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 Mn(4)Ca 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 I(0), eight successive steps of alternate proton and electron removal lead to I(8) 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.     

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].     

Low-barrier hydrogen bond plays key role in active photosystem II — A new model for photosynthetic water oxidation

The function and mechanism of Tyr_Z in active photosystem II (PSII) is one of the long-standing issues in the study of photosynthetic water oxidation. Based on recent investigations on active PSII and theoretical studies, a new model is proposed, in which D1-His190 acts as a bridge, to form a low-barrier hydrogen bond (LBHB) with Tyr_Z, and a coordination bond to Mn or Ca ion of the Mn-cluster. Accordingly, this new model differs from previous proposals concerning the mechanism of Tyr_Z function in two aspects. First, the LBHB plays a key role to decrease the activation energy for Tyr_Z oxidation and Tyr_Z^· reduction during photosynthetic water oxidation. Upon the oxidation of Tyr_Z, the hydrogen bond between Tyr_Z and His190 changes from a LBHB to a weak hydrogen bond, and vice versa upon Tyr_Z^· reduction. In both stages, the electron transfer and proton transfer are coupled. Second, the positive charge formed after Tyr_Z oxidation may play an important role for water oxidation. It can be delocalized on the Mn-cluster, thus helps to accelerate the proton release from substrate water on Mn-cluster. This model is well reconciled with observations of the S-state dependence of Tyr_Z oxidation and Tyr_Z^· reduction, proton release, isotopic effect and recent EPR experiments. Moreover, the difference between Tyr_Z and Tyr_D in active PSII can also be readily rationalized. The His190 binding to the Mn-cluster predicted in this model is contradictious to the recent structure data, however, it has been aware that the crystal structure of the Mn-cluster and its environment are significantly modified by X-ray due to radiation damage and are different from that in active PSII. It is suggested that the His190 may be protonated during the radiation damage, which leads to the loss of its binding to Mn-cluster and the strong hydrogen bond with Tyr_Z. This type of change arising from radiation damage has been confirmed in other enzyme systems.     

Proton transport facilitating water-oxidation: the role of second sphere ligands surrounding the catalytic metal cluster

The ability of PSII to extract electrons from water, with molecular oxygen as a by-product, is a remarkable biochemical and evolutionary innovation. From an evolutionary perspective, the invention of PSII approximately 2.7 Ga led to the accelerated accumulation of biomass in the biosphere and the accumulation of oxygen in the atmosphere, a combination that allowed for the evolution of a much more complex and extensive biosphere than would otherwise have been possible. From the biochemical and enzymatic perspective, PSII is remarkable because of the thermodynamic and kinetic obstacles that needed to have been overcome to oxidize water as the ultimate photosynthetic electron donor. This article focuses on how proton release is an integral part of how these kinetic and thermodynamic obstacles have been overcome: the sequential removal of protons from the active site of H₂O-oxidation facilitates the multistep oxidation of the substrate water at the Mn₄CaO _x , the catalytic heart of the H₂O-oxidation reaction. As noted previously, the facilitated deprotonation of the Mn₄CaO _x cluster exerts a redox-leveling function preventing the accumulation of excess positive charge on the cluster, which might otherwise hinder the already energetically difficult oxidation of water. Using recent results, including the characteristics of site-directed mutants, the role of the second sphere of amino acid ligands and the associated network of water molecules surrounding the Mn₄CaO _x is discussed in relation to proton transport in other systems. In addition to the redox-leveling function, a trapping function is assigned to the proton release step occurring immediately prior to the dioxygen chemistry. This trapping appears to involve a yet-to-be clarified gating mechanism that facilitates to coordinated release of a proton from the neighborhood of the active site thereby insuring that the backward charge-recombination reaction does not out-compete the forward reaction of dioxygen chemistry during this final step of H₂O-oxidation.     

Evaluation of photosynthetic activities in thylakoid membranes by means of Fourier transform infrared spectroscopy

Light-induced Fourier transformed infrared (FTIR) difference spectroscopy is a powerful method to study the structures and reactions of redox cofactors involved in the photosynthetic electron transport chain. So far, most of the FTIR studies of the reactions of oxygenic photosynthesis have been performed using isolated photosystem I (PSI) and photosystem II (PSII) preparations, which, however, could be modified during isolation procedures. In this study, we developed a methodology to evaluate the photosynthetic activities of thylakoids using FTIR spectroscopy. FTIR difference spectra upon successive flashes using thylakoids from spinach exhibited signals typical of the S-state cycle at the Mn₄CaO₅ cluster and Q_B reactions in PSII with period-four and -two oscillations, respectively. Similar measurement in the presence of an artificial quinone as an exogenous electron acceptor showed features specific to the S-state cycle. Simulations of the oscillation patterns provided the quantum efficiencies of the S-state cycle and electron transfer in PSII. Moreover, FTIR measurement under continuous illumination on thylakoids in the presence of DCMU showed signals due to Q_A reduction and P700 oxidation simultaneously. From the relative amplitudes of marker bands of Q_A^? and P700⁺, the molar ratio of photoactive PSII and PSI centers in thylakoids was estimated. FTIR analyses of the photo-reactions in thylakoids, which are more intact than isolated photosystems, will be useful in investigations of the photosynthetic mechanism especially by genetic modification of photosystem proteins.     

Interaction between tyrosineZ and substrate water in active photosystem II

In the field of photosynthetic water oxidation it has been under debate whether Tyrosine_Z (Tyr_Z) acts as a hydrogen or an electron acceptor from water. In the former concept, direct contact of Tyr_Z with substrate water has been assumed. However, there is no direct evidence for the interaction between Tyr_Z and substrate water in active Photosystem II (PSII), instead most experiments have been performed on inhibited PSII. Here, this problem is tackled in active PSII by combining low temperature EPR measurements and quantum chemistry calculations. EPR measurements observed that the maximum yield of Tyr_Z oxidation at cryogenic temperature in the S₀ and S₁ states was around neutral pH and was essentially pH-independent. The yield of Tyr_Z oxidation decreased at acidic and alkaline pH, with pKs at 4.7-4.9 and 7.7, respectively. The observed pH-dependent parts at low and high values of pH can be explained as due to sample inactivation, rather than active PSII. The reduction kinetics of Tyr_Z^· in the S₀ and S₁ states were pH independent at pH range from 4.5 to 8. Therefore, the change of the pH in bulk solution probably has no effect on the Tyr_Z oxidation and Tyr_Z^· reduction at cryogenic temperature in the S₀ and S₁ states of the active PSII. Theoretical calculations indicate that Tyr_Z becomes more difficult to oxidize when a H₂O molecule interacts directly with it. It is suggested that Tyr_Z is probably located in a hydrophobic environment with no direct interaction with the substrate H₂O in active PSII. These results provide new insights on the function and mechanism of water oxidation in PSII.     

Considerations on the mechanism of photosynthetic water oxidation – dual role of oxo-bridges between Mn ions in (i) redox-potential maintenance and (ii) proton abstraction from substrate water

Two mechanistic problems of photosynthetic water oxidation at the Mn complex of Photosystem II (PS II) are considered. (I) In the four Mn-oxidizing transitions, any pure Mn oxidation is predicted to cause an increase in redox potential that renders subsequent oxidation steps impossible (redox-potential problem). Formation of unprotonated oxo-bridges may counteract the potential increase. (II) The O–O formation step without any high-pK bases acting as proton acceptors is energetically unfavorable (acceptor-base problem). The pK of oxides in a bridging position between Mn ions may increase drastically upon reduction of Mn in the water-oxidation step (>10 units), thus rendering them favorable proton acceptors. It is proposed that in PS II, in the course of the four oxidizing transitions at least two unprotonated oxo-bridges are formed. Thereby (i) a redox potential increase is prevented and (ii) proton acceptors are prepared for the O–O formation step. Water oxidation in the O–O bond formation step is facilitated by simultaneous Mn reduction and proton transfer to bridging oxides amounting to hydrogen atom or hydride transfer from substrate water to the Mn-oxo core of the Mn complex of PS II.     

How chloride functions to enable proton conduction in photosynthetic water oxidation: Time-resolved kinetics of intermediates (S-states) in vivo and bromide substitution

Chloride (Cl⁻) is essential for O₂ evolution during photosynthetic water oxidation. Two chlorides near the water-oxidizing complex (WOC) in Photosystem II (PSII) structures from Thermosynechococcus elongatus (and T. vulcanus) have been postulated to transfer protons generated from water oxidation. We monitored four criteria: primary charge separation flash yield (P* → P⁺Q_A⁻), rates of water oxidation steps (S-states), rate of proton evolution, and flash O₂ yield oscillations by measuring chlorophyll variable fluorescence (P* quenching), pH-sensitive dye changes, and oximetry. Br-substitution slows and destabilizes cellular growth, resulting from lower light-saturated O₂ evolution rate (−20 %) and proton release (−36 % ΔpH gradient). The latter implies less ATP production. In Br- cultures, protonogenic S-state transitions (S₂ → S₃ → S₀’) slow with increasing light intensity and during O₂/water exchange (S₀’ → S₀ → S₁), while the non-protonogenic S₁ → S₂ transition is kinetically unaffected. As flash rate increases in Cl⁻ cultures, both rate and extent of acidification of the lumen increase, while charge recombination is suppressed relative to Br⁻. The Cl⁻ advantage in rapid proton escape from the WOC to lumen is attributed to correlated ion-pair movement of H₃O⁺Cl⁻ in dry water channels vs. separated Br⁻ and H⁺ ion movement through different regions (>200-fold difference in Bronsted acidities). By contrast, at low flash rates a previously unreported reversal occurs that favors Br⁻ cultures for both proton evolution and less PSII charge recombination. In Br⁻ cultures, slower proton transfer rate is attributed to stronger ion-pairing of Br⁻ with AA residues lining the water channels. Both anions charge-neutralize protons and shepherd them to the lumen using dry aqueous channels.     

The mechanism of photosynthetic water splitting.

Oxygenic photosynthesis, which provides the biosphere with most of its chemical energy, uses water as its source of electrons. Water is photochemically oxidized by the protein complex photosystem II (PSII), which is found, along with other proteins of the photosynthetic light reactions, in the thylakoid membranes of cyanobacteria and of green plant chloroplasts. Water splitting is catalyzed by the oxygen-evolving complex (OEC) of PSII, producing dioxygen gas, protons and electrons. O(2) is released into the atmosphere, sustaining all aerobic life on earth; product protons are released into the thylakoid lumen, augmenting a proton concentration gradient across the membrane; and photo-energized electrons pass to the rest of the electron-transfer pathway. The OEC contains four manganese ions, one calcium ion and (almost certainly) a chloride ion, but its precise structure and catalytic mechanism remain unclear. In this paper, we develop a chemically complete structure of the OEC and its environment by using molecular mechanics calculations to extend and slightly adjust the recently-obtained X-ray crystallographic model with reference to this structure and to some important recent experimental results.     

PSII manganese cluster: Protonation of W2, O5, O4 and His337 in the S1 state explored by combined quantum chemical and electrostatic energy computations

Photosystem II (PSII) is a membrane-bound protein complex that oxidizes water to produce energized protons, which are used to built up a proton gradient across the thylakoidal membrane in the leafs of plants. This light-driven reaction is catalyzed by withdrawing electrons from the Mn₄CaO₅-cluster (Mn-cluster) in four discrete oxidation steps [S₁ − (S₄ / S₀)] characterized in the Kok-cycle. In order to understand in detail the proton release events and the subsequent translocation of such energized protons, the protonation pattern of the Mn-cluster need to be elucidated. The new high-resolution PSII crystal structure from Umena, Kawakami, Shen, and Kamiya is an excellent basis to make progress in solving this problem. Following our previous work on oxidation and protonation states of the Mn-cluster, in this work, quantum chemical/electrostatic calculations were performed in order to estimate the pKa of different protons of relevant groups and atoms of the Mn-cluster such as W2, O4, O5 and His337. In broad agreement with previous experimental and theoretical work, our data suggest that W2 and His337 are likely to be in hydroxyl and neutral form, respectively, O5 and O4 to be unprotonated. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Reprint of PSII Manganese Cluster: Protonation of W2, O5, O4 and His337 in the S1 state explored by combined quantum chemical and electrostatic energy computations

Photosystem II (PSII) is a membrane-bound protein complex that oxidizes water to produce energized protons, which are used to built up a proton gradient across the thylakoidal membrane in the leafs of plants. This light-driven reaction is catalyzed by withdrawing electrons from the Mn₄CaO₅-cluster (Mn-cluster) in four discrete oxidation steps [S₁ − (S₄ / S₀)] characterized in the Kok-cycle. In order to understand in detail the proton release events and the subsequent translocation of such energized protons, the protonation pattern of the Mn-cluster need to be elucidated. The new high-resolution PSII crystal structure from Umena, Kawakami, Shen, and Kamiya is an excellent basis to make progress in solving this problem. Following our previous work on oxidation and protonation states of the Mn-cluster, in this work, quantum chemical/electrostatic calculations were performed in order to estimate the pKa of different protons of relevant groups and atoms of the Mn-cluster such as W2, O4, O5 and His337. In broad agreement with previous experimental and theoretical work, our data suggest that W2 and His337 are likely to be in hydroxyl and neutral form, respectively, O5 and O4 to be unprotonated. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Oxidation state changes of the Mn<Subscript>4</Subscript>Ca cluster in Photosystem II

A detailed electronic structure of the Mn₄Ca cluster is required before two key questions for understanding the mechanism of photosynthetic water oxidation can be addressed. They are whether all four oxidizing equivalents necessary to oxidize water to O₂ accumulate on the four Mn ions of the oxygen-evolving complex, or do some ligand-centered oxidations take place before the formation and release of O₂ during the S₃ → [S₄] → S₀ transition, and what are the oxidation state assignments for the Mn during S-state advancement. X-ray absorption and emission spectroscopy of Mn, including the newly introduced resonant inelastic X-ray scattering spectroscopy have been used to address these questions. The present state of understanding of the electronic structure and oxidation state changes of the Mn₄Ca cluster in all the S-states, particularly in the S₂ to S₃ transition, derived from these techniques is described in this review.     

The tetra-manganese complex of photosystem II during its redox cycle – X-ray absorption results and mechanistic implications

Using X-ray absorption spectroscopy (XAS), relevant information on structure and oxidation state of the water-oxidizing Mn complex of photosystem II has been obtained for all four semi-stable intermediate states of its catalytic cycle. We summarize our recent XAS results and discuss their mechanistic implications. The following aspects are covered: (a) information content of X-ray spectra (pre-edge feature, edge position, extended X-ray absorption fine-structure (EXAFS), dichroism in the EXAFS of partially oriented samples); (b) S₁-state structure; (c) X-ray edge results on oxidation state changes; (d) EXAFS results on structural changes during the S-state cycle; (e) a structural model for the Mn complex in its S₃-state; (f) XAS-based working model for the S₂–S₃ transition; (g) XAS-based working model for the S₀–S₁ transition; (h) potential role of hydrogen atom abstraction by the Mn complex. Finally, we present a specific hypothesis on the mechanism of dioxygen formation during the S₃–(S₄)–S₀ transition. According to this hypothesis, water oxidation is facilitated by manganese reduction that is coupled to proton transfer from a substrate water to bridging oxides.     

Lysine 362 in cytochrome c oxidase regulates opening of the K-channel via changes in pKA and conformation

The metabolism of aerobic life uses the conversion of molecular oxygen to water as an energy source. This reaction is catalyzed by cytochrome c oxidase (CcO) consuming four electrons and four protons, which move along specific routes. While all four electrons are transferred via the same cofactors to the binuclear reaction center (BNC), the protons take two different routes in the A-type CcO, i.e., two of the four chemical protons consumed in the reaction arrive via the D-channel in the oxidative first half starting after oxygen binding. The other two chemical protons enter via the K-channel in the reductive second half of the reaction cycle. To date, the mechanism behind these separate proton transport pathways has not been understood.In this study, we propose a model that can explain the reaction-step specific opening and closing of the K-channel by conformational and pK_A changes of its central lysine 362. Molecular dynamics simulations reveal an upward movement of Lys362 towards the BNC, which had already been supposed by several experimental studies. Redox state-dependent pK_A calculations provide evidence that Lys362 may protonate transiently, thereby opening the K-channel only in the reductive second half of the reaction cycle. From our results, we develop a model that assigns a key role to Lys362 in the proton gating between the two proton input channels of the A-type CcO.     

Bicarbonate rescues damaged proton-transfer pathway in photosystem II

The membrane-protein complex photosystem II (PSII) catalyzes photosynthetic water oxidation. Proton transfer plays an integral role in the catalytic cycle of water oxidation by maintaining charge balance to regulate and ensure the efficiency of the process. The hydrogen-bonded amino-acid residues that surround the oxygen-evolving complex (OEC) provide an efficient pathway for proton removal. Hence, it is crucial to identify these pathways to provide deeper insights into the proton-transfer mechanisms. In this study, we have used bicarbonate as a mobile exogenous proton-transfer reagent to recover the activity lost by site-directed mutations in order to identify amino-acid residues participating in the proton-transfer pathway. We find that bicarbonate restores efficient S-state cycling in D2-K317A PSII core complexes, but not in D1-D61A and CP43-R357K PSII core complexes, indicating that bicarbonate chemical rescue can be used to differentiate single-point mutations affecting the pathways of proton transfer from mutations that affect other aspects of the water-oxidation mechanism.     

Calcium exchange and structural changes during the photosynthetic oxygen evolving cycle

PSII catalyzes the oxidation of water and reduction of plastoquinone in oxygenic photosynthesis. PSII contains an oxygen-evolving complex, which is located on the lumenal side of the PSII reaction center and which contains manganese, calcium, and chloride. Four sequential photooxidation reactions are required to generate oxygen. This process produces five Sn-states, where n refers to the number of oxidizing equivalents stored. Calcium is required for oxygen production. Strontium is the only divalent cation that replaces calcium and maintains activity. In our previous FT-IR work, we assessed the effect of strontium substitution on substrate-limited PSII preparations, which were inhibited at the S3 to S0 transition. In this work, we report reaction-induced FT-IR studies of hydrated PSII preparations, which undergo the full S-state cycle. The observed difference FT-IR spectra reflect long-lived photoinduced conformational changes in the oxygen-evolving complex; strontium exchange identifies vibrational bands sensitive to substitutions at the calcium site. During the S1' to S2' transition, the data are consistent with an electrostatic or structural perturbation of the calcium site. During the S3' to S0' and S0' to S1' transitions, the data are consistent with a perturbation of a hydrogen bonding network, which contains calcium, water, and peptide carbonyl groups. To explain our data, persistent shifts in divalent cation coordination must occur when strontium is substituted for calcium. A modified S-state model is proposed to explain these results and results in the literature.     

Protons bound to the Mn cluster in photosystem II oxygen evolving complex detected by proton matrix ENDOR

Protons in the vicinity of the oxygen-evolving manganese cluster in photosystem II were studied by proton matrix ENDOR. Six pairs of proton ENDOR signals were detected in both the S₀ and S₂ states of the Mn-cluster. Two pairs of signals that show hyperfine constants of 2.3/2.2 and 4.0 MHz, respectively, disappeared after D₂O incubation in both states. The signals with 2.3/2.2 MHz hyperfine constants in S₀ and S₂ state multiline disappeared after 3 h of D₂O incubation in the S₀ and S₁ states, respectively. The signal with 4.0 MHz hyperfine constants in S₀ state multiline disappeared after 3 h of D₂O incubation in the S₀ state, while the similar signal in S₂ state multiline disappeared only after 24 h of D₂O incubation in the S₁ state. The different proton exchange rates seem to be ascribable to the change in affinities of water molecules to the variation in oxidation state of the Mn cluster during the water oxidation cycle. Based on the point dipole approximation, the distances between the center of electronic spin of the Mn cluster and the exchangeable protons were estimated to be 3.3/3.2 and 2.7 Å, respectively. These short distances suggest the protons belong to the water molecules ligated to the manganese cluster. We propose a model for the binding of water to the manganese cluster based on these results.     

The terminal reaction cascade of water oxidation: Proton and oxygen release

In cyanobacteria, algae and plants Photosystem II produces the oxygen we breathe. Driven and clocked by light quanta, the catalytic Mn₄Ca-tyrosine centre accumulates four oxidising equivalents before it abstracts four electrons from water, liberating dioxygen and protons. Aiming at intermediates of the terminal four-electron cascade, we previously have suppressed this reaction by elevating the oxygen pressure, thereby stabilising one redox intermediate. Here, we established a similar suppression by increasing the proton concentration. Data were analysed in terms of only one (peroxy) redox intermediate between the fourfold oxidised Mn₄Ca-tyrosine centre and oxygen release. The surprising result was that the release into the bulk of one proton per dioxygen is linked to the first and rate-limiting electron transfer in the cascade rather than to the second which produces free oxygen. The penultimate intermediate might thus be conceived as a fully deprotonated peroxy-moiety.     

Mono-manganese mechanism of the photosytem II water splitting reaction by a unique Mn4Ca cluster

The molecular mechanism of the water oxidation reaction in photosystem II (PSII) of green plants remains a great mystery in life science. This reaction is known to take place in the oxygen evolving complex (OEC) incorporating four manganese, one calcium and one chloride cofactors, that is light-driven to cycle four intermediates, designated S₀ through S₄, to produce four protons, five electrons and lastly one molecular oxygen, for indispensable resources in biosphere. Recent advancements of X-ray crystallography models established the existence of a catalytic Mn₄Ca cluster ligated by seven protein amino acids, but its functional structure is not yet resolved. The ¹⁸O exchange rates of two substrate water molecules were recently measured for four S_i-state samples (i = 0-3) leading to ³⁴O₂ and ³⁶O₂ formations, revealing asymmetric substrate binding sites significantly depending on the S_i-state. In this paper, we present a chemically complete model for the Mn₄Ca cluster and its surrounding enzyme field, which we found out from some possible models by using the hybrid density functional theoretic geometry optimization method to confirm good agreements with the 3.0 Å resolution PSII model [B. Loll, J. Kern, W. Saenger, A. Zouni , J. Biesiadka, Nature 438 (2005) 1040-1044] and the S-state dependence of ¹⁸O exchange rates [W. Hillier and T. Wydrzynski, Phys. Chem. Chem. Phys. 6 (2004) 4882-4889]. Furthermore, we have verified that two substrate water molecules are bound to asymmetric cis-positions on the terminal Mn ion being triply bridged (μ-oxo, μ-carboxylato, and a hydrogen bond) to the Mn₃CaO₃(OH) core, by developing a generalized theory of ¹⁸O exchange kinetics in OEC to obtain an experimental evidence for the cross exchange pathway from the slow to the fast exchange process. Some important experimental data will be discussed in terms of this model and its possible tautomers, to suggest that a cofactor, Cl⁻ ion, may be bound to CP43-Arg357 nearby Ca²⁺ ion and that D1-His337 may be used to trap a released proton only in the S₂-state.     

Towards the mechanism of proton pumping by the haem-copper oxidases

The haem-copper oxidases comprise a large family of enzymes that is widespread among aerobic organisms. These remarkable membrane-bound proteins catalyse the respiratory reduction of dioxygen to water, and conserve free energy from this reaction by operating as proton pumps. The mechanism of redox-dependent proton translocation has been elusive despite the availability of high resolution crystal structures from several oxidases. Here, we discuss some recent as well as some older results that may shed light on this mechanism. We conclude that proton-pumping is initiated by vectorial proton transfer from a conserved glutamic acid (Glu242 in the bovine enzyme) to a proton acceptor above the haem groups, and that this primary event is mechanistically coupled to electron transfer from haem a to the binuclear haem a₃/Cu_B centre. Subsequently, Glu242 is reprotonated from the negatively charged side of the membrane. Next this proton is transferred to the binuclear site to complete the chemistry, Glu242 is reprotonated once more, and the “prepumped” proton is ejected on the opposite side of the membrane. The different kinetics of electron-coupled proton transfer in different steps of the catalytic cycle may be related to differences in the driving force due to different E_m values of the electron acceptor in the binuclear site.