Calcium ligation in photosystem II under inhibiting conditions

In oxygenic photosynthesis, PSII carries out the oxidation of water and reduction of plastoquinone. The product of water oxidation is molecular oxygen. The water splitting complex is located on the lumenal side of the PSII reaction center and contains manganese, calcium, and chloride. Four sequential photooxidation reactions are required to generate oxygen from water; the five sequentially oxidized forms of the water splitting complex are known as the Sn states, where n refers to the number of oxidizing equivalents stored. Calcium plays a role in water oxidation; removal of calcium is associated with an inhibition of the S state cycle. Although calcium can be replaced by other cations in vitro, only strontium maintains activity, and the steady-state rate of oxygen evolution is decreased in strontium-reconstituted PSII. In this article, we study the role of calcium in PSII that is limited in water content. We report that strontium substitution or 18OH2 exchange causes conformational changes in the calcium ligation shell. The conformational change is detected because of a perturbation to calcium ligation during the S1 to S2 and S2 to S3 transition under water-limited conditions.     

Novel Features of Eukaryotic Photosystem II Revealed by Its Crystal Structure Analysis from a Red Alga

Photosystem II (PSII) catalyzes light-induced water splitting, leading to the evolution of molecular oxygen indispensible for life on the earth. The crystal structure of PSII from cyanobacteria has been solved at an atomic level, but the structure of eukaryotic PSII has not been analyzed. Because eukaryotic PSII possesses additional subunits not found in cyanobacterial PSII, it is important to solve the structure of eukaryotic PSII to elucidate their detailed functions, as well as evolutionary relationships. Here we report the structure of PSII from a red alga Cyanidium caldarium at 2.76 Å resolution, which revealed the structure and interaction sites of PsbQ′, a unique, fourth extrinsic protein required for stabilizing the oxygen-evolving complex in the lumenal surface of PSII. The PsbQ′ subunit was found to be located underneath CP43 in the vicinity of PsbV, and its structure is characterized by a bundle of four up-down helices arranged in a similar way to those of cyanobacterial and higher plant PsbQ, although helices I and II of PsbQ′ were kinked relative to its higher plant counterpart because of its interactions with CP43. Furthermore, two novel transmembrane helices were found in the red algal PSII that are not present in cyanobacterial PSII; one of these helices may correspond to PsbW found only in eukaryotic PSII. The present results represent the first crystal structure of PSII from eukaryotic oxygenic organisms, which were discussed in comparison with the structure of cyanobacterial PSII.     

The S3 state of photosystem II: differences between the structure of the manganese complex in the S2 and S3 states determined by X-ray absorption spectroscopy

O2-evolving photosystem II (PSII) membranes from spinach have been cryogenically stabilized in the S3 state of the oxygen-evolving complex. The cryogenic trapping of the S3 state was achieved using a double-turnover illumination of dark-adapted PSII preparations maintained at 240 K. A double turnover of PSII was accomplished using the high-potential acceptor, Q400, which is the high-spin iron of the iron-quinone acceptor complex. EPR spectroscopy was the principal tool establishing the S-state composition and defining the electron-transfer events associated with a double turnover of PSII. The inflection point energy of the Mn X-ray absorption K-edge of PSII preparations poised in the S3 state is the same as for those poised in the S2 state. This is surprising in light of the loss of the multiline EPR signal upon advancing to the S3 state. This indicates that the oxidative equivalent stored within the oxygen-evolving complex (OEC) during this transition resides on another intermediate donor which must be very close to the manganese complex. An analysis of the Mn extended X-ray absorption fine structure (EXAFS) of PSII preparations poised in the S2 and S3 states indicates that a small structural rearrangement occurs during this photoinduced transition. A detailed comparison of the Mn EXAFS of these two S states with the EXAFS of four multinuclear mu-oxo-bridged manganese compounds indicates that the photosynthetic manganese site most probably consists of a pair of binuclear di-mu-oxo-bridged manganese structures. However, we cannot rule out, on the basis of the EXAFS analysis alone, a complex containing a mononuclear center and a linear trinuclear complex. The subtle differences observed between the S states are best explained by an increase in the spread of Mn-Mn distances occurring during the S2----S3 state transition. This increased disorder in the manganese distances suggests the presence of two inequivalent di-mu-oxo-bridged binuclear structures in the S3 state.     

Symbols,système international (SI) units,abbreviations, conversion factors and special instructions to be used in photosynthesis research

Biochemical techniques now exist to produce the oxygen-evolving complex of photosystem II (PSII) and its associated photochemical redox reactions in various states of purity. These preparations permit one to assess the structural roles of polypeptides in promoting activity by using selective extraction techniques which remove certain polypeptides, to carry out reconstitution studies which re-establish activity, and, in the case of more recently developed, highly purified preparations discussed in this overview, to identify the minimal polypeptide complement necessary for photosynthetic oxygen evolution activity. These comparative investigations also suggest a tentative structure for an oxygen-evolving PSII core complex whose primary constituents are a hydrophobic complex of polypeptide, manganese, calcium and chloride, and the 33 kDa extrinsic polypeptide.Abbreviations DCBQ 2,6 dichloro-p-benzoquinone - Chl chlorophyll - LHCP light-harvesting chlorophyll proteins - PS photosystem Presented at the Japan/US Binational Seminar on Energy Conversion: Photochemical Reaction Centers and Oxygen-Evolving Complexes of Plant Photosynthesis, March 17–21, 1987. See conference report by G. Renger in Photosynthesis Research, in press, 1987...Editor.     

Sucrose and glycerol effects on photosystem II

Photosystem II catalyzes the oxidation of water and the reduction of plastoquinone. The active site cycles among five oxidation states, which are called the S(n) states. PSII purification procedures include the use of the cosolvents, sucrose and/or glycerol, to stabilize water splitting activity and for cryoprotection. In this study, the effects of sucrose and glycerol on PSII were investigated. Sucrose addition was observed to stimulate the steady-state rate of oxygen evolution in the range from 0 to 1.35 M. Glycerol addition was observed to stimulate oxygen evolution in the range from 0 to 30%. Both cosolvents were observed to be inhibitory at higher concentrations. Sucrose addition was shown to have no effect on the rate of Q(A)(-) oxidation or on the K(M) for exogenous acceptor. PSII was then treated to remove extrinsic proteins. In these samples, sucrose addition stimulated activity, but glycerol addition was inhibitory at concentrations higher than approximately 0.5 M. This inhibitory effect of glycerol at relatively low concentrations is attributed to glycerol binding to the active site, when extrinsic subunits are not present. Reaction induced FTIR spectra, associated with the S(1) to S(2) transition of the water-oxidizing complex, exhibited significant differences throughout the 1,800-1,200 cm(-1) region, when glycerol- and sucrose-containing samples were compared. These measurements suggest a cosolvent-induced shift in the pK(A) of an aspartic or glutamic acid side chain, as well as structural changes at the active site. These structural alterations are attributed to a change in preferential hydration of the oxygen-evolving complex.     

Photosynthetic generation of oxygen

The oxygen in the atmosphere is derived from light-driven oxidation of water at a catalytic centre contained within a multi-subunit enzyme known as photosystem II (PSII). PSII is located in the photosynthetic membranes of plants, algae and cyanobacteria and its oxygen-evolving centre (OEC) consists of four manganese ions and a calcium ion surrounded by a highly conserved protein environment. Recently, the structure of PSII was elucidated by X-ray crystallography thus revealing details of the molecular architecture of the OEC. This structural information, coupled with an extensive knowledge base derived from a wide range of biophysical, biochemical and molecular biological studies, has provided a framework for understanding the chemistry of photosynthetic oxygen generation as well as opening up debate about its evolutionary origin.     

Light-induced FTIR difference spectroscopy of the S(2)-to-S(3) state transition of the oxygen-evolving complex in Photosystem II

We have applied flash-induced FTIR spectroscopy to study structural changes upon the S(2)-to-S(3) state transition of the oxygen-evolving complex (OEC) in Photosystem II (PSII). We found that several modes in the difference IR spectrum are associated with bond rearrangements induced by the second laser flash. Most of these IR modes are absent in spectra of S(2)/S(1), of the acceptor-side non-heme ion, of Yradical(D)/Y(D) and of S(3)'/S(2)' from Ca-depleted PSII preparations. Our results suggest that these IR modes most likely originate from structural changes in the oxygen-evolving complex itself upon the S(2)-to-S(3) state transition in PSII.     

pH dependence of the flash-induced S-state transitions in the oxygen-evolving center of photosystem II from Thermosynechoccocus elongatus as revealed by Fourier transform infrared spectroscopy

pH dependence of the efficiencies of the flash-induced S-state transitions in the oxygen-evolving center (OEC) was studied by means of Fourier transform infrared (FTIR) difference spectroscopy using photosystem II (PSII) core complexes from the thermophilic cyanobacterium Thermosynechoccocus elongatus. The PSII core complexes dark-adapted at different pHs in the presence of ferricyanide as an electron acceptor were excited by four consecutive saturating laser flashes, and FTIR difference spectra induced by each flash were recorded in the region of 1800-1200 cm(-1). Each difference spectrum was fitted with a linear combination of standard spectra measured at pH 6.0, which represent the spectra upon individual S-state transitions, and the transition efficiencies were estimated from the fitting parameters. It was found that the S1 --> S2 transition probability is independent of pH throughout the pH region of 3.5-9.5, while the S2 --> S3, S3 --> S0, and S0 --> S1 transition probabilities decrease at acidic pH with pK values of 3.6 +/- 0.2, 4.2 +/- 0.3, and 4.7 +/- 0.5, respectively. These findings, i.e., the pH-independent S1 --> S2 transition probability and the pK values for the inhibition in the acidic range of the other three transitions, were in good agreement with recent results obtained by electron paramagnetic resonance measurements for PSII-enriched membranes of spinach [Bernát, G., Morvaridi, F., Feyziyev, Y., and Styring, S. (2002) Biochemistry 41, 5830-5843]. On the basis of this correspondence for quite different types of PSII preparations exhibiting marked difference in the pH dependence of the apparent proton release pattern, it is concluded that the inhibition of the S2 --> S3, S3 --> S0, and S0 --> S1 transitions in the acidic region is an inherent property of the OEC. This feature probably reflects proton release from substrate water in these three transitions. On the other hand, all of the S-state transitions remained generally efficient up to pH 9.5 in the alkaline region, except for a slight decrease of the S3 --> S0 transition probability above pH 8 (pK approximately 10). This observation partly differs from the tendency reported for spinach preparations, suggesting that a mechanism different from that in the acidic region is responsible for the transition efficiencies in the alkaline region.     

Photoreduction of Molecular Oxygen in Preparations of Photosystem II under Photoinhibitory Conditions

Preparations of photosystem II (PSII) from pea (Pisum sativum L.) leaves were used to study the evolution and reduction of molecular oxygen under photoinhibitory conditions. Under these conditions, the photoinduced oxygen uptake did not exceed 10% of the total oxygen-evolving activity in PSII preparations. Both the Hill and the Mehler reactions were found to occur simultaneously under long-term illumination of PSII preparations with high-intensity light in the presence of potassium ferricyanide. During this light treatment in the presence of potassium ferricyanide, the rate of oxygen uptake increased gradually reaching 30% of the oxygen-evolving activity. The photogeneration of superoxide anion radical at increasing light intensities followed a typical light-response curve with a light saturation at 800 W/m². The results provide evidence that the Mehler reaction is the major source for superoxide and hydrogen peroxide in PSII preparations under photoinhibitory conditions and that the Mehler reaction in PSII proceeds more effectively at high light intensities. The relatively low and sustained rate of oxygen photoreduction in PSII preparations under photoinhibitory conditions substantiates the hypothesis on the involvement of Mehler reaction in cell signaling and regulation.     

Time-resolved X-ray spectroscopy leads to an extension of the classical S-state cycle model of photosynthetic oxygen evolution

In oxygenic photosynthesis, a complete water oxidation cycle requires absorption of four photons by the chlorophylls of photosystem II (PSII). The photons can be provided successively by applying short flashes of light. Already in 1970, Kok and coworkers [Photochem Photobiol 11:457-475, 1970] developed a basic model to explain the flash-number dependence of O2 formation. The third flash applied to dark-adapted PSII induces the S3-->S4-->S0 transition, which is coupled to dioxygen formation at a protein-bound Mn4Ca complex. The sequence of events leading to dioxygen formation and the role of Kok's enigmatic S4-state are only incompletely understood. Recently we have shown by time-resolved X-ray spectroscopy that in the S3-->S0 transition an interesting intermediate is formed, prior to the onset of O-O bond formation [Haumann et al. Science 310:1019-1021, 2005]. The experimental results of the time-resolved X-ray experiments are discussed. The identity of the reaction intermediate is considered and the question is addressed how the novel intermediate is related to the S4-state proposed in 1970 by Bessel Kok. This leads us to an extension of the classical S-state cycle towards a basic model which describes sequence and interplay of electron and proton abstraction events at the donor side of PSII [Dau and Haumann, Science 312:1471-1472, 2006].     

S-state dependence of the calcium requirement and binding characteristics in the oxygen-evolving complex of photosystem II

The functional role of the Ca (2+) ion in the oxygen-evolving complex of photosystem II is not yet clear. Current models explain why the redox cycle of the complex would be interrupted after the S 3 state without Ca (2+), but the literature shows that it is interrupted after the S 2 state. Reinterpretation of the literature on methods of Ca (2+) depletion [Miqyass, M., van Gorkom, H. J., and Yocum, C. F. (2007) Photosynth. Res. 92, 275-287] led us to propose that all S-state transitions require Ca (2+). Here we confirm that interpretation by measurements of flash-induced S-state transitions in UV absorbance. The results are explained by a cation exchange at the Ca (2+) binding site that, in the absence of the extrinsic PsbP and PsbQ polypeptides, can occur in minutes in low S-states and in seconds in high S-states, depending on the concentration of the substituting cation. In the S 2(K (+)) or S 2(Na (+)) state a slow conformational change occurs that prevents recovery of the slow-exchange situation on return to a lower S-state but does not inhibit the S-state cycle in the presence of Ca (2+). The ratio of binding affinities for monovalent vs divalent cations increases dramatically in the higher S-states. With the possible exception of S 0 to S 1, all S-state transitions specifically require Ca (2+), suggesting that Ca (2+)-bound H 2O plays an essential role in a H (+) transfer network required for H (+)-coupled electron transfer from the Mn cluster to tyrosine Z.     

Low-temperature photochemistry in photosystem II from Thermosynechococcus elongatus induced by visible and near-infrared light

The active site for water oxidation in photosystem II (PSII) consists of a Mn4Ca cluster close to a redox-active tyrosine residue (TyrZ). The enzyme cycles through five sequential oxidation states (S0 to S4) in the water oxidation process. Earlier electron paramagnetic resonance (EPR) work showed that metalloradical states, probably arising from the Mn4 cluster interacting with TyrZ., can be trapped by illumination of the S0, S1 and S2 states at cryogenic temperatures. The EPR signals reported were attributed to S0TyrZ., S1TyrZ. and S2TyrZ., respectively. The equivalent states were examined here by EPR in PSII isolated from Thermosynechococcus elongatus with either Sr or Ca associated with the Mn4 cluster. In order to avoid spectral contributions from the second tyrosyl radical, TyrD., PSII was used in which Tyr160 of D2 was replaced by phenylalanine. We report that the metalloradical signals attributed to TyrZ. interacting with the Mn cluster in S0, S1, S2 and also probably the S3 states are all affected by the presence of Sr. Ca/Sr exchange also affects the non-haem iron which is situated approximately 44 A units away from the Ca site. This could relate to the earlier reported modulation of the potential of QA by the occupancy of the Ca site. It is also shown that in the S3 state both visible and near-infrared light are able to induce a similar Mn photochemistry.     

Biosynthetic Ca2+/Sr2+ exchange in the photosystem II oxygen-evolving enzyme of Thermosynechococcus elongatus

The thermophilic cyanobacterium, Thermosynechococcus elongatus, has been grown in the presence of Sr2+ instead of Ca2+ with the aim of biosynthetically replacing the Ca2+ of the oxygen-evolving enzyme with Sr2+. Not only were the cells able to grow normally with Sr2+, they actively accumulated the ion to levels higher than those of Ca2+ in the normal cultures. A protocol was developed to purify a fully active Sr(2+)-containing photosystem II (PSII). The modified enzyme contained a normal polypeptide profile and 1 strontium/4 manganese, indicating that the normal enzyme contains 1 calcium/4 manganese. The Sr(2+)- and Ca(2+)-containing enzymes were compared using EPR spectroscopy, UV-visible absorption spectroscopy, and O2 polarography. The Ca2+/Sr2+ exchange resulted in the modification of the EPR spectrum of the manganese cluster and a slower turnover of the redox cycle (the so-called S-state cycle), resulting in diminished O2 evolution activity under continuous saturating light: all features reported previously by biochemical Ca2+/Sr2+ exchange in plant PSII. This allays doubts that these changes could be because of secondary effects induced by the biochemical treatments themselves. In addition, the Sr(2+)-containing PSII has other kinetics modifications: 1) it has an increased stability of the S3 redox state; 2) it shows an increase in the rate of electron donation from TyrD, the redox-active tyrosine of the D2 protein, to the oxygen-evolving complex in the S3-state forming S2; 3) the rate of oxidation of the S0-state to the S1-state by TyrD* is increased; and 4) the release of O2 is slowed down to an extent similar to that seen for the slowdown of the S3TyrZ* to S0TyrZ transition, consistent with the latter constituting the limiting step of the water oxidation mechanism in Sr(2+)-substituted enzyme as well as in the normal enzyme. The replacement of Ca2+ by Sr2+ appears to have multiple effects on kinetics properties of the enzyme that may be explained by S-state-dependent shifts in the redox properties of both the manganese complex and TyrZ as well as structural effects.     

Eight steps preceding O-O bond formation in oxygenic photosynthesis—A basic reaction cycle of the Photosystem II manganese complex

In oxygenic photosynthesis, water is split at a Mn₄Ca complex bound to the proteins of photosystem II (PSII). Powered by four quanta of visible light, four electrons and four protons are removed from two water molecules before dioxygen is released. By this process, water becomes an inexhaustible source of the protons and electrons needed for primary biomass formation. On the basis of structural and spectroscopic data, we recently have introduced a basic reaction cycle of water oxidation which extends the classical S-state cycle [B. Kok, B. Forbush, M. McGloin, Cooperation of charges in photosynthetic O2 evolution- I. A linear four-step mechanism, Photochem. Photobiol. 11 (1970) 457-475] by taking into account also the role and sequence of deprotonation events [H. Dau, M. Haumann, Reaction cycle of photosynthetic water oxidation in plants and cyanobacteria, Science 312 (2006) 1471-1472]. We propose that the outwardly convoluted and irregular events of the classical S-state cycle are governed by a simple underlying principle: protons and electrons are removed strictly alternately from the Mn complex. Starting in I₀, eight successive steps of alternate proton and electron removal lead to I₈ and only then the O-O bond is formed. Thus not only four oxidizing equivalents, but also four bases are accumulated prior to the onset of dioxygen formation. After reviewing the kinetic properties of the individual S-state transition, we show that the proposed basic model explains a large body of experimental results straightforwardly. Furthermore we discuss how the I-cycle model addresses the redox-potential problem of PSII water oxidation and we propose that the accumulated bases facilitate dioxygen formation by acting as proton acceptors.     

High-resolution structure of the photosynthetic Mn4Ca catalyst from X-ray spectroscopy

The application of high-resolution X-ray spectroscopy methods to study the photosynthetic water oxidizing complex, which contains a unique hetero-nuclear catalytic Mn4Ca cluster, is described. Issues of X-ray damage, especially at the metal sites in the Mn4Ca cluster, are discussed. The structure of the Mn4Ca catalyst at high resolution, which has so far eluded attempts of determination by X-ray diffraction, X-ray absorption fine structure (EXAFS) and other spectroscopic techniques, has been addressed using polarized EXAFS techniques applied to oriented photosystem II (PSII) membrane preparations and PSII single crystals. A review of how the resolution of traditional EXAFS techniques can be improved, using methods such as range-extended EXAFS, is presented, and the changes that occur in the structure of the cluster as it advances through the catalytic cycle are described. X-ray absorption and emission techniques (XANES and Kbeta emission) have been used earlier to determine the oxidation states of the Mn4Ca cluster, and in this report we review the use of X-ray resonant Raman spectroscopy to understand the electronic structure of the Mn4Ca cluster as it cycles through the intermediate S-states.     

Light-induced FTIR difference spectroscopy as a powerful tool toward understanding the molecular mechanism of photosynthetic oxygen evolution

The molecular mechanism of photosynthetic oxygen evolution remains a mystery in photosynthesis research. Although recent X-ray crystallographic studies of the photosystem II core complex at 3.0-3.5 A resolutions have revealed the structure of the oxygen-evolving center (OEC), with approximate positions of the Mn and Ca ions and the amino acid ligands, elucidation of its detailed structure and the reactions during the S-state cycle awaits further spectroscopic investigations. Light-induced Fourier transform infrared (FTIR) difference spectroscopy was first applied to the OEC in 1992 as detection of its structural changes upon the S(1)-->S(2) transition, and spectra during the S-state cycle induced by consecutive flashes were reported in 2001. These FTIR spectra provide extensive structural information on the amino acid side groups, polypeptide chains, metal core, and water molecules, which constitute the OEC and are involved in its reaction. FTIR spectroscopy is thus becoming a powerful tool in investigating the reaction mechanism of photosynthetic oxygen evolution. In this mini-review, the measurement method of light-induced FTIR spectra of OEC is introduced and the results obtained thus far using this technique are summarized.     

Very strong UV-A light temporally separates the photoinhibition of photosystem II into light-induced inactivation and repair

When organisms that perform oxygenic photosynthesis are exposed to strong visible or UV light, inactivation of photosystem II (PSII) occurs. However, such organisms are able rapidly to repair the photoinactivated PSII. The phenomenon of photoinactivation and repair is known as photoinhibition. Under normal laboratory conditions, the rate of repair is similar to or faster than the rate of photoinactivation, preventing the detailed analysis of photoinactivation and repair as separate processes. We report here that, using strong UV-A light from a laser, we were able to analyze separately the photoinactivation and repair of photosystem II in the cyanobacterium Synechocystis sp. PCC 6803. Very strong UV-A light at 364 nm and a photon flux density of 2600 μmol photons m⁻² s⁻¹ inactivated the oxygen-evolving machinery and the photochemical reaction center of PSII within 1 or 2 min before the first step in the repair process, namely, the degradation of the D1 protein, occurred. During subsequent incubation of cells in weak visible light, the activity of PSII recovered fully within 30 min and this process depended on protein synthesis. During subsequent incubation of cells in darkness for 60 min, the D1 protein of the photoinactivated PSII was degraded. Further incubation in weak visible light resulted in the rapid restoration of the activity of PSII. These observations suggest that very strong UV-A light is a useful tool for the analysis of the repair of PSII after photoinactivation.     

QM/MM computational studies of substrate water binding to the oxygen-evolving centre of photosystem II

This paper reports computational studies of substrate water binding to the oxygen-evolving centre (OEC) of photosystem II (PSII), completely ligated by amino acid residues, water, hydroxide and chloride. The calculations are based on quantum mechanics/molecular mechanics hybrid models of the OEC of PSII, recently developed in conjunction with the X-ray crystal structure of PSII from the cyanobacterium Thermosynechococcus elongatus. The model OEC involves a cuboidal Mn3CaO4Mn metal cluster with three closely associated manganese ions linked to a single mu4-oxo-ligated Mn ion, often called the 'dangling manganese'. Two water molecules bound to calcium and the dangling manganese are postulated to be substrate molecules, responsible for dioxygen formation. It is found that the energy barriers for the Mn(4)-bound water agree nicely with those of model complexes. However, the barriers for Ca-bound waters are substantially larger. Water binding is not simply correlated to the formal oxidation states of the metal centres but rather to their corresponding electrostatic potential atomic charges as modulated by charge-transfer interactions. The calculations of structural rearrangements during water exchange provide support for the experimental finding that the exchange rates with bulk 18 O-labelled water should be smaller for water molecules coordinated to calcium than for water molecules attached to the dangling manganese. The models also predict that the S1-->S2 transition should produce opposite effects on the two water-exchange rates.     

Trapping of metalloradical intermediates of the S-states at liquid helium temperatures. Overview of the phenomenology and mechanistic implications

The oxygen-evolving complex (OEC) of photosystem II (PSII) consists of a Mn cluster (believed to be tetranuclear) and a tyrosine (Tyr Z or Y(Z)). During the sequential absorption of four photons by PSII, the OEC undergoes four oxidative transitions, S(0) to S(1), ..., S(3) to (S(4))S(0). Oxygen evolves during the S(3) to S(0) transition (S(4) being a transient state). Trapping of intermediates of the S-state transitions, particularly those involving the tyrosyl radical, has been a goal of ultimate importance, as that can test critically models employing a role of Tyr Z in proton (in addition to electron) transfer, and also provide important clues about the mechanism of water oxidation. Until very recently, however, critical experimental information was lacking. We review and evaluate recent observations on the trapping of metalloradical intermediates of the S-state transitions, at liquid helium temperatures. These transients are assigned to Tyr Z(*) magnetically interacting with the Mn cluster. Besides the importance of trapping intermediates of this unique catalytic mechanism, liquid helium temperatures offer the additional advantage that proton motions (unlike electron transfer) are blocked except perhaps across strong hydrogen bonds. This paper summarizes the recent observations and discusses the constraints that the phenomenology imposes.     

Mechanism and energy diagram for O-O bond formation in the oxygen-evolving complex in photosystem II

The recent finding of a transition state with a significantly lower barrier than previously found, has made the mechanism for O-O bond formation in photosystem II much clearer. The full mechanism can be described in the following way. Electrons and protons are ejected from the oxygen-evolving complex (OEC) in an alternating fashion, avoiding unnecessary build-up of charge. The S0-S1 and S1-S2 transitions are quite exergonic, while the S2-S3 transition is only weakly exergonic. The strong endergonic S3-S4 transition is a key step in the mechanism in which an oxygen radical is produced, held by the dangling manganese outside the Mn3Ca cube. The O-O bond formation in the S4-state occurs by an attack of the oxygen radical on a bridging oxo ligand in the cube. The mechanism explains the presence of both a cube with bridging oxo ligands and a dangling manganese. Optimal orbital overlap puts further constraints on the structure of the OEC. An alternating spin alignment is necessary for a low barrier. The computed rate-limiting barrier of 14.7 kcal mol(-1) is in good agreement with experiments.