 being an indispensable structural component of the assembled WOC. To uphold the idea that Asp A170 is a metal ligand, structural changes in the Val, Ile and Leu mutants have been invoked that would allow another residue or water molecule to act as an alternative ligand (259, 262).
To further deal with the ligation problem, the D(A170)H mutant was investigated by using EPR and ESEEM spectroscopy in X-band (263). The basic idea is that if Asp A170 is a ligand to Mn and as such is functionally replaced with His in the mutant, the hfc between one of the histidyl nitrogen nuclei and the unpaired electrons of a Mn ion should show up in spectra of the S1 or S2 states by analogy with His A332 (see subsection 5.5.3). However, no additional signal was detected. This result is in line with studies using FTIR spectrosopy (reviewed by Noguchi (264, 265)), which showed that all Sn+1-minus-Sn FTIR difference spectra (n = 0 - 2) of D(A170)H are essentially unchanged from that of wild type (262, 266, 267). In FTIR spectroscopy, one expects a frequency shift of characteristic vibrational modes of a carboxyl group, if it ligates a Mn ion that undergoes a change of its oxidation state or charge density during a Sn ® Sn+1 transition. The corresponding feature in the Sn+1-minus-Sn FTIR difference spectrum should vanish, if the carboxyl group is replaced with a histidyl group. As discussed by Debus (262), there are several explanations for the lack of any such spectral changes: (i) Asp A170 ligates a Mn ion that does not increase its charge or oxidation state during any of the S0 ® S1, S1 ® S2, or S2 ® S3 transitions. (ii) Charge delocalization in the Mn4Ca-cluster as suggested by RXES (see subsection 4.2) renders the carboxyl modes of ligating residues insensitive to oxidation state changes in the WOC. (iii) Asp A170 does not ligate the assembled Mn4Ca-cluster. (iv) Asp A170 in the wild type ligates a Mn ion, but His A170 in the mutant does not. This explanation would explain the lack of nitrogen hfc signals in the ESEEM experiments, but would work for the FTIR data only, if points (i) and/or (ii) apply. (v) Deprotonation events or changes in tautomeric equilibria compensate for the charge changes in the vicinity of Asp A170 due to oxidation of the WOC.
We would like to add two further points: (vi) Asp A170 ligates the Ca2+-ion. This possibility is suggested by model IIa of Yano et al. (22) when placed into the 2.9 Å resolution structure of Guskov et al. (15) and would explain the lack of signals in both FTIR and ESEEM spectroscopy. (vii) Asp A170 functions as a proton acceptor, possibly forming a hydrogen bond to either a terminal water ligand or a �-hydroxo bridge. Note that points (vi) and (vii) are not mutually exclusive. Also, a role of Asp A170 as proton transporter does not exclude the possibility of Mn-ligation as suggested by DFT-calculations of Siegbahn (256).
5.5.2. Glu A189
At 3.0 and 2.9 Å resolution (14, 15), Glu A189 is modeled as a bridging ligand between the Ca2+-ion and the Mn-ion that is also ligated by His A332 (Figures 7A, B). This motif has been adopted by Siegbahn in his DFT-model (254). In contrast, Glu A189 is a unidentate ligand to a Mn-ion only in the heterocubane-models (Figure 8B) by Ferreira et al. (12) and Sproviero et al. (233). The EXAFS-models suggest that Glu A189 could be a unidentate (model IIa, Figure 8A) or bidentate (model III) ligand to Ca2+.
Site-directed mutagenesis in Synechocystis sp. PCC 6803 revealed a situation of similar complexity as found for Asp A170 (see subsection 5.5.1). Only the mutations of Glu A189 to either Gln, Lys, Arg, Leu, or Ile do not abolish photoautotrophic growth (268). In all other cases, there is essentially no O2 evolution, although heterotrophically grown cells still synthesize PSIIcc with a significant amount of photooxidizable Mn ions. The fact that O2 is still evolved, when Glu A189 is replaced with the non-ligating residues Leu or Ile (40 - 60% of wild type), argues against a structure-stabilizing role of Mn-ligation by this residue. Consequently, the interaction of Glu A189 with a Mn-ion inferred from the electron density in this region has been proposed to be an artifact of the radiation-induced reduction of the Mn4Ca-cluster (262). It is also striking that the mutants with the highest O2-evolution are those in which the side chain of Glu A189 is exchanged to the large basic side chains of Lys (~ 80% activity) or Arg (~ 70%). The highly active Gln mutant (~ 70%) exhibits normal EPR multiline signals in S1 and S2 (268), as well as normal kinetics of O2 release and of electron transfer from YZ to PD1 and from the Mn4Ca-cluster to YZ during the S1 ® S2, S2 ® S3, and S3 ® S0 transitions (269). The Lys and Arg mutants still exhibit normal rates of electron transfer from YZ to PD1 and from the Mn4Ca-cluster to YZ during the S1 ® S2 and S2 ® S3 transitions (269).
Inspired by the crystallographic studies, two groups used FTIR difference spectroscopy to assess the ligation problem. Kimura et al. (270) detected effects of the E(A189)Q mutant, which they interpreted as evidence for a ligation of Mn by Glu A189. On the other hand, Strickler et al. (271) presented evidence that these changes arise from indirect structural perturbations and do not indicate loss of a metal-ligation. Instead, they demonstrated that like the D(A170)H-mutation, neither of mutations E(A189)Q or E(A189)R eliminates any carboxylate modes from any of the difference spectra pertaining to the S0 ® S1, S1 ® S2, or S2 ® S3 transitions. Again, there are several possibilities to explain these data: (i) Glu A189 does not ligate Mn, but only Ca2+. (ii) Glu A189 ligates a Mn-ion that does not change its oxidation state during the S0 ® S1, S1 ® S2, or S2 ® S3 transitions. (iii) The carboyxlate mode of Glu A189 is insensitive to oxidation state changes of the Mn4Ca-cluster, presumably because of charge delocalization.
5.5.3. His A332
His A332 is a ligand to the same Mn-ion as Glu A189 in all crystallographic (12, 14, 15) and DFT-based (233, 254) models (Figures 7B, 9B), but this ligation is more difficult to accomplish with the EXAFS-models (22) placed into the latest crystal structure (15) with Ne-Mn distances of 3.5 - 3.7 Å. Direct experimental evidence for Mn-ligation by at least one histidine was provided earlier on the basis of X-band ESEEM studies on PSIIcc preparations containing 15N-labeled histidine (272).
The importance of His A332 for water oxidation is evident from the fact that of the many mutants at position A332 (made in Synechocystis sp. PCC 6803), none is able to promote photoautotrophic growth (273). Only the mutants H(A332)Q and H(A332)S, when cultured heterotrophically, evolve O2, but only at 10 - 15% of the wild type rates. Among the non-O2-evolving mutants, H(A332)D and H(A332)E still biosynthesize large amounts of PSIIcc (273), which can be isolated for spectroscopic studies. An X-band ESEEM study of the H(A332)E mutant was interpreted as providing evidence that the histidyl ligand to Mn is indeed due to His A332 (274). The diminished amplitude of a characteristic histidyl nitrogen modulation in the mutant compared to wild type can be explained straightfowardly by the loss of metal-ligation in the mutant or the replacement of the ligating nitrogen with a glutamate oxygen. However, the possibility was not ruled out that His A332 does not ligate the Mn4Ca-cluster and that the mutation merely modifies another His-Mn interaction by structural perturbations. A possible second candidate for a histidyl ligand is His A337 (see subsection 5.5.5). We note that according to the crystal structure (15), His A332 and His A337 are close to each other with a distance between the Ne-atoms of ~ 4 Å, so that such a perturbation is conceivable. More recent ESEEM studies of 14N- and 15N-containing PSIIcc in Ka - and Q-band support the idea that there is at least one histidine ligating Mn, and the N-Mn distance was estimated to be in the range of 2.00 - 2.92 Å (172, 173).
FTIR experiments were conducted on spinach PSII-membranes containing 15N and Synechocystis PSIIcc with 15N-labeled histidine, and histidine signals were identified in S2-minus-S1 FTIR difference spectra (275). The vibrational signature indicated that this histidine is likely protonated at its Nd-atom (also called Np) and that this proton is involved in hydrogen bonding. Indeed, the crystal structure (15) suggests a hydrogen bond between Nd of His A332 and the backbone carbonyl of Glu A329. The measurable influence of the S1 ® S2 transition on the vibrations of this histidine was interpreted as a possible evidence for this residue to ligate the Mn4Ca-cluster (262, 275). This ligation then would be accomplished via the Ne-atom (also called Nt) in agreement with the crystal structures (12, 14, 15) and the DFT-based models (233, 254). A later study interpreted Sn+1-minus-Sn FTIR difference spectra (n = 0 - 2) as indicating that a single histidine residue coupled with structural changes of the WOC during the S-state cycle is responsible for the observed bands and that it is a Mn-ligand (276).
More recent mutagenesis experiments employ the thermophilic cyanobacterium T. elongatus (277, 278). The two mutants H(A332)Q and H(A332)S were constructed and characterized by different techniques (279). In striking contrast to the corresponding mutants of Synechocystis sp. PCC 6803, both T. elongatus mutants were able to grow photoautotrophically, and PSIIcc could be isolated with an O2-evolution capacity corresponding to ~ 80% of the wild type value. As in the case of Ca/Sr-exchange (see subsection 5.3), the decrease in activity was traced back to kinetic limitations, while absorbance difference spectroscopy indicated normal cycling through the S-states. Among the various experiments performed was an X-band ESEEM study of the H(A332)S-mutant revealing no significant difference to wild type. Consequently, it was conluded that the observed nitrogen signals do not arise from His A332 and that the effects of the H(A332)E mutant of Synechocystis originate from structural perturbations of another His-Mn interaction corresponding to the second possible interpretation considered by Debus et al. (274). So, there is spectroscopic evidence that a Mn in the WOC is ligated by a histidine, but no clear evidence that it is His A332.
5.5.4. Glu A333
In all crystallographic structures, Glu A333 is a ligand to Mn, either unidentate (12) or as a bidentate bridge (14, 15). Interestingly, it is also a bidentate bridge in the heterocubane R-QM/MM model (233) and Siegbahn�s DFT-model (254). In the EXAFS-models (22), placed into the 2.9 Å resolution structure, it is close enough to Mn to be a unidentate ligand to Mn3 (models IIa and III) or probably even a bidentate bridge between Mn3 and Mn4 (model IIa, Figure 8A). Mutations at position A333 have been constructed in Synechocystis sp. PCC 6803 (273, 280). Remarkably, only the E(A333)Q mutant supports photoautotrophic growth and shows O2-evolution at a non-negligible rate (20 - 30% of wild type). No FTIR results of a Glu A333 mutant have yet been reported. On the basis of this limited information, we conclude that Glu A333 is an indispensible structural component of the Mn4Ca-cluster, likely a metal ligand, and can only partly be replaced by Gln in its function.
5.5.5. His A337
His A337 is close to His A332 (see subsection 5.5.3.), but is not a ligand to any metal ion of the Mn4Ca-cluster in any crystal structure (12, 14, 15) or any DFT-based model (233, 254). However, the EXFAS-models (22) combined with the 2.9 Å resolution structure (15) suggest that it could be a ligand to Mn2 (Figure 8A) with the Ne-Mn distance of 2.8 - 3.0 Å being significantly shorter than that of His A332. At the mutagenesis side, the situation is again complicated: Phototrophic growth is supported only by Arg and weakly so by Gln, Asn, and Phe (273). O2 is evolved to a significant extent (> 5% of wild type) in the cases of Arg (~ 51 %), Gln (30 - 40%), Phe (~ 40%) as well as Glu, Asn, and Leu (10 - 20%). In all His A337 mutants, 10 - 50% of PSIIcc lack photooxidizable Mn ions in vivo (273). Therefore, His A337 has been considered as a possble Mn-ligand, but also as a possible hydrogen bond donor. To our knowledge, no spectroscopic study of any His A337 mutant has yet been reported. We note that according to the crystal structure (15), the Nd-atom of His A337 is likely engaged in hydrogen bonding to the backbone O of Glu A333. Thus, the observations made by Noguchi et al. (275) discussed in subsection 5.5.3. in the context of His A332 could likewise apply to His A337.
5.5.6. Asp A342
Asp A342, close to the C-terminus of PsbA, is suggested to be a ligand to Mn in all crystal structures and DFT-based models, either as bidentate bridge (14, 15, 254) or unidentate (12, 233). EXFAS-model IIa suggests a unidentate ligation to Mn2 (Figure 8A). Five site-directed mutations of Asp A342 in Synechocystis sp. PCC 6803 have been reported (262, 273). Among these, only D(A342)E supports photoautotrophic growth, and O2-evolution is possible in D(A342)E, D(A342)N, and D(A342)H at rates corresponding to ~ 20%, ~ 30%, and ~ 6%, respectively, of the wild type. No O2-evolution is possible, if Asp A342 is replaced with the non-ligating residues Ala or Val. Furthermore, 20 - 50 % of PSIIcc in theAsp A342 mutants lack photooxidizable Mn ions. This surprisingly clear situation strongly suggests a role for Asp A342 as Mn-ligand. However, Sn+1-minus-Sn FTIR difference spectra (n = 0 - 2) of the D(A342)N mutant are essentially identical to that of the wild type (281). This again suggests that either Asp A342 is not a Mn-ligand or it is a ligand, but insensitive to the oxidation of the Mn4Ca-cluster.
5.5.7. C-terminal Ala A344
Ala A344 is unique among the ligands to the Mn4Ca-cluster as it has no ligating side chain, but uses the C-terminal carboxyl group of PsbA instead. In most organisms, PsbA (D1) is biosynthesized as a precursor protein (termed pD1) with a C-terminal extension that has to be cleaved to allow for a functional assembly of the WOC (282-284). In the 3.5 Å resolution structure (12), the C-terminal residue of PsbA is located close to the Ca2+-ion. Consequently, it was considered as ligand to Ca2+ (Figure 8C) in the R-QM/MM-model (233). At 3.0 and 2.9 Å resolution (14, 15), the C-terminus of PsbA adopts a different conformation, so that a bridging, bidentate ligation of Mn and Ca2+ becomes possible (Figures 7A, B, C). The EXAFS-models suggest a close proximity of the C-terminal carboxyl group to Mn1 (Figure 8A), and Siegbahn models Ala A344 as a unidentate ligand to Mn (254).
In contrast to the Asp and Glu residues discussed above, FTIR difference spectroscopy provided clear evidence for a close interaction of Ala A344 with the Mn4Ca-cluster. To this end, Sn+1-minus-Sn FTIR difference spectra were recorded of PSIIcc from Synechocystis sp. PCC 6803 either unlabeled or labeled with L-(1-13C)-alanine in two independent studies (285, 286). A band characteristic of a symmetric carboxylate stretching vibration was found to be downshifted during the S1 ® S2 transition and restored during S3 ® S0. The data were interpreted in terms of Ala A344 being a unidentate ligand to a Mn-ion that is oxidized (or whose charge increases) during S1 ® S2.
The publication of the 3.5 Å resolution structure (12) triggered a re-investigation of the possibility that Ala A344 ligates Ca2+. Inspired by the work of Boussac et al. (251), cells of Synechocystis sp. PCC 6803 were cultured in a medium containing SrCl2 instead of the normally used CaCl2 to achieve biosynthetic Ca/Sr exchange (287). In addition, cells were labeled with L-(1-13C)-alanine. The data demonstrated that several symmetric and asymmetric carboxylate stretching modes, that show up in Sn+1-minus-Sn FTIR difference spectra (n = 0 - 2), are affected by Ca/Sr exchange, but not that of the C-terminal carboxyl group of Ala A344. This was taken as evidence that Ala A344 does not ligate Ca2+. Also, the modes that are perturbed by Ca/Sr exchange are neither due to Asp A170 nor Glu A189 or Asp A342, as these residues are insensitive to the investigated S-state changes (see above). This implies that any changes of carboxylate modes of Asp A170 or Glu A189 due to Ca/Sr exchange, that would be expected, if any of these two residues ligated Ca2+, would likely escape detection, so that a Ca2+-ligation by these groups can not be ruled out.
More recently, 13C-ENDOR spectroscopy was applied to the S2-state of L-(1-13C)-alanine labeled PSIIcc from Synechocystis sp. PCC 6803, and a strong magnetic interaction of a 13C-nucleus with unpaired electrons of the WOC was detected (288). This is further clear evidence that Ala A344 ligates a Mn-ion.
5.5.8. Glu C354
The only potential ligand to the Mn4Ca-cluster that is not from PsbA is Glu C354. It is suggested to be a unidentate ligand to a Mn-ion (Figure 8B) in the heterocubane structures (12, 233), but a bidentate, bridging ligand between two Mn-ions in the 3.0 and 2.9 Å structures (14, 15) as well as in the DFT-model of Siegbahn (254). In the EXAFS-models, it is at a position, which would allow for a bridging ligation between Mn1 and Mn2 (Figure 8A) according to models IIa and III or between Mn2 and Mn3 (see Figure 4 in (18)) according to model IIa.
So far, only the mutant E(C354)Q has been constructed in Synechocystis sp. PCC 6803 and investigated spectroscopically (289-291). According to an earlier study, this mutant does not support photoautotrophic growth, evolves no O2, and is not even able to accumulate PsbC (CP43) in the thylakoid membrane (289). The more recent studies report different phenotypes. Strickler et al. (290) were able to isolate PSIIcc from thylakoid membranes of their E(C354)Q mutant that evolved O2 at 10 - 18% compared to wild type. The mutant PSIIcc was able to normally cycle through the S-states, but apparently only in a small fraction of centers. However, about 75% of centers were able to reach the S2-state upon flash illumination, but were unable to advance beyond the S2- or S3-states. The S2-minus-S1 FTIR difference spectrum indicated a significant effect of the E(C354)Q mutation on a number of modes in the mid-frequency region. The data were taken as evidence that Glu C354 strongly interacts with the Mn4Ca-cluster. One possible interpretation suggested by Strickler et al. (290) is that Glu C354 is a bridging ligand between two metal ions in the S1-state, but changes to a unidentate ligand to a single metal ion in the S2-state. However, other interpretations are possible due to the complexity of spectral changes induced by the mutation, so that further work is necessary to arrive at a better understanding of structural and functional implications.
Shimada et al. (291), in an independent study, were also able to isolate PSIIcc from the E(C354)Q mutant and found 21% O2-evolution activity. Their data indicated a slightly lower efficiency of the S2 ® S3 transition compared to wild type and significantly retarded transitions beyond the S3-state. The S2-minus-S1 FTIR difference spectrum of Shimada et al. is similar to that of Strickler et al., but Shimada et al. arrived at a different interpretation: They proposed that Glu C354 changes from a bridging ligand between two Mn-ions in the S1-state to a chelating bidentate ligand to a single Mn-ion in the S2-state. They also reported a S3-minus-S2 FTIR difference spectrum and concluded that none of the modes affected by the S2 ® S3 transition is due to Glu C354. This likely implies that the mode of ligation of a Mn-ion by Glu C354 is not changed during S2 ® S3 and that this Mn-ion does not change its oxidation state in this transition. Shimada et al. also reported evidence for an effect of the E(C354)Q mutation on vibrations of a water molecule.
6. EDUCT AND PRODUCT CHANNELS
6.1. Channel proposals
The crystal structures show that the WOC is buried within the protein (Figure 1) suggesting that there are channels within the protein matrix that allow for the delivery of substrate water (the educt) to the Mn4Ca-cluster as well as the removal of protons and dioxygen (the products). Even prior to the crystal structures, the existence of such channels was proposed; for a recent review, see (292). One line of argumentation originates from earlier efforts to identify a possible metal peroxide intermediate in the reaction cycle of the WOC. It was recognized that certain treatments of plant PSIIcc, involving inter alia depletion of extrinsic subunits (293) or Cl- (294, 295), stimulate the formation of hydrogen peroxide (H2O2) instead of O2. Besides singlet oxygen, H2O2 is among the reactive oxygen species (ROS) that cause photo-oxidative damage of PSIIcc (296-298) and might be responsible for the requirement of a fast PsbA turnover (for reviews on photoinhibition, see (299-302)). The apparent correlation of H2O2 formation with an O2-production deficit led Wydrzynski et al. (303) to formulate the "water accessibility hypothesis". According to this hypothesis, the accessibility of water to the WOC is controlled by the protein matrix and determines the reaction path of water oxidation. Depletion of extrinsic subunits or Cl- disturbs the structural integrity of this protein matrix and leads to an uncontrolled water access to the WOC, which opens alternative reaction paths that result in an incomplete water oxidation with H2O2 as the product.
A second line of evidence has its roots in studies with substrate water analogs. Radmer and Ollinger (304) investigated the activity of substituted hydroxylamines as reductants of the WOC and interpreted the observed dependence of the activity on the size of the reductant molecule as evidence that the substrate water binding sites reside in a cleft. Later work confirmed the size dependence and showed that the inhibitory effect of the reductants is diminished by the presence of extrinsic subunits (243) as well as Cl- (243, 305) or Ca2+ (157, 242, 244), suggesting an influence of these components of PSIIcc on the integrity and/or width of inlet channels or on the accessibility of the Mn ions. Force et al. (306) studied the interaction of deuterated alcohols with the WOC by using ESEEM spectrosocopy to detect alkyl deuterons in the vicinity of Mn. They found evidence that the smaller alcohols methanol and ethanol and to a certain extent the slim n-propanol bind near the Mn4Ca cluster, but the bulkier 2-propanol and dimethyl sulfoxide (DMSO) do not. On the basis of these data, they considered the existence of an access channel of limited size.
Complementing the water accessibility hypothesis, an "oxygen accessibility hypothesis" has been formulated (307, 308). According to this hypothesis, O2 formed by the Mn4Ca-cluster is directed into the lumen by a specific pathway whose purpose is to prevent the PSII-RC from coming into contact with O2. The reason, why such a contact has to be avoided, is the occurrence of charge recombination reactions in the RC. Since the quinone exchange reactions in the RC are slow compared to EET and primary ET, it may happen under high-light conditions that the foward ET is blocked, e.g., QA reduced and the QB-site empty. Under these circumstances, the radical pair PPheo is still formed and lives long enough to be to a certain extent converted from its original singlet form 1(PPheo) to its triplet form 3(PPheo). The latter recombines to a Chl triplet state 3Chl, which is likely localized on ChlD1 and PD1 at ambient temperatures (37). Neither 3ChlD1 nor 3PD1 can be quenched by carotenoids, as these are too far away for triplet - triplet energy transfer (Figure 2). However, there is evidence that the radical anion Q
is able to act as triplet quencher (309, 310). Nonetheless, 3Chl formation in the RC could promote photosensitized production of singlet oxygen (311). As this process requires contact between 3Chl in the RC with the triplet oxygen produced by the WOC, rapid egress of O2 into the lumen would avoid singlet oxygen formation. Since singlet oxygen is the major ROS being responsible for photo-oxidative damage (297, 298), a special O2-egress channel would contribute to the protection of PSIIcc.
Another aspect is the possible reversibility of the last, O2-producing step of water oxidation, i.e., (S4) ® S0 + O2 + n H+, to be further discussed in subsection 10.3. Clausen et al. (312, 313) reported evidence that this step has a low driving force and, hence, can be suppressed or reversed at increased O2-pressure. The significance and implications of this finding were debated in the literature (314-318). The important point in the present context is that reversibility of the last step would necessitate efficient removal of the product O2 from the catalytic site and protection of this site against the atmospheric O2-pressure. Both tasks could be perfomed by a specially designed transport channel, which directs O2 outward, while the surrounding protein matrix prevents O2 diffusion towards the Mn4Ca cluster. In other words, the protein has to be "O2-proof", but at the same time, has to act as an O2-outlet valve. As suggested by Renger (102), an oxygen channel could help to prevent a reversal of electron transport that would turn the WOC in an "oxidase reaction mode".
Like an electron, the product "H+" of water oxidation is an elementary particle and no molecule, so that it needs a carrier molecule rather than a channel. Therefore, it is more appropriate to talk about "H+ exit pathways". On the other hand, these pathways are likely associated with channels, in which water molecules are placed. The widely accepted mechanism of proton transfer along water chains is that one proton "hops" from a water molecule to the next, then another proton from the second water molecule to the third and so forth. This is usually called the "Grotthuss mechanism", although it differs somewhat from de Grotthuss� original ideas (319, 320). Illustrations of this mechanism, including also protonatable amino acid side chains that contribute to proton transfer in proteins, can be found in earlier reviews (292, 321-323). The Grotthuss mechanism requires the involved water molecules and amino acid side chains to be arranged in an optimal way to promote proton transfer. Moreover, the side chains have to have suitable pKa values to provide the driving force for directed proton transport (324). Also, the inscribed water molecules may have a tuned pKa due to their interaction with the protein. These arrangements of proton carriers have to work under conditions of varying electric field (e.g., charge accumulation in the Mn4Ca cluster, see subsection 4.6) and against the proton activity in the lumen, which is increased by the action of the WOC itself. It is, therefore, beyond dispute that specific H+ exit pathways exist in PSIIcc.
6.2. Channel calculations
With the appearance of more detailed crystal structures, attempts have been made to identify possible educt and product channels of water oxidation by structure-based calculations. Different methods have been applied to the structures at 3.5 Å (325), 3.0 Å (324, 326, 327), and 2.9 Å (15, 200) resolution. Methods and work up to 2008 have been reviewed (292) and a brief comparison of this work with studies at 2.9 Å resolution given (18). In the following, we concentrate on the most recent work applying cavity search algorithms (200) and molecular dynamics (MD) simulations (327). Proton exit is further discussed in section 9.
In the cavity search algorithm applied, routes that a (spherical) particle of a given size can take from the interior of the protein to the bulk solvent are identified (328). For application to PSIIcc, the Mn, Ca2+ and Cl- ions are removed, and possible trajectories for a sphere of specified radius starting from the WOC-site are evaluated (15, 325). A drawback of this method is that calculations are carried out on a static crystal structure and protein dynamics such as breathing motions that might be of importance for molecule transport through channels (292) are not considered. Therefore, the cavity search algorithm only allows for an initial characterization of possible channels. The most recent results obtained in this way on the basis of the 2.9 Å resolution structure (200) are summarized in Figure 9. Nine channels were identified and classified according to their width, length and hydrophobicity. The wider channels A1, A2, B1 and B2 were assigned to possible molecule (H2O, O2) transport channels, whereas the narrower channels C - G (yellow) were considered too tight for molecule transport and assigned to possible proton exit pathways (see the discussion below). The channels C - G form a complex network merging into three different exits to the lumen (Figure 9B), but only into two channels approaching the Mn4Ca-cluster (Figure 9C). To demonstrate the principle ability of channels C - G to act as proton exit pathways, Gabdulkhakov et al. (200) modeled water molecules into these channels. One water molecule is situated between Mn4 and Cl-, which is also "seen" as a blob of electron density in the 2.9 Å structure (Figure 7D). However, it is not clear from this analysis, whether all of these channels are really proton exit pathways. Moreover, there might be additional pathways that can not be traced by the cavity search algorithm simply because they involve mainly amino acid side chains or water molecules situated in cavities that are not connected by a trajectory with the WOC-site. Finally, it should be recalled that the calculations were performed on one PSIIcc-monomer. In the PSIIcc-dimer, the opening of channels CD, which points towards the monomer-monomer interface, is largely blocked by the second dimer half. This is not the case for the other channels.
The wider channels A1, A2, B1 and B2 all originate from one region on the other side of the WOC and allow access to Ca2+, Mn1 and Mn2. Channels A1 and A2 were tentatively assigned to water inlet channels and B1 and B2 to O2 egress channels (200), but this assignment is merely based on the assumption that O2 transport requires a broader channel profil. Such a distinction is difficult on the basis of a static structure and given the coordinate error of about 0.26 Å. The channel B2 is actually blocked by Lys U134, but it could be demonstrated that a conformational change of this residue would open it (200).
Channels A2 and B1 are the most suitable candidates for entry paths allowing access of small molecules to the WOC. Their limited diameters provide a straightfoward explanation for the results of Force et al. (306) concerning the interaction of deuterated alcohols of different size with the Mn4Ca-cluster (see subsection 6.1). Removal of Ca2+ would open an additional path from the wider channels to Mn4 and thus likely increase the susceptibility of the WOC against reductants in accordance with experimental observations (157). These channels could also provide size-limited pathways for Ca2+ exchange, which is slow in intact PSIIcc (237). The larger Sr2+ would probably be even slower, if not arrested, providing an explanation for the apparent inability to exchange biosynthetically incorporated Sr2+ (see subsection 5.3). We note that the channels in the vicinity of the WOC are determined by conserved protein subunits and thus are likely similar in cyanobacterial and plant PSIIcc.
To obtain a more realistic picture of possible water flow in PSIIcc, Vassiliev et al. (327) performed MD simulations based on the 3.0 Å structure (14) supplemented with more than 900 buried water molecules and the quantum chemical WOC-model of Sproviero et al. (329). Note that this model contains a Cl- ion as ligand to Ca2+, which may have an influence on charge distribution and dynamics. The ingenious idea of Vassiliev et al. was to employ mathematical techniques originating from fluid dynamics and used in magnetic resonance imaging to analyse water tracts in tissues (330). They applied these techniques to information on water diffusion in PSIIcc obtained from the MD simulation in order to identify highly anisotropic motions called streamlines. Water streamlines were found in spaces free of protein backbone atoms and in most cases also free of heavy atoms of amino acid side chains. These streamlines are generally located in the same regions as the channels suggested by analyses based on the static structure, indicating that the latter are a reasonable first approximation. However, there are also streamlines in spaces transiently occupied by amino acid side chains, demonstrating that side chain movements can promote water diffusion through regions, which from the viewpoint of the cavity search algorithms appear "waterproof". Also, thermal motions of the protein can open and close channels.
One major streamline region connects the "calcium side" of the WOC via channels A1 and A2 with the protein surface close to the membrane, supporting the idea that these are water inlet channels. The streamlines appear broader than the channels, indicating that water can leak through the channel walls because of thermal motion. These differences point to the limits of analyses based on static structures. Details of how the streamlines approach the Mn4Ca-cluster may depend on the WOC-model used and are difficult to assess in the absence of a clear-cut structure. Therefore, the significance of differences between streamlines and channels in this region remain unclear. A second major streamline region is associated with channels C and G. Interestingly, these streamlines do not reach the protein surface, but seem to be blocked by narrow regions of channels C, D, and G (although in the case of channel G not at the narrowest pass). In particular, there are no streamlines in channel D and in channel C beyond the narrow pass at Glu A65. This residue is likely of importance for proton transfer as discussed in section 9. In contrast, there is significant water flow in the major part of channel G and in regions around channel G and the WOC. It is important to note that the Cl- ion is lacking in the calculations based on the 3.0 Å structure. As hypothesized earlier (200), the Cl- in this position could block water flow. In any case, the channels C, D, and G appear to be less suitable for water inlet than the region around channels A1 and A2.
The MD simulations showed an abundance of mobile water molecules around channels B1 and B2 with several entry possibilities. One entry coincides with the mouth of channel B2, but there are also other paths due to protein dynamics. Interestingly, there is no significant water stream in the opening region of the widest channel, B1. In contrast to the suggestions based on cavity analysis, there is a significant water migration in the EF channel system reaching the WOC region. In addition, there are shortcuts between the EF and B2 channels allowing water to bypass the barrier made up in the B2 channel by the salt-bridge between Lys U134 and Val V79.
To summarize, we present an updated working model of educt and product channels in Figure 10, merging the ideas from ref (200) and (327). According to this model, there are two major pathways for water inlet approaching the WOC from different sides. The details of how the water streams make contact with the Mn4Ca-cluster can not be fixed at present due to the lack of a reliable structure in this region. The main entry points are around the mouths of channels A1, A2, B2, and EF. Although, there might be additional water motions in the inner parts of channels B1 and B2, we hypothesize channel B1 to be a likely O2 egress pathway in agreement with earlier suggestions (200, 326). Channels CD are the prototype H+ exit pathways, but there might by additional paths not associated with them. The G channel is indicated as an additional proton path, but water motion in this region can not be ruled out depending on the effect of the Cl- ion on the dynamics.
6.3. Noble gas pressurization
To identify possible hydrophobic pathways for dioxygen transfer, PSIIcc crystals have been investigated with X-ray diffraction that were derivatized with Xe (15, 200, 331) or Kr (200) under pressure. Noble gas pressurization is a well-known tool in X-ray crystallography to explore hydrophobic sites (332, 333) and especially to track O2 transport channels (334, 335) in proteins. The idea behind Xe derivatization is that the Xe atom has a van-der-Waals radius of 2.16 Å (336) similar to that of the O2 molecule (2.13 Å along the O=O bond) and is likewise nonpolar. Therefore, Xe should prefer the same environment as O2, but can be detected by means of anomalous X-ray scattering (cf. subsection 5.1). However, the Xe-O2-analogy is limited by two facts: (i) O2 is not spherical, but has a smaller effective radius of 1.5 - 1.7 Å normal to the double bond axis (336). Hence, it could in principle enter channels from which Xe is excluded. (ii) Binding of atoms and nonpolar molecules to proteins depends not only on size, but also on polarizability (332, 333). As Xe has a significantly higher polarizability (4.04 Å3 (337)) than O2 (1.58 Å3 (338)), it may prefer different binding niches. Moreover, there is recent evidence that O2 may also bind to a polar site (339). In view of these facts, it is actually not surprising that no Xe atom was found in any of the proposed channels (15, 200, 331). Instead, Xe atoms mostly occupied niches amongst the lipid acyl chains in the membrane spanning part of PSIIcc. These sites were interpreted in terms of hypothetical pathways allowing O2 to escape from the lumen via the membrane phase (200).
The pressurization experiments were repeated with Kr with the hope that the smaller radius of this atom (2.02 Å (336)) would allow for a tracking of channels (200). Despite the smaller anomalous signal of Kr, 23 binding sites could be identified in the PSIIcc dimer (11 per monomer and one on the C2-axis). Whereas most Kr atoms again occupied the lipid phase of PSIIcc, one was found in channel B1 (close to Phe C419) and another one in the inner part of channel B2 (close to Ile V71). In addition, PSIIcc was cocrystallized with DMSO, and two DMSO molecules were found in these channels close to the Kr sites (200). These data provide the first crystallographic evidence that channel B1 is accessible to small molecules and a likely pathway to approach the Mn4Ca cluster or to egress.
7. CHLORIDE BINDING SITES
The requirement of Cl- for activity of the WOC is known for quite some time and has been studied extensively; for reviews, see (157, 340). The earlier studies resulted in two general hypotheses on the role of Cl- in water oxidation: (i) Cl- ions are ligands to Mn, either as a bridging ligand (341, 342) or as an exchangeable anion helping to maintain charge neutrality during the S-state cycle (343). The idea of manganese ligation by chloride has been taken up in recent theoretical studies (344). Since the identification of calcium as a constituent of the WOC, a variant of the charge neutralization hypothesis has been suggested, in which Cl- is a ligand to Ca2+ (232, 329, 345). The presently available crystallographic information including native (15) (see above) as well as bromide-substituted PSIIcc (15, 346, 347) (see below) provides no evidence for chloride ligating any metal ion of the WOC. (ii) Cl- ions are bound to specific sites in the protein matrix close to the WOC and serve to facilitate deprotonation of water (348-350). The latter hypothesis was also incorporated into theoretical work (212, 256) and is more in line with the recent crystallographic studies (15, 346, 347).
The evidence from the earlier biochemical work for a physiological role of chloride in water oxidation was questioned, because the methods used for Cl- depletion at that time were suspected to cause irreversible changes in the properties of PSIIcc (351). The seminal work of Andréasson and co-workers provided a clearer picture (350, 352-354). They labeled spinach PSII membranes with radioactive 36Cl- and applied extensive dialysis for Cl- depletion to preserve the integrity of the protein matrix. In this way, they could demonstrate the following: (i) There is about one slowly exchanging Cl- per WOC and no non-exchangeable Cl- bound. (ii) Cl- is needed to establish full water oxidation activity, but 35 - 55 % oxygen evolution is still obtained after Cl- depletion by dialysis. (iii) The reduced activity can be traced back to a retardation of the S-state transitions reminiscent of the effect of Ca/Sr-exchange (see subsection 5.3), that is, full S-state cycling is possible in the absence of Cl- (see also (355)). (iv) Oxygen evolution activity can be immediately (< 15 s) restored to 85 - 90% of the original value by addition of high concentrations of Cl- corresponding to a dissociation constant of Kd = 0.5 - 0.8 mM (depending on whether sucrose or glycerol is present in the buffer), but is rapidly lost in Cl--free media. Long-term incubation in Cl- results in a conversion to slow Cl- exchange behavior (t1/2 = 1 h) characterized by a dissociation constant of Kd = 13 - 20 �M. The interconversion takes place on a time scale of minutes. These results have been interpreted in terms of a "one-site two-state" model (354), in which there is one Cl- binding site close to the WOC that can exist in an "open" conformation (O-state) characterized by low affinity and rapid exchange and after Cl- binding is transformed into a "closed" conformation (C-state) characterized by high affinity and slow exchange. Alternatively, one may consider a "two-site" model (355), in which one site is weakly binding and rapidly exchanging, while the other is tightly binding and only slowly exchanging. In this case, the interconversion between the two forms would likely occur by diffusion of Cl- through the protein rather than dissociation and rebinding, since the interconversion is faster than the exchange at the high-affinity site. (v) In accordance with earlier studies (119, 356), Cl- is found to be required for the formation of the S2-state multiline signal at g = 2.0 (see subsection 4.3). In fact, binding of Cl- in both modes (O- and C-state) stabilizes the multiline signal, whereas Cl- depletion shifts the equilibrium towards the g = 4.1 signal (350, 354, 355). This result seems to indicate that in both modes Cl- binds to the same site, in which it influences the magnetic properties of the WOC in a specific manner, in line with the one-site two-state model. On the other hand, the two-site model would require to postulate that Cl- binding to both sites affects the spin system in the WOC in the same way. (vi) The slowly exchanging Cl- binding was found to depend on pH with an apparent pKa of ~ 7.5 (353). Earlier work has indicated the presence of lysine residues in PSIIcc with an anomalously low pKa near 7.8 (357-359). So, it was suggested that Cl- may participate in a structure involving such a lysine (350). We note that all these experiments have been performed on plant thylakoid membranes. The situation is less clear in the case of cyanobacterial PSIIcc. In the following, we shall first discuss these data on the basis of the single "native" Cl- binding site identified in the 2.9 Å resolution structure (15) and later turn to the possibility of additional halide binding sites following from work on Br--substituted PSIIcc (346, 347). For consistency, we shall refer to the former Cl- site as "Cl1-site".
Assuming that the Cl- binding behavior observed in plant PSIIcc resembles that of cyanobacterial PSIIcc, it is tempting to assign the Cl1-site to the slowly exchanging high-affinity Cl- binding site. There are three aspects that support such an assignment: (i) Cl1 is bound to Lys D317 (Figure 7). According to the electrostatic calculations performed by Ishikita et al. (324) on the basis of the 3.0 Å resolution structure, this lysine has a pKa value of 1.5 - 9.6 in the absence of Cl- depending on the charge distribution assigned to the Mn4Ca cluster. Thus, it likely falls into the category of low pKa lysines and its deprotonation could significantly destabilize Cl- binding. (ii) Cl1 resides deeply buried in the protein, and chloride exchange requires a channel. A possible candidate is channel G. The MD simulations of Vassiliev et al. (327) show that channel G is rather flexible in the absence of Cl- allowing for some water flow that could support fast chloride transport. It would be interesting to repeat these calculations with chloride bound to the Cl1-site in order to reveal a possible influence on the protein dynamics that could result in a retardation of water and chloride transport. Also, an evaluation of Cl--induced pKa shifts of nearby residues and concomitant structural changes would be important to develop a molecular basis of the one-site two-state model. (iii) Cl1 is bound to the backbone of Glu A333, which is a ligand to Mn, and a water molecule in contact with Mn (Figure 7D). Removal of chloride from this position would change the charge distribution in this region and also likely cause structural alterations of at least Mn4 and its environment. These changes could be responsible for the shift from the g = 2.0 multiline to the g = 4.1 signal in the S2-state and also cause a retardation of S-state cycling without a complete loss of water oxidation activity.
We now turn to the more complicated picture emerging from studies on bromide-substituted PSIIcc. A number of anions has been tested for the ability to compete with 36Cl- for the slowly exchanging chloride binding site in plant PSIIcc (353). Among the competing anions, only Br-, NO3-, and I- support an oscillation of the S2 multiline signal under a series of laser flashes. The oscillation pattern is somewhat weaker in the case of NO3- and I-, whereas Br- shows activation properties very similar to Cl- (350). As an alternative to the chemical exchange procedure, Boussac and co-workers (360) developed a biosynthetic method for Cl/Br-exchange in cyanobacterial PSIIcc related in spirit to the Ca/Sr-exchange technique (see subsection 5.3) and allowing also for a combination of the two replacements. They confirmed that the ion replacements resulted in a fully intact but kinetically limited WOC and showed that the effects of the Ca2+/Sr2+ and Cl-/Br- exchanges were additive. These data clearly indicate the importance of both, Ca2+ and Cl-, for optimizing the photosynthetic water oxidation.
The possibility to replace Cl- with functionally competent Br- paved the way to employ X-ray techniques for a further study of the anion under physiologically relevant conditions. XAS studies at the Br K-edge in Cl- depleted and Br- substituted PSII membranes from spinach excluded the presence of metal ions in the first and second coordination sphere of Br- (361). EXAFS analysis provided tentative evidence of at least one metal ion at a distance of about 5 Å from Br- and a Br-O/N distance of ~ 3.3 Å (361), which would be in reasonable agreement with a bromide ion occupying the Cl1-site (henceforth referred to as "Br1"). X-ray crystallography exploiting the anomalous dispersion of Br- was applied by three independent groups resulting in partially conflicting results: The first publication was by Murray et al. (346), who employed both, chemical (Br-infiltrated) and biosynthetical (Br-physiologically) exchange to PSIIcc from T. elongatus and found two bromide sites, Br1 and Br2, close to the WOC. This result was confirmed shortly thereafter by Kawakami et al. (347) based on chemical Cl/Br exchange in PSIIcc from T. vulcanus (Figure 11B). The Br1-site is identical to Cl1. The anion in Br2 is ligated by the backbone NH-groups of Glu C354 (likely ligating Mn, see above) as well as Asn A338 and might be stabilized by interaction with the OH-dipole of Thr C355. In contrast, Guskov et al. (15) used biosynthetical exchange with PSIIcc from T. elongatus and found clear evidence for Br- in the difference density map only for the Br1-site as shown explicitly in Figure 11A. On the basis of these data and the 2.9 Å resolution structure, the presence of a second Cl- binding site in native PSIIcc was questioned (18). However, irrespective of the existence of a Cl2-site, the conflicting data obtained with bromide-substituted PSIIcc hint at the possibility of several halide binding sites that are differentially occupied under various conditions. The challenge for future research will be to find out, which factors control the occupation of the sites and which sites are of functional relevance.
8. REDOX ACTIVE TYROSINES
It is known for a long time that there is an ET intermediate between the WOC and the RC of PSIIcc (362) that retains an unpaired electron after being oxidized, giving rise to a signal detectable by EPR techniques (118). The signal has the characteristics of an organic radical and has been identified as a tyrosine radical on the basis of isotopic labelling experiments (103). The matter is complicated by the fact that there are two redox active tyrosines in PSIIcc, YZ and YD, which are located at symmetry-related positions close to the RC (Figure 2, for a review, see (363)). Only YZ (Tyr A161) is situated between PD1 and the WOC (Figure 12), whereas YD (Tyr D160) sits at a significant distance to the latter.
8.1. YZ
The identity of Tyr A161 with YZ was demonstrated by site-directed mutagenesis (364-366) long before its location in the vicinity of the Mn4Ca cluster became apparent from the crystal structures. Besides the role of YZ as the intermediate electron acceptor between the WOC and PD1, other functions have been attributed to it such as being a hydrogen abstractor (367), an electrostatic promoter of water splitting (368) or involved in proton release from the WOC (369). At present, these additional roles can not be settled. Here, we focus on the effect of hydrogen bonding between Tyr A161 and His A190.
In aqueous solution, the mid-point potential Em of the phenolic side chain of tyrosine depends on pH (370, 371), since the group is deprotonated with different pKa values pKred and pKox in the reduced and oxidized form, respectively (Figure 12A). The oxidation of the neutral protonated tyrosine YH to the neutral deprotonated tyrosine radical Y· can proceed via two principle routes involving either the anion Y- or the radical cation YH· + depending on whether deprotonation or oxidation occurs first. The free energy differences for the four individual processes are determined by the redox mid-point potentials Em(YH· +/YH) and Em(Y· /Y-) as well as the two dissociation constants Kred and Kox and are related to each other by the thermodynamic cycle depicted in Figure 12A. This cycle implies that if, e.g., Em(YH· +/YH), Kred, and Kox are given, Em(Y· /Y-) is uniquely determined. The pH-dependence of Em is shown in the Pourbaix diagram, i.e., a potential-pH-plot (372), in Figure 12B (black curve) and is given by the Nernst-Clark equation:
(1)
valid at room temperature. To obtain the behavior of the tyrosine side chain in aqueous solution, reactions of the backbone amino and carboxy groups have to be avoided. Therefore, measurements have been performed on the model compound N-acetyl-L-tyrosinamid resulting in Em(Y· /Y-) = +650 mV and pKred = 9.9 (373). The value of pKox is fairly low and more difficult to measure, but can be estimated on the basis of EPR experiments on phenol radical cations in aqueous sulphuric acid to be ~ -2 (374). The value of Em(YH· +/YH) is then fixed to about +1.38 V, which is higher than the estimated potential of PD1 (36). But what is the actual redox mid-point potential of YZ in PSIIcc?
The protein environment can have a strong influence on Em of YZ, in particular, a hydrogen bond to a nearby base B such as His A190 (Figures 7A, 12C). To describe such a scenario, we have to consider at least eight states: four with reduced ("red") and four with oxidized ("ox") YZ. The four states in each case are with both YZ and B deprotonated, both protonated as well as only YZ or only B protonated. For simplicity, we assume His A190 to be protonatable only at Ne, while the proton at Nd is engaged in a stable hydrogen bond to Asn A298 (Figure 12C), and neglect the influence of other titratable groups. First, we consider the pH-dependent population of the four states pertaining to reduced YZ that are shown in the thermodynamic cycle of Figure 13A. Within this minimal model, the Tyr A161/His A190/Asn A298 system is described formally as a diprotic acid. YZ has two different pKa values (
and 
) depending on the protonation state of B and vice versa (
and 
; we dropped the index "red" in Figure 13 for simplicity). A titration curve of this system would exhibit two steps determined by the lowest and the highest of the four pKa values. Between these two pKa values, one of the titratable protons remains in the system and is distributed between the two moieties as determined by the free energy difference for proton transfer (PT) from B to Y, 
with 
. That is, Y is protonated for 
, and B is protonated for 
. These two cases are shown in Figures 13 B and C, respectively, where the mole fractions X of the individual states are plotted as a function of pH.
Experimental information on the protonation state of YZ may be obtained from spectroscopy (363, 371). While data from UV/VIS spectroscopy remained inconclusive, FTIR measurements argue strongly that YZ is protonated at pH 6 (375), implying . Very recently, Rappaport et al. (376) investigated PSIIcc, in which 3-fluorotyrosine (3F-Y) was incorporated biosynthetically. In aqueous solution, the pKa of the corresponding model compound N-acetyl-3-fluorotyrosinamid is shifted by -1.5 to 8.4 (377). Rappaport et al. investigated inter alia the pH-dependence of the kinetics of 
reduction in labeled and unlabeled, Mn-depleted PSIIcc from T. elongatus. As discussed below, their data suggest that 
> 12 for unlabeled YZ (at least in Mn-depleted samples). The electrostatic calculations of Ishikita and Knapp (378) suggest (according to our interpretation) that 
»
12.5, 
»
2.5, and 
= - 0.8. On the basis of these parameters, we calculated the titration curves in Figure 13B to illustrate a possible pH-dependence of the various states. Note in particular, that in the absence of a strong influence by another titratable group, the protonation states of both, Tyr A161 and His A190, are expected to be pH-independent in a wide range between and . If YZ is fluorinated, the pKa values of YZ are expected to decrease by 1.5, while those of B remain unaffected. If > -1.5 as in the present model, fluorination can be expected to change the protonation state of reduced YZ at physiological pH (Figure 13C). Therefore, FTIR experiments on such labeled samples could give information about pKPT.
Turning back to the redox potential, we can write down a scheme analogous to Figure 13A also for oxidized YZ with appropriate pKa values 
, 
, 
, 
, and 
, and connect the two schemes by mid-point potentials for the various redox couples. The Nernst-Clark equation of this minimal-model reads
(2)
Here, Em(YH·
+BH+/YHBH+) is the mid-point potential for the left redox couple in Figure 12A, but in the presence of a protonated (positively charged) His A190. The blue curve in Figure 12B respresents equation (2) in the relevant pH range for the parameters listed in the figure caption. We made use again of the calculations by Ishikita and Knapp (378), suggesting Em(Y·
BH+/Y-BH+) = 1216 mV (lower blue dashed line in Figure 12B). Furthermore, we assumed that the pKa shift due to the oxidation of YZ is the same in protein and solution, i.e., DpKa = -11.9, and that 
. With these settings, we can define the eight-state model completely. Clearly, these parameters are preliminary, and a more precise evaluation awaits a reliable WOC structure at a higher resolution and further experiments. Nonetheless, the following insight may be gained: (i) At physiological conditions, i.e., pH 6, the equilibrium mid-point potential of YZ is calculated to be +986 mV in agreement with Ishikita and Knapp (378) and with experimental estimates by Vass and Styring (379) placing the potential in the range of 950 - 990 mV (red double arrow in Figure 12B). The potential depends on pH as expected (dEm/d(pH) = -59 mV) between 
and 
. The situation may become more complicated, if the influence of other titratable residues (e.g., Arg C357) or an S-state dependence is taken into account. (ii) In the present model, the pKa-values of Tyr A161 are 
= 12.5 and 
= 0.6, which are increased by 2.6 compared to tyrosine in aqueous solution. This shift is not only due to the hydrogen bond to His A190, but also depends on other parts of PSIIcc as do the redox potentials. In particular, electrostatic calculations suggest that there is an influence of the protonation state of Arg C357 and the charges assigned to the WOC, where the former is affected by the latter (378). This implicates that pKa values and redox potentials may be S-state-dependent and influenced by Mn- or Ca-depletion procedures. Notably, the charge state of PD1 has only a small effect (< 10 mV). (iii) In the present model, < 0 and 
> 0, meaning that YZ becomes deprotonated and His A190 protonated upon oxidation of YZ in equilibrium. This result does not necessarily indicate that a concerted proton-electron transfer (CPET) takes place. (iv) If ET precedes PT (ET-PT mechanism) the relevant redox potential in the present model is Em(YH·
+B/YHB) = 1299 mV, which is higher than the estimated potential of the 
couple by about 50 mV. Given the uncertainty of these numbers, an ET-PT mechanism cannot be excluded on purely energetic grounds, at least within this model. (v) If proton transfer to His A190 precedes ET to 
(PT-ET mechanism), the relevant redox potential is Em(Y·
BH+/Y-BH+) = 1216 mV (378), and the driving force for ET is rather small (< 40 meV).
The kinetics of reduction by YZ is complex; for recent reviews, see (21, 380, 381). A reasonable description involves at least three exponential kinetics with a "fast" ns, a "slow" ns, and a �s component, and this general picture is obtained from experiments on a variety of sample and organism types (199, 277, 368, 376, 382-386). To unravel the complexity of YZ oxidation, experiments have been performed on intact and Mn-depleted PSIIcc. In intact PSIIcc, the amplitude of the "fast" ns phase is larger in S0 and S1 compared to S2 and S3, while that of the "slow" ns component is smaller, i.e., shows the opposite behavior (199, 382, 383). This S-state dependence could be due to an electrostatic effect of the presumed surplus charge on the WOC in S2 and S3 (see subsection 4.6) or originate from conformational changes (381). In contrast to the ns phases that lack any kinetic H/D isotope exchange effect (368, 383, 387, 388), the �s component is significantly different in H2O compared to D2O (383, 389). Therefore, the �s component is assigned to proton rearrangements in the neighborhood of YZ, while the "slow" ns component is ascribed to protein relaxation not involving proton shifts. The "fast" ns component is interpreted as reflecting the actual ET from YZ to , but the observed rate constant (35 - 70 ns) does not necessarily correspond to the instrinsic rate constant for forward ET as discussed in (386). The absence of an isotope effect indicates that if this ET is coupled to PT, the latter is not rate-limiting. Another interesting feature of the "fast" phase is its pH-dependence, i.e., the amplitude declines at acidic pH with an apparent pKa of 4.6 (368, 390-392) and in the alkaline range with pKa ~
8 (368). Since the population of the state YH B in the model outlined above shows a similar behavior (albeit with different pKa values used in the modeling, see Figure 13B), it is tempting to identify the two apparent pKa�s as 
= 4.6 and 
= 8.0. In particular, the identification of would be in line with the proposal of Kühn et al. (392) that protonation of the Tyr A161/His A190 pair at the Ne of the imidazole would inhibit YZ oxidation due to suppression of the necessary PT. However, such an interpretation is premature as other titratable groups could affect the kinetics (cf. discussion of YD below), and further studies are required to explain the pH-dependence at a molecular level. Mn-depletion causes a drastic change in the characteristic of reduction by YZ (381), and it remains unclear at present, what actually can be learned from these experiments about intact PSIIcc.
8.2. Metalloradical signals of YZ
During the last decade, EPR signals were detected that represent light-induced 
interacting magnetically with the Mn4Ca-cluster. These so-called metalloradical signals can be induced at helium temperatures by illumination with visible (VIS) or near-infrared (NIR) light; for reviews, see (393, 394). In this context, it should be recalled that S-state progression is inhibited at cryogenic temperatures with characteristic half-inhibition temperatures for the individual transitions between 135 and 235 K (395). Also, reduction of QB is blocked, so that VIS-illumination of the RC will create the state 
. Under these circumstances, 
is apparently able to oxidize YZ at helium temperatures, if PSIIcc is poised in the S0 or S1 state prior to freezing, but not in S2 or S3. However, YZ oxidation occurs only in 40 - 50% of the centers (396). The ability to oxidize YZ appears to be correlated with the charge state of the WOC, i.e., the presumed surplus charge in S2 and S3 (see subsection 4.6) seems to inhibit the reaction. More recently, it could be demonstrated that YZ oxidation by VIS-illumination in centers poised in the S2 state is possible at nitrogen temperatures (77 - 190 K) indicating a thermal barrier (397).
The Mn4Ca cluster poised in the S2 or S3 state is sensitive to NIR light (398-401). Illumination at 10 K in the presence of 
produces a metalloradical signal that resembles 
(402). Illumination of the state 
at 4 - 50 K also results in a metalloradical signal (169, 403, 404) with an action spectrum peaking around 760 nm (401) that is interpreted as 
, where 
is assigned to a proton-deficient variant of S2 based on its pH-dependence (405). The state recombines to 
, which in turn can be transformed into by VIS-illumination at helium temperatures (393, 405, 406). Note that NIR-illumination of 
and apparently leads to backward ET from YZ to the WOC. The suggested mechanisms of NIR sensitivity involve intervalence charge transfer transitions between MnIII and MnIV, NIR-induced spin state transitions or d-d transitions in MnIII causing a spin change (394). The presence of Mn-ions with the "correct" oxidation state seems to be crucial.
An important property of the metalloradical signals is the pH-dependence, which has beeen studied recently for S0 (407), S1 (408) and S3 (394). While the pH-dependence of S-state turnover at room temperature (409) is not necessarily indicative of YZ activity, formation of metalloradical signals is a more direct indicator. Indeed, the results from the different experiments do not always agree, Whereas both, S-state progression and metalloradical formation, decline at acidic pH with essentially the same apparent pKa for S0 and S3, this is not the case for S1. The S1 ®
S2 transition at ambient temperatures is pH-independent between pH 3.5 and 9.5 (409), but the formation of at cryogenic temperatures is inihibited at low pH with an apparent pKa of ~
4.7 (408). It should be noted that in all these low-temperature studies of PSIIcc, it is implicitly assumed that the protonation probabilities of groups are only weakly temperature-dependent. This assumption remains untested.
Havelius et al. (394, 407, 408) provided a tempting explanation for the apparent discrepancy between S1 ®
S2 transition activity and formation of at low pH: They interpreted the apparent pKa ~
4.7 of the metalloradical signal as in accordance with the interpretation of Kühn et al. (392) for the ns kinetics of YZ oxidation. At pH > , the state YH B prevails, and the normal proton-coupled ET proceeds. At pH < , the state YH BH+ is present, and oxidation of YZ is only possible, if the phenolic proton is taken up by another group, albeit slower (�s kinetics). The latter process is coupled to proton movements not confined to a single hydrogen bond. It, therefore, only proceeds at elevated temperatures and may be responsible for the H/D isotope exchange effect. The group accepting the proton is likely important for proton release from the WOC. Then, its protonation by YZ blocks proton release and consequently those S-state progressions that are accompanied by proton egress. Since no protons are expelled in the S1 ®
S2 transition (see section 9), the latter can proceed at high temperature at all pH, while YZ is oxidized fast at pH > and slow at pH < . Accordingly, the S0 ®
S1 and S3 ®
S0 transitions that require the proton acceptor are blocked below at any temperature. This model awaits further testing by studies of the pH-dependent formation of the metalloradical associated with the S2 state.
8.3. YD
We now turn to the second player in the tyrosine game. Despite its remote position relative to the WOC (Figure 2), YD (i.e., Tyr D160) is not unimportant (363, 410). In mutants of Synechocystis PCC 6803 with Tyr D160 replaced by Phe, photoautotrophic growth is slowed down (411), which may be related to a retardation and reduced efficiency of the photoassembly of the Mn4Ca-cluster (412). YD is apparently able to donate electrons to 
in Mn-depleted PSIIcc (413) and is in redox equilibrium with PD1, YZ and the WOC in intact PSIIcc (379). To disentangle the various contributions, Mn-depleted PSIIcc of a YZ-lacking mutant was investigated with time-resolved EPR and optical techniques (414). This study revealed electron donation of YD to with sub-�s kinetics that is pH-dependent, i.e., rises at high pH with an apparent pKa of 7.7. To explain the unexpectedly fast ET, an involvement of PD2 (i.e., partial delocalization of P+ on PD2) was proposed. The pH-dependence was confirmed in low-temperature studies showing that YD can be oxidized at 15 K (415). These results imply that there is a pH-dependent competition between YZ and YD as demonstrated recently in studies of the YZ metalloradical signals (394, 408).
In intact PSIIcc, oxidized YD (termed 
in the following, as it is likely a deprotonated radical, see below) is able to oxidize the S0 state of the Mn4Ca cluster up to S1 (Figure 4B) as shown by using EPR spectrosocpy (410, 416). This ET has a half life time between 2 and 50 min. depending on the species and sample type (416-418). It has been proposed that YD could serve to stabilize the WOC by maintaining it in a higher oxidation state (416). This also suggests a role of YD in the photoactivation process (412), in which Mn2+ is incorporated into apo-PSIIcc by light-triggered oxidation (238-241). Further support for this idea comes from the observation that is able to oxidize the overreduced states S-1 and S-2 of the Mn4Ca-cluster (183). However, there is also evidence that reduced YD is able to reduce the S2 and S3 states (419), thereby effectuating a back-stepping in the Kok-cycle down to S1 (Figure 4B). These processes are characterized by half life times between 0.8 and 5.5 s (417, 418). The redox mid-point potential of the 
couple has been estimated from direct chemical oxidation of Mn-depleted PSIIcc to be 760 mV (420), which is in agreement with estimates based on kinetic measurements on intact PSIIcc (379). The difference of ~
200 mV with respect to YZ is also reproduced by electrostatic calculations (378) and can be considered as compatible with the structural information about PSIIcc.
Like YZ, YD is believed to be protonated in its reduced form and to deliver a proton to a nearby base upon oxidation ("proton rocking motion" model (421)). EPR spectroscopy combined with site-directed mutagenesis provided evidence that this base is His D189 (422-424), and ENDOR data indicated the presence of an exchangeable proton hydrogen-bonded to (425) and bound to Ne of His A189 (426). Such a hydrogen bond is also in agreement with FTIR data (375). The crystal structure confirms the presence of His D189 in the vicinity of Tyr D160 (Figure 14). Unlike its counterpart in PsbA, His D189 is, at the Nd-side, not hydrogen bonded to an asparagine side chain, but to the protonatable side chain of Arg D294, which in turn is in hydrogen bonding distance to a chain of titratable residues. Moreover, there is a hydrogen bond between two amide side chains close to and "parallel" to the hydrogen bond between Tyr D160 and His D189. It remains to be clarified, how this arrangement affects the dynamics of proton-coupled ET. A recent FTIR study (427) indicates that reduced YD remains protonated between pH 6 and 10, i.e., the state YDH B prevails. However, in the oxidized form , His D189 remains neutral and its protonation state is not responsible for the pH-dependence of YD oxidation. The measured apparent pKa of 7.7 (see above) is, therefore, to be assigned to other groups. A likely explanation is that after accepting a proton from Tyr D160 at Ne, His D198 deprotonates at Nd by virtue of its interaction with Arg D294, which in turn delivers a proton to the hydrogen bonded network of titratable groups (Figure 14). This explanation is in agreement with very recent QM/MM computations (428) suggesting that the measured hfc of are more in line with hydrogen bonding to a neutral than to a charged histidine. However, the functional role of the hydrogen-bond network around YD remains still elusive.
9. PROTON RELEASE
A key aspect in the understanding of biological water oxidation is the proton release pattern, i.e., the number of protons released in each of the S-state transitions. Accordingly, there have been many studies aiming at a determination of the proton release stoichiometry applying various techniques (371, 429-431). The pioneering work of Fowler (432) indicated a release pattern for S0 ® S1 ® S2 ® S3 ® S0 of 0.75 : 0 : 1.25 : 2 measured by using a pH electrode. This principle pattern was confirmed by other groups using optical techniques (433-437) and more recently by Schlodder and Witt (438) using a pH electrode and by Suzuki et al (431) using FTIR spectrosopy (see Figure 4A). Based on the expectation that the proton stoichiometry should be represented by a set of integers, these results were interpreted as a pattern of 1 : 0 : 1 : 2. Other studies on various sample types challenged this view and arrived at a simple stoichiometry of 1 : 1 : 1 : 1 (429, 439-441). However, one has to distinguish carefully between the actual number of protons expelled in each S-state transition (intrinsic proton release pattern) and the number of protons reaching the outer medium and being detectable by pH-electrodes, indicator dyes etc. (extrinsic proton release pattern) (442).
In the following, we shall concentrate on the more recent data obtained with isolated PSIIcc from T. elongatus (431, 438). The first problem to deal with is the proton uptake by the artificial electron acceptor (e.g., dichloro-p-benzoquinone, DCBQ) used to maintain light-induced RC turnover. This can be circumvented by the addition of (non-protonatable) ferricyanide as final electron acceptor, ensuring reoxidation of DCBQ (438). Then, two complications remain: (i) The sequence of proton release as a function of flash number n is given by (437)
(3)
where ei is the number of protons liberated in the Si ® Si+1 transition and Ti(n) the weight of transition Si ® Si+1 upon flash n. As discussed in subsection 4.1., Ti(n) depends on the Kok-parameters, i.e., the number of "double-hits" and "misses". These parameters have to be determined by analysis of an independent observable or by fitting. (ii) If we denote the number of protons expelled from the WOC in the Si ® Si+1 transition with ki (intrinsic proton release pattern), then in general, ei ¹ ki, i.e. the extrinsic proton release pattern may be different. This can be understood by considering the presence of a titratable amino acid side chain in the vicinity of the WOC. Similar to the effect of YZ oxidation on the pKa of His A190 discussed in subsection 8.1, oxidation of the WOC may shift the pKa of this nearby group by virtue of their electrostatic interaction. If the pKa shift is large enough, S-state progression may cause the group to deprotonate and hence a proton to be liberated even though ki = 0. Under these conditions, one expects the ei to be pH-dependent and not necessarily integer numbers.
Schlodder and Witt (438) performed experiments on crystallizable PSIIcc from T. elongatus and found a pH-dependent proton release pattern. While e0 = e2 = 1 at all pH between 5.5 and 7.5, e1 = m and e3 = 2 - m , where m is approximately a sigmoidal function of pH with an inflection point around 5.7. As a consequence, the pattern is 1 : 0 : 1 : 2 at pH 7.5, but tends towards 1 : 1 : 1 : 1 below pH 5.5. The simplest interpretation of this feature is that there is one titratable group in the vicinity of the WOC (called AH in Figure 4C) with a pKa ~ 5.7 in the S0 and S1 states and a shifted pKa < 5.5 in states S2 and S3, where the pKa shift is caused by the positive surplus charge in the latter two states (see subsection 4.6). Then, m is the protonation probability of AH. On the basis of these data, it is believed that the intrinsic proton release pattern is indeed 1 : 0 : 1 : 2 and the deviations of the extrinsic pattern originate from deprotonation of (at least) one amino acid side chain.
A problem of the earlier proton release measurements is that they are conducted at low or even zero buffer concentration. Under these conditions, protons may be trapped by buffering groups of the protein, which could disturb the determination of liberated protons. Suzuki et al. (431) circumvented this problem by using high buffer concentrations to ensure trapping of all liberated protons by the buffer and exploited the FTIR-response of isotopically labeled buffer to quantitate the proton uptake. In this way, they comfirmed the results of Schlodder and Witt for pH 6 and approved the reliability of the data. The FTIR-data also indicated that the amino acid residue responsible for the pH-dependence is neither Asp, Glu, His nor Cys, and left only Arg, Lys and Tyr as candidates. The only groups of this kind within ~ 10 Å of the Mn4Ca-cluster are Arg A334, Arg C357, Lys D317, and Tyr A161. The latter is YZ discussed in section 8 and is unlikely to titrate at pH 5.7. Also, the interaction with His A190 suggests a response of His vibrations upon deprotonation that is not observed. Lys D317 interacts with the chloride ion Cl1 (see section 7), which likely upshifts its pKa. However, a loss of chloride from the Cl1-site or a reorganiztaion of the protein in this region as suggested by the "one-site two-state" model could change the titration behavior of Lys D317 and/or the nearby Arg A334. The currently most interesting candidate is Arg C357, which is located very close to the Mn4Ca-cluster (see Figure 15) and has been proposed to be directly involved in the reaction mechanism of water oxidation, viz. as proton abstractor (23, 443). Mutants of Synechocystis sp. PCC 6803 have been constructed, in which Arg C357 is replaced with Ser (444, 445) or Lys (446). Both mutations cause a severe impairment of O2 evolution, but still contain 60 - 80 % of PSIIcc. Most strikingly, the R(C357)K mutant evolves ~ 10 % O2 compared to wild type and does not show any oscillations of O2 yield as a function of flash number (446). This feature has been interpreted tentatively as representing a "miss" factor of 46 % indicating that Arg C357 is extremely important for S-state turnover and can not be fully replaced in its function with the different base Lys. These findings are principally in agreement with a role of Arg C357 as proton abstractor, but do not yet prove it. Electrostatic calculations confirm qualitatively that the protonation state of Arg C357 depends on the net charge of the Mn4Ca-cluster and demonstrate an influence of this protonation state on the redox mid-point potential of YZ (378). So, in principle, Arg C357 could be responsible for the pH-dependence of the proton release pattern, the "fast" ns phase of YZ oxidation and the S-state turnover for S2 to S0 (see section 8). However, according to our present knowledge, the apparent pKa values of all these processes are different, so that Arg C357 is probably not the sole origin of the pH-dependence.
As apparent from section 6, proton egress pathways connecting the Mn4Ca-cluster with the lumen have not yet been identified. However, proposals for such pathways have been made based on visual inspection of the crystal structure (12, 447-449), channel calculations as described in subsection 6.2, electrostatic calculations of pKa values (324), and QM/MM calculations (232, 329). The first proposals were based upon the 3.5 Å structure and assigned an important role in proton egress to titratable residues of PsbO (blue arrow in Figure 15). Later electrostatic calculations based upon the 3.0 Å structure confirmed this assignment by revealing a gradient of pKa values to be established by these residues, but also identfied additional possible pathways (orange arrows in Figure 15). On the basis of their QM/MM calculations using the heterocubane structure, Sproviero et al. (232, 329) suggested proton exit from the Mn4Ca-cluster via Asp A61 and Glu A65 (red arrow in Figure 15) leading to the originally proposed pathway involving PsbO. Glu A65 was also discussed by Gabdulkhakov et al. (200), as it was found to be located close to a narrow pass of a designated "proton channel" (Figure 10). They demonstrated in particular, that channel C (Figure 9) can be filled with water molecules connecting Mn4 (with one water molecule detected experimentally, see Figure 7D) via the chloride ion in the Cl1-site with Glu A65, and proposed this as a possible proton egress pathway in agreement with earlier suggestions concerning the role of Cl- (350). Note that this pathway also leads to the originally proposed PsbO-path, but that the PsbO-path deviates from the further joint course of channels C and D (Figure 15).
Central residues in these paths are Asp A61, Glu A65, and Glu D312 (Figure 15). Asp A61 was mutated in Synechocystis sp. PCC 6803 to either Glu, Asn or Ala (259). The mutants D(A61)N and D(A61)A exhibit only slow photoautotrophic growth and 20 % O2-evolution activity despite a high apparent PSIIcc-content. In contrast, the mutation D(A61)E grows normally and evolves O2 at 60 % of the wild type level. A similar feature was obtained with Glu A65, where the mutation to either Gln or Ala slowed down the photoautotrophic growth and reduced the O2-evolution to 20 %, whereas the mutation to Asp allowed for normal growth and 90 % O2-activity (259). These data suggest that titratable residues are required in these positions in line with their presumed roles as intermediate proton acceptors. Very recently, Glu D312 has been mutated to Ala (450). This mutant is photoautotrophic and evolves O2 at 30 % of the wild type level. Notably, the three mutations D(A61)A, E(A65)A and E(D312)A perturb the properties of the Mn4Ca-cluster far more than many mutations of the putative ligands discussed in subsection 5.5. In all three mutants, the efficiency of the S2 ® S3 transition was found to be lower than in the wild type, and the effciency of the S3 ® S0 transition was even more substantially lowered (450). Since according to the likely intrinsic proton release pattern discussed above, the S1 ® S2 transition requires no proton abstraction, whereas the S2 ® S3 and S3 ® S0 transitions do (cf. Figure 4C), this finding is consistent with a role of the three residues in proton egress.
10. WATER BINDING, WATER CONSUMPTION, AND OXYGEN RELEASE
10.1. Water binding sites
The binding of substrate water to the WOC has been reviewed recently (451), so that we give only a brief overwiew here, focussing on two points: spectroscopic analysis and mass spectrometry. One spectroscopic approach to water in PSIIcc is vibrational spectroscopy, in particular FTIR (265, 452), and will be discussed in subsection 10.2. The second important group of spectroscopic experiments is based on magnetic resonance. Earlier NMR experiments demonstrated an S-state dependence of proton spin relaxation rates ascribed to different oxidation states of bound manganese, but it was not possible to distinguish water protons from other exchangeable protons (453). The S2-state multiline EPR signal (see subsection 4.3) exhibits line broadening upon incubation of PSIIcc with H217O due to hyperfine coupling with the I = 5/2 nucleus 17O, indicating binding of water to the catalytic center in the S0, S1 or S2 state with oxygen coordinating Mn (454). The 17O-Mn interaction was later confirmed by ESEEM (455) and a pulse EPR technique known as hyperfine sublevel correlation (HYSCORE) spectroscopy (456).
ESEEM was also used to study manganese-deuterium hfc and the ability of d3-labeled methanol to displace D2O (457). Four classes of exchangeable deuterons were identified in S2 and S0. The two classes with the strongest hfc were interpreted as one water molecule that is closely bound to a Mn and is not displaced by methanol. The other two classes represent water molecules that are not closely bound to Mn (Mn-D distance ~ 3.7-4.7 Å) and protein matrix protons (Mn-D distance ~ 4 Å), where the latter are variably exchanged in different preparations. The effect of H/D-exchange on Mn-H hfc was studied with ENDOR and evidence was presented for different proton exchange rates in different S-states ascribed to a variation of water binding affinities with the oxidation state of the Mn4Ca-cluster (458).
A more direct approach to ligand exchange at the Mn4Ca-cluster applies mass spectrometry (451). In this type of experiment, the incorporation of 18O from isotopically labeled water into the product O2 of water oxidation is monitored by detecting the various formed O2 species using a mass spectrometer. Crucial to the success of this method was the development of a rapid mixing system by Wydrzynski, Messinger and co-workers, setting the limit for detectable exchange rates to 175 s-1 (459, 460). In this way, it could be demonstrated that there is a slow (t1/2 » 500 ms at 10 �C) and a fast (t1/2 < 25 ms at 10 �C) exchanging substrate water in the S3 state. Note that this result implies that 18O-labeled water injected after poising the WOC in the S3-state can still exchange oxygen isotopes with the substrate water that finally gives rise to the produced dioxygen. The two different rates indicate that there are two inequivalent sites concerning this exchange process. Later work uncovered the complete S-state dependence of the exchange rates (461). The two sites are inequivalent throughout the whole catalytic cycle. The rapidly exchanging substrate water is progressively more tightly bound in S1, S2, and S3, but can be kinetically resolved only in the S2 and S3 states. On the other hand, the slowly exchanging substrate water is tightly bound in S1, less so in S2 and S3, and most loosely bound in S0 (451). Also, the two substrate water molecules do not exhibit interconversion between their sites. A comparison with exchange rates in di-�-oxo dimanganese complexes suggests that these two binding sites do not correspond to the position of a �-oxo bridge between two manganese ions at least in S0, S2 and S3 (462, 463). However, the data are consistent with the slowly exchanging substrate being a bridge in S1. Another hint to the slow-binding site is the effect of chemical Ca2+/Sr2+ exchange, which consistently increases the exchange rate in S1, S2, and S3 (464). A possible explanation is that this slow-exchange site corresponds to a terminal or bridging ligand to Ca2+.
Recently, the 18O exchange rates have been measured in two site-directed mutants (465). Upon replacement of Asp A170, one of the likely ligands to Mn4 (see subsection 5.5), with His, the exchange rates in S3 are only marginally affected. This was interpreted as indication that Mn4 does not bind substrate water, but binding of non-substrate water to this Mn ion was not excluded. Replacement of Asp A61 with Asn, known to suppress S-state advancement beyond S2 (see section 9), causes a decrease of both exchange rates in S3. This result suggests an interaction of Asp A61 with both substrate water molecules, probably via a hydrogen bonding network.
10.2. Water insertion and consumption
FTIR spectroscopy is a suitable tool to investigate water reactions (265, 452). Water molecules in biological systems can always be expected to be engaged in hydrogen bonding to a certain extent. These hydrogen bonds affect the stretching vibrations of the OH groups. The vibrations of weakly hydrogen bonded groups occur in the range 3700 - 3500 cm-1 and are thus well separated from other vibrations (466). However, strongly hydrogen bonded OH groups vibrate at lower frequencies (3500 - 3000 cm-1) and are thus superimposed to NH stretching vibrations of the peptide backbone (467), which makes the analysis more difficult. Analysis of Si+1 - Si difference FTIR spectra in the region of weakly hydrogen bonded OH stretching modes revealed significant differences between the transitions (468). In the S1 ® S2 transition one observes a shift of a frequency from 3588 to 3617 cm-1, which is assigned to a water molecule with an asymmetry in hydrogen bonding that becomes more pronounced upon formation of the S2 state. In contrast, the Si ® Si+1 transitions for i = 0, 2 as well as the S3 ® S0 transition exhibit a clear decrease in OH oscillator absorption intensity, which is twice as large in the latter as in the two former transitions. The negative signal can be interpreted in any of the following two ways: (i) A weak hydrogen bond of water or hydroxid turns into a strong one or (ii) a proton is released from a weakly hydrogen bonded OH group. The intensities suggest that two weakly hydrogen bonded OH groups undergo a reaction in the S3 ® S0 transition, whereas only one weakly hydrogen bonded OH group reacts in any of the transitions S0 ® S1 and S2 ® S3. If we interpret the changes in the vibrational spectra as deprotonation, they are in nice agreement with the intrinsic proton release pattern of 1 : 0 : 1 : 2 discussed in section 9 (see Figure 4C for m = 0). Thus, it seems that FTIR monitors the deprotonation of substrate water. Note that this interpretation implies that a hydroxid ion is bound to the Mn4Ca-cluster in S1 and S2. The possible lack of a Mn-centered oxidation in the S2 ® S3 transition could be related to the oxidation of a water or hydroxid ligand accompanied by deprotonation of this ligand.
One way to analyse water insertion is to probe the need of the individual S-state transitions for water. This has been done in an FTIR study by variation of the hydration extent of a T. elongatus PSIIcc film sample through a change of the relative humidity in a sealed infrared cell (469). In the range of 73 - 99% relative humidity, the efficiencies of the S2 ® S3 and S3 ® S0 transitions were found to decrease faster than those of the S1 ® S2 and S0 ® S1 transitions upon lowering the water content, which was interpreted as indication of water insertion into the WOC in the former two transitions (452). At lower relative humidities, all S-state transitions are severely impaired, demonstrating that one has to be careful with procedures involving dehydration of samples. The interpretation of the effects of humidity suggests that one substrate water molecule is already inserted into the WOC early in the catalytic cycle, that is, prior to the formation of the S0 state (and presumably after O2 release from the preceding cycle). This is in line with the interpretation of the proton release during the S0 ® S1 transition as reflecting deprotonation of substrate water. However, there is a problem with the second water molecule: The humidity data seem to imply that it is inserted during the S2 ® S3 transition. This is in conflict with the detection of two inequivalent 18O exchange rates in the S2 state (see subsection 10.1), suggesting that the second water molecule is already bound at this stage (470).
Another indicator of water reactions could be the HOH bending mode appearing around 1640 cm-1. However, the absorption of this mode shows only weak intensity relative to the OH stretching vibration (471) and is overlapping with peptide backbone vibrations. In D2O, the DOD bending mode is shifted to ~ 1200 cm-1, where the protein does not absorb strongly. Therefore, D2O bound to the WOC was investigated with FTIR in the bending region (1100 - 1300 cm-1) as well as in the region of the OD stretch (2530 - 2730 cm-1) (472). All amenable difference spectra exhibited six to eigth peaks in the bending region, indicating that at least two fully deuterated D2O molecules are coupled to each S-state transition. Besides peak shifts, some signals represented loss of absorption intensity in agreement with water consumption. A negative peak at ~ 1240 cm-1 was used to identify the S2 ® S3, S3 ® S0, and S0 ® S1 transitions as possible substrate insertion steps. Analysis of the OD stretching region showed the presence of a negative peak at 2694 cm-1 mainly due to the S2 ® S3 and S3 ® S0 transitions in agreement with the idea that these are substrate insertion steps. Altogether, these data suggest that there are always more than two water molecules bound to the WOC, and that some substrate water may be inserted in one cycle, but is not consumed until the next cycle. This would also help to reconcile the FTIR data with the 18O exchange experiments.
10.3. Dioxygen formation and release
As stated by McEvoy and Brudvig (23), there are so many proposals for the mechanism of water oxidation that it is impossible to examine or even mention them all. For completeness, we give here a brief summary of aspects concerning the question of how dioxygen is actually formed. The hypothetical mechanisms can be grouped roughly into "radical mechanisms", "coupling of oxo-ligands", "nucleophilic attack", and "S3 state peroxide".
The possibility that the S2 ® S3 transition does not involve oxidation of Mn triggered mechanistic proposals, in which the oxidation is oxygen-centered and the formed oxyl radical attacks a second oxygen species in the S4 state (473-475). The radical could actually be a �-oxo bridge linking Mn and Ca2+ ions, which would explain the more pronounced changes in metal distances upon S3 formation as observed with EXAFS (253).
"Coupling of oxo-ligands" here refers to studies of Mn4O4 cubane structures (476-478) advocating a model, in which two oxo-vertices of the cubane are covalently linked by an intermolecular pathway, and the release of the two bridges as O2 effectuates an opening of the cubane to a "butterfly" structure. Since the opening implies a relaxation of strain in the structure, this is also referred to as the "Jack-in-the-Box" hypothesis of dioxygen formation (23). Although the original mechanism is unlikely to describe biological water oxidation, the "Jack-in-the-Box" aspect is intriguing. If at least one of the substrate water molecules ultimately forms a �-oxo bridge as discussed above, and provided the remaining metal ligands (i.e., the other �-oxo bridges and the amino acid side chains) impose a strain on the metal cluster, the stepwise oxidation/deprotonation of the cluster could weaken this bridge, until it is finally too weak to resist the strain, and the cluster is opened under ejection of O2. Relaxation of strain could be an important aspect of dioxygen release, making a back reaction unfavorable.
Based on their 18O exchange data (see subsection 10.1), Wydrzynski and coworkers (461) proposed that both substrate water molecules are terminal ligands to manganese and are bound in the form of hydroxide ligands to MnIII in the S3 state. After a further oxidation/deprotonation event in the S3 ® S4 transition, one of the hydroxides turns into a terminal oxo-ligand that by virtue of its intercation with MnIV is electron-depleted and thus allows for a "nucleophilic attack" by the second, neighboring hydroxide to form an O-O bond. In variants of this mechanism, the attacking hydroxide is bound to Ca2+ (479), or a Ca2+-bound water molecule or hydroxide ion attacks an oxo-ligand to a MnV (480, 481). However, at present, it seems rather unlikely that there is a MnV in the WOC.
A common feature of these mechanisms is that O-O bond formation is associated with the kinetically elusive S4 state. In contrast, there are also mechanistic proposals, in which this linkage is already established in the S3 state in the form of a bound peroxide, i.e., O-O bond formation precedes the final deprotonation steps. We refer the reader to the review by Renger (380) for an in-depth discussion of these variants.
Since the formulation of the S-state model by Kok and co-workers (99, 100), there has been a quest for the S4 state, which remained clouded in secrecy. Formally, such a state has to exist, as four oxidation equivalents are needed to produce O2 from H2O, but apparently, this state is not stable. One has to distinguish carefully between the states 
, in which YZ is oxidized, and S4YZ, in which ET from the WOC to YZ has taken place. In the following, we concentrate on experiments that aimed at the identification of a reaction intermediate between 
and S0YZ that could be S4YZ.
The temperature dependence of S-state advancement was investigated by following the kinetics with UV spectroscopy. In contrast to the S1 ® S2 and S2 ® S3 transitions, the S3 ® S0 transition showed a pronounced break in the Arrhenius plot for PSIIcc from T. vulcanus (482). Similar results were obtained with different preparations from spinach (483, 484), but no break could be detected in other studies employing pea (388) and Synechocystis sp. (485). Therefore, the general significance of the non-Arrhenius behavior remains unclear. As a possible explanation, it has been proposed that the break point represents the presence of different redox isomers of the S3 state (482-484) rather than a redox intermediate and that it is species dependent (109).
A metastable intermediate (S4YZ) that has different spectroscopic properties than the final state (S0YZ) should eventually be detectable as a lag phase in the time course of a spectroscopic signal. Rappaport et al. (486) investigated absorption transients at 295 nm to monitor ET to 
and at 424 - 440 nm interpreted as probing ET and proton release. They found a simple monophasic kinetics for 
and 
, but more complicated biphasic transients for 
and 
. We note that a similarly complex kinetics indicating a lag phase in had been observed earlier (482, 487, 488). Rappaport et al. (486) interpreted the lag phase (30 �s) as a proton release (or OH- binding) step preceding the actual ET, so that it is circumstantial evidence for a reaction intermediate, but not necessarily for a redox intermediate. Razeghifard and Pace (489) measured the dioxygen release kinetics by using time-resolved EPR oximetry. In this technique, the paramagnetic O2 is detected by virtue of its effect on the EPR spectra of stable radicals (e.g., deuterated tempone). The O2 release kinetics was directly compared with the EPR-detected reduction of 
. The data were interpreted in terms of a sequential reaction scheme 
®
intermediate ®
, where the intermediate could be either S4YZ or the modified S3 state as proposed by Rappaport et al. (486). Razeghifard and Pace (489) came to the conclusion that the most likely explanation for the delayed O2 release (50 - 200 �s) is the modification (e.g., deprotonation) of S3, implying again that S4 remained kinetically elusive.
Another way to detect O2 is polarography. Clausen et al. (485) detected a lag phase (450 �s) in the polarographic transient that was, however, not detectable in the UV kinetics. They concluded that the lag phase was not indicative of S4, but rather due to O2 transiently bound to the protein. Haumann et al. (110) used time-resolved XAS to monitor shifts of the Mn K-edge during the S state transitions. For Si ®
Si+1 with i = 0, 1, 2, an absorption decrease is observed in agreement with oxidation of the Mn4Ca cluster by . In the S3 ®
S0 transition, the absorption increases as expected for Mn reduction to S0, but with a lag phase of about 250 �s. Since the reduction of in this step is slower, it was concluded that the Mn4Ca-cluster does not change its redox state during the lag phase. As an explanation, earlier proposals of a preceding proton release were taken up. The deprotonated S3 state was originally termed "S4", which is somewhat misleading as discussed in subsection 4.1. In fact, the XAS transient after the third flash is essentially biphasic and besides the lag phase shows no sign of any redox intermediate that could be assigned to S4.
If the final step of dioxygen production is reversible, an increase of the O2 pressure should shift the equilibrium towards educts and intermediate. This idea was exploited by Clausen and Junge (312, 490), who analyzed UV transients and reported evidence for the O2-induced stabilization of an intermediate B. Their data yielded a free energy change for the final, O2-releasing step of DGBC = - 8.6 kJ mol-1. It should be noted that the nature of B was not strictly specified and that there is no evidence for an additional oxidation step of the Mn4Ca cluster. Instead, the data were analysed by assuming B = S2YZH2O2, where H2O2 is a hypothetical peroxide intermediate. These data were later supported by delayed fluorescence measurements, where B was called 
(313). The results were debated in the literature (314-316). As pointed out by Penner-Hahn and Yocum (315), the interpretation 
is difficult to reconcile with the data of Haumann et al. (110), as there is no evidence for a redox intermediate, neither with oxidized nor reduced Mn. Of course, there could be more intermediates, and any of these could have too short a life time to be detected. This debate triggered a study of the O2-pressure effect with time-resolved XAS (318). It was concluded that elevated O2 pressure rather causes an artificial Mn oxidation (e.g., by ROS) than a thermodynamic product inhibition. This result calls into question the formerly estimated DGBC. A recent study on whole cells, leaves and thylakoid membranes also questions the reversibility of the last, O2-forming step that would allow for product inhibition (317). We conclude, that there is presently no evidence for an S4 state in terms of a resolvable redox intermediate, and it is still unknown, how O2 is actually formed in photosynthetic water oxidation.
11. CONCLUSION AND PERSPECTIVES
PSIIcc is a complex molecular machinery. This complexity is intriguing, but also quite demanding. In the present review, we have made an attempt to collect experimental facts that along with insight from theoretical computations may help to uncover the operating principles of Nature�s water oxidation device on the basis of recent crystallographic information. Clearly, the growth of our knowledge about PSIIcc before and after the advent of crystal structures is enormous, but there are still too many white spots on the map. The ultimate goal is to further improve the quality of the crystal structure, which might be attained soon (491). However, this structure will not mark the end of the struggle for understanding, but rather open a new stage. The focus will then likely shift to obtain direct structural information about different functional states of PSIIcc, viz. the S-states. As X-rays will always cause some damage (at least Mn-reduction), X-ray crystallography will have ultimate limits in this respect, but nonetheless important insight may be gained, not the least concerning the surrounding of the WOC (e.g., the chloride cofactors). Spectroscopic methods such as FTIR, EXAFS or EPR/ENDOR will remain important. A challenge for future research will be to improve crystals and/or technical facilities to allow for the study of PSIIcc by using neutron diffraction. This non-invasive technique could bring us the desired mechanistic details of efficient light-induced water oxidation and the necessary inspiration for the design of commercially useful catalysts. In any case, PSIIcc will keep us busy.
12. ACKNOWLEDGEMENTS
We thank A. Gabdulkhakov for providing figure material and G. Renger for critial reading of the manuscript. This work benefitted from financial support by the Deutsche Forschungsgemeinschaft through Sfb 429 (to F. M.) and Center of Excellence "UniCat" coordinated by the Technische Universität Berlin (to A. Z.).
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Key Words: Electron transfer, EPR, ENDOR, FTIR, proton transfer, X-ray crystallography, X-ray spectroscopy, Review
Send correspondence to: Athina Zouni, Max-Volmer-Laboratorium für Biophysikalische Chemie, Technische Universität Berlin, Strasse des 17. Juni 135, D-10623 Berlin, Germany, Tel: 493031425650, Fax: 493031421122, E-mail:athina.zouni@tu-berlin.de