Reaction pattern and mechanism of light induced oxidative water splitting in photosynthesis

This mini review is an attempt to briefly summarize our current knowledge on light driven oxidative water splitting in photosynthesis. The reaction leading to molecular oxygen and four protons via photosynthesis comprises thermodynamic and kinetic constraints that require a balanced fine tuning of the reaction coordinates. The mode of coupling between electron (ET) and proton transfer (PT) reactions is shown to be of key mechanistic relevance for the redox turnover of Y_Z and the reactions within the WOC. The WOC is characterized by peculiar energetics of its oxidation steps in the WOC. In all oxygen evolving photosynthetic organisms the redox state S₁ is thermodynamically most stable and therefore this general feature is assumed to be of physiological relevance. Available information on the Gibbs energy differences between the individual redox states S_i+1 and S_i and on the activation energies of their oxidative transitions are used to construct a general reaction coordinate of oxidative water splitting in photosystem II (PS II). Finally, an attempt is presented to cast our current state of knowledge into a mechanism of oxidative water splitting with special emphasis on the formation of the essential O-O bond and the active role of the protein environment in tuning the local proton activity that depends on time and redox state S_i. The O-O linkage is assumed to take place within a multistate equilibrium at the redox level of S₃, comprising both redox isomerism and proton tautomerism. It is proposed that one state, S₃(P), attains an electronic configuration and nuclear geometry that corresponds with a hydrogen bonded peroxide which acts as the entatic state for the generation of complexed molecular oxygen through S₃(P) oxidation by Y_Z^ox.     

Water cleavage by solar radiation—an inspiring challenge of photosynthesis research

Solar energy exploitation by photosynthetic water cleavage is of central relevance for the development and sustenance of all higher forms of living matter in the biosphere. The key steps of this process take place within an integral protein complex referred to as Photosystem II (PS II) which is anisotropically incorporated into the thylakoid membrane. This minireview concentrates on mechanistic questions related to i) the generation of strongly oxidizing equivalents (holes) at a special chlorophyll a complex (designated as P680) and ii) the cooperative reaction of four holes with two water molecules at a manganese containing unit WOC (water oxidizing complex) resulting in the release of molecular oxygen and four protons. The classical work of Pierre Joliot and Bessel Kok and their coworkers revealed that water oxidation occurs via a sequence of univalent oxidation steps including intermediary redox states S_i (i = number of accumulated holes within the WOC). Based on our current stage of knowledge, an attempt is made a) to identify the nature of the redox states S_i, b) to describe the structural arrangement of the (four) manganese centers and their presumed coordination and ligation within the protein matrix, and c) to propose a mechanism of photosynthetic water oxidation with special emphasis on the key step, i.e. oxygen-oxygen bond formation. It is assumed that there exists a dynamic equilibrium in S₃ with one state attaining the nuclear geometry and electronic configuration of a complexed peroxide. This state is postulated to undergo direct oxidation to complexed dioxygen by univalent electron abstraction with Y_Z ^ox and simultaneous internal ligand to metal charge transfer.Key questions on the mechanism will be raised. The still fragmentary answers to these questions not only reflect our limited knowledge but also illustrate the challenges for future research.Abbreviations b559 cytochrome b559 - BChl bacteriochlorophyll - Chl chlorophyll - CP47 Chl a containing a 47 kDa polypeptide - D1/D2 polypeptides of the PS II reaction center - ENDOR electron nuclear double resonance - EPR electron paramagnetic resonance - ESEEM electron spin echo envelope modulation - EXAFS extended X-ray absorption fine structure - FTIR Fourier transform infrared - NMR nuclear magnetic resonance - P680, P700 photoactive Chl a of PS II and PS I, respectively - PS II Photosystem II - Q_A special plastoquinone of PS II - S_i redox states of WOC - WOC water oxidizing complex - WOS water oxidizing site - UV/VIS ultraviolet/visible - Y_D, Y_Z redox active tyrosines of polypeptides D2 and D1, respectively     

Mechanistic and structural aspects of photosynthetic water oxidation

Conclusions on the functional and structural organisation of photosynthetic water oxidation are gathered from a critical survey of the wealth of data reported in the literature and author's own experimental research: (1) the water oxidising complex (WOC) contains a tetranuclear manganese cluster of dimer of dimers' structure and functional heterogeneity of the metal centers, (2) the four step univalent oxidative pathway leading to water oxidation into molecular oxygen and four protons comprises only manganese, tyrosine Y_Z of polypeptide Dl and the substrate as redox active species, (3) the redox transitions S₀→ S₁ and S₁→ S₂ are manganese centered whereas S₂→ S₃ is most likely a ligand-centered reaction, (4) there exist several lines of evidence for a marked structural change that accompanies the redox transition S₂→ S₃, (5) one Ca²⁺ is an indispensible constituent of a functionally competent WOC while the role of Cl is much less clear and a direct participation disputable, (6) substrate water is most likely bound in all redox states S₀,…,S₃ and exchangeable with the bulk phase. The protonation state is determined by the redox state S₁ and the protein microenvironment. A mechanism is proposed for water oxidation in the WOC that is based on three key postulates: (1) water oxidation takes place in the first coordination sphere of one manganese dimer [Mn_aMn_b]; (2) the essential O-O bond is preformed in S₃ as part of a rapid redox isomerism S₃(I)→S₃(II) where in S₃(II) a nuclear geometry and electronic configuration is attained that corresponds to a peroxidic-type species; and (3) S₃(II) is an ‘entatic state’ for the formation of complexed dioxygen triggered by Y_Z^OX induced electron abstraction from the WOC and electronic redistribution to S₀(O₂).     

Application of the Marcus theory for analysis of the temperature dependence of the reactions leading to photosynthetic water oxidation: results and implications

The temperature dependence of donor side reactions was analysed within the framework of the Marcus theory of nonadiabatic electron transfer. The following results were obtained for PS II membrane fragments from spinach: (1) the reorganisation energy of P680^+? reduction by Y_Z is of the order of 0.5?eV in samples with a functionally fully competent water oxidising complex (WOC); (2) destruction of the WOC by Tris-washing gives rise to a drastic increase of λ to values of the order of 1.6?eV; (3) the reorganisation energies of the oxidation steps in the WOC are dependent, on the redox states S _i with values of about 0.6?eV for the reactions Y_Z ^OX S ₀→Y_Z S ₁ and Y_Z ^OX S ₁→Y_Z S ₂, 1.6?eV for the reaction Y_Z ^OX S ₂→Y_Z S ₃ and 1.1?eV (above a characteristic temperature u_c of about 6??°C) for the reaction Y_Z ^OX S ₃→→Y_Z S ₀+O₂. Using an empirical rate constant-distance relationship, the van der Waals distance between Y_Z and P680 was found to be about 10?Å, independent of the presence or absence of the WOC, whereas the distance between Y_Z and the manganese cluster in the WOC was ≥15?Å. Based on the calculated activation energies the environment of Y_Z is inferred to be almost "dry" and hydrophobic when the WOC is intact but becomes enriched with water molecules after WOC destruction. Furthermore, it is concluded that the transition S ₂→S ₃ is an electron transfer reaction gated by a conformational change, i.e. it comprises significant structural changes of functional relevance. Measurements of kinetic H/D isotope exchange effects support the idea that none of these reactions is gated by the break of a covalent O-H bond. The implications of these findings for the mechanism of water oxidation are discussed.     

Photosystem II: Structure and mechanism of the water:plastoquinone oxidoreductase

This mini-review briefly summarizes our current knowledge on the reaction pattern of light-driven water splitting and the structure of Photosystem II that acts as a water:plastoquinone oxidoreductase. The overall process comprises three types of reaction sequences: (a) light-induced charge separation leading to formation of the radical ion pair P680^+•Q_A^−•; (b) reduction of plastoquinone to plastoquinol at the Q_B site via a two-step reaction sequence with Q_A^−• as reductant and (c) oxidative water splitting into O₂ and four protons at a manganese-containing catalytic site via a four-step sequence driven by P680^+• as oxidant and a redox active tyrosine Y_Z acting as mediator. Based on recent progress in X-ray diffraction crystallographic structure analysis the array of the cofactors within the protein matrix is discussed in relation to the functional pattern. Special emphasis is paid on the structure of the catalytic sites of PQH₂ formation (Q_B-site) and oxidative water splitting (Mn₄O _x Ca cluster). The energetics and kinetics of the reactions taking place at these sites are presented only in a very concise manner with reference to recent up-to-date reviews. It is illustrated that several questions on the mechanism of oxidative water splitting and the structure of the catalytic sites are far from being satisfactorily answered.     

Studies on the electron transfer from Tyr-161 of polypeptide D-1 to P680+ in PS II membrane fragments from spinach

The functional connection between redox component Y _z identified as Tyr-161 of polypeptide D-1 (Debus et al. 1988) and P680⁺ was analyzed by measurements of laser flash induced absorption changes at 830 nm in PS II membrane fragments from spinach. It was found that neither DCMU nor the ADRY agent 2-(3-chloro-4-trifluoromethyl) anilino-3,5-dinitrothiophene (ANT 2p) affects the rate of P680⁺ reduction by Y _z under conditions where the catalytic site of water oxidation stays in the redox state S₁. In contrast to that, a drastic retardation is observed after mild trypsin treatment at pH=6.0. This effect which is stimualted by flash illumination can be largely reversed by Ca²⁺. The above mentioned data lead to the following conclusions: (a) the segment of polypeptide D-1 containing Tyr-161 and coordination sites of P680 is not allosterically affected by structural changes due to DCMU binding at the Q_B-site which is also located in D-1. (b) ANT 2p as a strong protonophoric uncoupler and ADRY agent does not modify the reaction coordinate of P680⁺ reduction by Y _z , and (c) Ca²⁺ could play a functional role for the electronic and vibrational coupling between the redox groups Y _z and P680. The electron transport from Y _z to P680⁺ is discussed within the framework of a nonadiabatic process. Based on thermodynamic considerations the reorganization energy is estimated to be in the order of 0.5 V.Abbreviations ADRY acceleration of the deactivation reactions of the water splitting enzyme system Y - ANT 2p 2-(3-chloro-4-trifluoromethyl)anilino-3,5 dinitrothiophene - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - MES 2[N-Morpholino]ethanesulfonic acid - PS II photosystem II - Q_A, Q_B primary and secondary plastoquinone acceptor of photosystem II - S _i redox states of the catalytic site of water oxidation - Y _z redox active Tyr-161 of polypeptide D-1     

Electron transfer from Cyt b 559 and tyrosine-D to the S2 and S3 states of the water oxidizing complex in photosystem II at cryogenic temperatures

The Mn₄CaO₅ cluster of photosystem II (PSII) catalyzes the oxidation of water to molecular oxygen through the light-driven redox S-cycle. The water oxidizing complex (WOC) forms a triad with Tyrosine_Z and P₆₈₀, which mediates electrons from water towards the acceptor side of PSII. Under certain conditions two other redox-active components, Tyrosine_D (Y_D) and Cytochrome b ₅₅₉ (Cyt b ₅₅₉) can also interact with the S-states. In the present work we investigate the electron transfer from Cyt b ₅₅₉ and Y_D to the S₂ and S₃ states at 195 K. First, Y_D ^? and Cyt b ₅₅₉ were chemically reduced. The S₂ and S₃ states were then achieved by application of one or two laser flashes, respectively, on samples stabilized in the S₁ state. EPR signals of the WOC (the S₂-state multiline signal, ML-S₂), Y_D ^? and oxidized Cyt b ₅₅₉ were simultaneously detected during a prolonged dark incubation at 195 K. During 163 days of incubation a large fraction of the S₂ population decayed to S₁ in the S₂ samples by following a single exponential decay. Differently, S₃ samples showed an initial increase in the ML-S₂ intensity (due to S₃ to S₂ conversion) and a subsequent slow decay due to S₂ to S₁ conversion. In both cases, only a minor oxidation of Y_D was observed. In contrast, the signal intensity of the oxidized Cyt b ₅₅₉ showed a two-fold increase in both the S₂ and S₃ samples. The electron donation from Cyt b ₅₅₉ was much more efficient to the S₂ state than to the S₃ state.     

Models for Proton-Coupled Electron Transfer in Photosystem II

The coupling of proton and electron transfers is a key part of the chemistry of photosynthesis. The oxidative side of photosystem II (PS II) in particular seems to involve a number of proton-coupled electron transfer (PCET) steps in the S-state transitions. This mini-review presents an overview of recent studies of PCET model systems in the authors’ laboratory. PCET is defined as a chemical reaction involving concerted transfer of one electron and one proton. These are thus distinguished from stepwise pathways involving initial electron transfer (ET) or initial proton transfer (PT). Hydrogen atom transfer (HAT) reactions are one class of PCET, in which H⁺ and e ⁻ are transferred from one reagent to another: AH+B→A+BH, roughly along the same path. Rate constants for many HAT reactions are found to be well predicted by the thermochemistry of hydrogen transfer and by Marcus Theory. This includes organic HAT reactions and reactions of iron-tris(α-diimine) and manganese-(μ-oxo) complexes. In PS II, HAT has been proposed as the mechanism by which the tyrosine Z radical (Y_Z) oxidizes the manganese cluster (the oxygen evolving complex, OEC). Another class of PCET reactions involves transfer of H⁺ and e ⁻ in different directions, for instance when the proton and electron acceptors are different reagents, as in AH–B+C⁺→A–HB⁺+C. The oxidation of Y_Z by the chlorophyll P680 + has been suggested to occur by this mechanism. Models for this process – the oxidation of phenols with a pendent base – are described. The oxidation of the OEC by Y_Z could also occur by this second class of PCET reactions, involving an Mn–O–H fragment of the OEC. Initial attempts to model such a process using ruthenium-aquo complexes are described. An erratum to this article can be found at     

Reaction pattern and mechanism of light induced oxidative water splitting in photosynthesis

This mini review is an attempt to briefly summarize our current knowledge on light driven oxidative water splitting in photosynthesis. The reaction leading to molecular oxygen and four protons via photosynthesis comprises thermodynamic and kinetic constraints that require a balanced fine tuning of the reaction coordinates. The mode of coupling between electron (ET) and proton transfer (PT) reactions is shown to be of key mechanistic relevance for the redox turnover of Y(Z) and the reactions within the WOC. The WOC is characterized by peculiar energetics of its oxidation steps in the WOC. In all oxygen evolving photosynthetic organisms the redox state S(1) is thermodynamically most stable and therefore this general feature is assumed to be of physiological relevance. Available information on the Gibbs energy differences between the individual redox states S(i+1) and S(i) and on the activation energies of their oxidative transitions are used to construct a general reaction coordinate of oxidative water splitting in photosystem II (PS II). Finally, an attempt is presented to cast our current state of knowledge into a mechanism of oxidative water splitting with special emphasis on the formation of the essential O-O bond and the active role of the protein environment in tuning the local proton activity that depends on time and redox state S(i). The O-O linkage is assumed to take place within a multistate equilibrium at the redox level of S(3), comprising both redox isomerism and proton tautomerism. It is proposed that one state, S(3)(P), attains an electronic configuration and nuclear geometry that corresponds with a hydrogen bonded peroxide which acts as the entatic state for the generation of complexed molecular oxygen through S(3)(P) oxidation by Y(Z)(ox).     

The reduced S<Subscript>−1</Subscript> and S<Subscript>−2</Subscript> oxidation states of the O<Superscript>2</Superscript>-evolving complex of photosystem II: An EPR microwave power saturation study

Progressive microwave power saturation (P_1/2) measurements have been performed on the tyrosine D radical (Y_D ^•) of photosystem II (PSII) in order to examine its relaxation enhancement by the oxygen-evolving complex (OEC) poised to the reduced S₋₁ and S₋₂ oxidation states by NO treatment. Analysis of the power saturation curves showed that the S₋₁ oxidation state of the OEC does not enhance the relaxation of Y_D ^•: it therefore possesses a diamagnetic ground state. In contrast, the Mn(II)-Mn(III) multiline electron paramagnetic resonance (EPR) signal characteristic of the S₋₂ oxidation state of the OEC was shown to provide a relaxation enhancement pathway for Y_D ^•, however less efficient relative to the one provided by the S₂-state multiline EPR signal. We also examined the Y_D ^• relaxation enhancement characteristics of the EPR-silent oxidation state produced after brief (1–5 min) dark incubation at 0°C of a PSII sample poised to the EPRactive S₋₂ state. This EPR-silent oxidation state denoted as “0°C incubation” state was shown to possess remarkably similar P_1/2 values with the EPR-active S₋₂ state in the overall examined temperature range (6–20 K). In addition, these values remained unchanged after successive cycles of the OEC between the EPR-active S₋₂ state and the “0°C incubation” state. The data presented in this work point to the conclusion that the “0°C incubation” state is indeed an S₋₂ oxidation state with half-integer spin.     

Interactions of photosystem II with bicarbonate, formate and acetate

In this study, we probe the effects of bicarbonate (hydrogencarbonate), BC, removal from photosystem II in spinach thylakoids by measuring flash-induced oxygen evolution patterns (FIOPs) with a Joliot-type electrode. For this we compared three commonly employed methods: (1) washing in BC-free medium, (2) formate addition, and (3) acetate addition. Washing of the samples with buffers depleted of BC and CO₂ by bubbling with argon (Method 1) under our conditions leads to an increase in the double hit parameter of the first flash (β₁), while the miss parameter and the overall activity remain unchanged. In contrast, addition of 40–50 mM formate or acetate results in a significant increase in the miss parameter and to an ∼50% (formate) and ∼10% (acetate) inhibition of the overall oxygen evolution activity, but not to an increased β₁ parameter. All described effects could be reversed by washing with formate/acetate free buffer and/or addition of 2–10 mM bicarbonate. The redox potential of the water-oxidizing complex (WOC) in samples treated by Method 1 is compared to samples containing 2 mM bicarbonate in two ways: (1) The lifetimes of the S₀, S₂, and S₃ states were measured, and no differences were found between the two sample types. (2) The S₁, S₀, S₋₁, and S₋₂ states were probed by incubation with small concentrations of NH₂OH. These experiments displayed a subtle, yet highly reproducible difference in the apparent S_i/S_−i state distribution which is shown to arise from the interaction of BC with PSII in the already reduced states of the WOC. These data are discussed in detail by also taking into account the CO₂ concentrations present in the buffers after argon bubbling and during the measurements. These values were measured by membrane-inlet mass spectrometry (MIMS).     

On the chlorophyll a fluorescence yield in chloroplasts upon excitation with twin turnover flashes (TTF) and high frequency flash trains

Chlorophyll fluorescence is routinely taken as a quantifiable measure of the redox state of the primary quinone acceptor Q_A of PSII. The variable fluorescence in thylakoids increases in a single turnover flash (STF) from its low dark level F _o towards a maximum F _m^STF when Q_A becomes reduced. We found, using twin single turnover flashes (TTFs) that the fluorescence increase induced by the first twin-partner is followed by a 20–30% increase when the second partner is applied within 20–100 μs after the first one. The amplitude of the twin response shows a period-of-four oscillation associated with the 4-step oxidation of water in the Kok cycle (S states) and originates from two different trapped states with a life time of 0.2–0.4 and 2–5 ms, respectively. The oscillation is supplemented with a binary oscillation associated with the two-electron gate mechanism at the PSII acceptor side. The F(t) response in high frequency flash trains (1–4 kHz) shows (i) in the first 3–4 flashes a transient overshoot 20–30% above the F _m^STF = 3*F _o level reached in the 1st flash with a partial decline towards a dip D in the next 2–3 ms, independent of the flash frequency, and (ii) a frequency independent rise to F _m = 5*F _o in the 3–60 ms time range. The initial overshoot is interpreted to be due to electron trapping in the S₀ fraction with Q_B-nonreducing centers and the dip to the subsequent recovery accompanying the reoxidation of the double reduced acceptor pair in these RCs after trapping. The rise after the overshoot is, in agreement with earlier findings, interpreted to indicate a photo-electrochemical control of the chlorophyll fluorescence yield of PSII. It is anticipated that the double exciton and electron trapping property of PSII is advantageous for the plant. It serves to alleviate the depression of electron transport in single reduced Q_B-nonreducing RCs, associated with electrochemically coupled proton transport, by an increased electron trapping efficiency in these centers.     

Theoretical study of the hydroxylation of phenolates by the Cu2O2(N,N′-dimethylethylenediamine)2 2+ complex

Tyrosinase catalyzes the ortho hydroxylation of monophenols and the subsequent oxidation of the diphenolic products to the resulting quinones. In efforts to create biomimetic copper complexes that can oxidize C–H bonds, Stack and coworkers recently reported a synthetic μ-η²:η²-peroxodicopper(II)(DBED)₂ complex (DBED is N,N′-di-tert-butylethylenediamine), which rapidly hydroxylates phenolates. A reactive intermediate consistent with a bis-μ-oxo-dicopper(III)-phenolate complex, with the O–O bond fully cleaved, is observed experimentally. Overall, the evidence for sequential O–O bond cleavage and C–O bond formation in this synthetic complex suggests an alternative mechanism to the concerted or late-stage O–O bond scission generally accepted for the phenol hydroxylation reaction performed by tyrosinase. In this work, the reaction mechanism of this peroxodicopper(II) complex was studied with hybrid density functional methods by replacing DBED in the μ-η²:η²-peroxodicopper(II)(DBED)₂ complex by N,N′-dimethylethylenediamine ligands to reduce the computational costs. The reaction mechanism obtained is compared with the existing proposals for the catalytic ortho hydroxylation of monophenol and the subsequent oxidation of the diphenolic product to the resulting quinone with the aim of gaining some understanding about the copper-promoted oxidation processes mediated by 2:1 Cu(I)O₂-derived species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

EPR studies of the water oxidizing complex in the S1 and the higher S states: the manganese cluster and YZ radical

The parallel polarization electron paramagnetic resonance (EPR) method has been applied to investigate manganese EPR signals of native S₁ and S₃ states of the water oxidizing complex (WOC) in photosystem (PS) II. The EPR signals in both states were assigned to thermally excited states with S=1, from which zero-field interaction parameters D and E were derived. Three kinds of signals, the doublet signal, the singlet-like signal and g=11–15 signal, were detected in Ca²⁺-depleted PS II. The g=11–15 signal was observed by parallel and perpendicular modes and assigned to a higher oxidation state beyond S₂ in Ca²⁺-depleted PS II. The singlet-like signal was associated with the g=11–15 signal but not with the Y_Z (the tyrosine residue 161 of the D1 polypeptide in PS II) radical. The doublet signal was associated with the Y_Z radical as proved by pulsed electron nuclear double resonance (ENDOR) and ENDOR-induced EPR. The electron transfer mechanism relevant to the role of Y_Z radical was discussed.     

Isotope Systematics of Sulfate-oxygen and Sulfate-sulfur in Six European Peatlands

Oxygen (O) and sulfur (S) isotope systematics in bog water sulfates were determined for six Sphagnum dominated wetlands located in the British Isles and the Czech Republic, Central Europe. Comparison of a polluted and unpolluted site showed that 4 times higher atmospheric S inputs led to 3 times higher bog water sulfate concentrations and substrate S concentrations, 3 times increased ranges of substrate S concentrations, and 3 times increased ranges of δ³⁴S values. Sites with elevated atmospheric S inputs exhibited greater geochemical variability in wetland S species. Sulfate O–S isotope composition of bog pore water at a depth of 40 cm below surface differed from that of surface bog water, indicating that dissimilatory bacterial sulfate reduction, a process known to discriminate against the heavier isotopes ¹⁸O and ³⁴S, occurred in surface peat layers. While bacterial sulfate reduction remained to be one of the main isotope-selective processes for sulfate in peat, it could not fully explain the O–S isotope systematics of peat waters. The ‘residual’ sulfate was not simultaneously enriched in the heavier isotopes ¹⁸O and ³⁴S. Mixing of residual sulfate following bacterial sulfate reduction with the product of S²⁻ reoxidation, cleavage of esters, and isotope exchange reactions may have contributed to the decoupling of the δ³⁴S_so4 and δ¹⁸S_so4 values. Large within-site differences in δ¹⁸S_so4 and δ³⁴S_so4 (up to 13 and 15‰, respectively) indicated little communication between the 0 and 40 cm peat depth at some sites. Extremely high δ¹⁸S_so4 and δ³⁴S_so4 values found in several peat bog water samples from Connemara (Ireland), Thorne Moors (England) and Ocean (Czech Republic) were not seen in streams draining the wetlands. Direct runoff of atmogenic sulfate constituted a significant portion of the bog outflow. At the wetland scale, zones of dissimilatory bacterial sulfate reduction form pockets whose lateral hydrological fluxes are small.     

Volume changes and electrostriction in the primary photoreactions of various photosynthetic systems: estimation of dielectric coefficient in bacterial reaction centers and of the observed volume changes with the Drude–Nernst equation

Photoacoustics (PA) allows the determination of enthalpy and volume changes of photoreactions in photosynthetic reaction centers on the 0.1–10 μs time scale. These include the bacterial centers from Rb. sphaeroides, PS I and PS II centers from Synechocystis and in whole cells. In vitro and in vivo PA data on PS I and PS II revealed that both the volume change (–26 A³) and reaction enthalpy (–0.4 eV) in PS I are the same as those in the bacterial centers. However the volume change in PS II is small and the enthalpy far larger, –1 eV. Assigning the volume changes to electrostriction allows a coherent explanation of these observations. One can explain the large volume decrease in the bacterial centers with an effective dielectric coefficient of ∼4. This is a unique approach to this parameter so important in estimation of protein energetics. The value of the volume contraction for PS I can only be explained if the acceptor is the super- cluster (Fe₄S₄)(Cys₄) with charge change from –1 to –2. The small volume change in PS II is explained by sub-μs electron transfer from Y_Z anion to P₆₈₀ cation, in which charge is only moved from the Y_Z anion to the Q_A with no charge separation or with rapid proton transfer from oxidized Y_Z to a polar region and thus very little change in electrostriction. At more acid pH equally rapid proton transfer from a neighboring histidine to a polar region may be caused by the electric field of the P₆₈₀ cation. This revised version was published online in June 2006 with corrections to the Cover Date.     

The origin of split EPR signals in the Ca2+-depleted Photosystem II

A light-driven reaction model for the Ca²⁺-depleted Photosystem (PS) II is proposed to explain the split signal observed in electron paramagnetic resonance (EPR) spectra based on a comparison of EPR assignments with recent x-ray structural data. The split signal has a splitting linewidth of 160 G at around g = 2 and is seen upon illumination of the Ca²⁺-depleted PS II in the S₂ state associated with complete or partial disappearance of the S₂ state multiline signal. Another g=2 broad ESR signal with a 110 G linewidth was produced by 245 K illumination for a short period in the Ca²⁺-depleted PS II in S₁ state. At the same time a normal Y_Z^· radical signal was also efficiently trapped. The g=2 broad signal is attributed to an intermediate S₁X^· state in equilibrium with the trapped Y_Z^· radical. Comparison with x-ray structural data suggests that one of the split signals (doublet signal) is attributable to interaction between His 190 and the Y_Z^· radical, and other signals is attributable to interaction between His 337 and the manganese cluster, providing further clues as to the mechanism of water oxidation in photosynthetic oxygen evolution.     

Involvement of the donor tyrosine-D1 (Yd) in Photosystem II electron transport in the green alga, Chlamydobotrys stellata

The light-induced oxidation of the accessory donor tyrosine-D (Y_D) has been studied by measurements of the EPR Signal II_slow at room temperature in the autotrophically and photoheterotrophically cultivated alga Chlamydobotrys stellata. After illumination and dark adaptation, Y_D Signal II_slow was observed only in autotrophic algae, i.e. under conditions of a linear photosynthetic electron transfer from water to NADP⁺. The addition of artificial electron acceptors phenyl-p-benzoquinone (PPQ) or dichloro-p-benzoquinone (DCQ) to the autotrophic cells caused an almost negligible increase of this signal. When photosynthetic electron flow and oxygen evolution were diminished by removal of the carbon source CO₂ and addition of acetate (photoheterotrophy), a pronounced Y_D Signal II_slow was seen only in presence of DCQ or PPQ. Several possibilities are discussed to explain the absence of Y_D Signal II_slow in photoheterotrophic Chl. stellata such as the existence of a cyclic PS II electron flow very effectively reducing P₆₈₀ and thereby preventing the possibility of Y_D oxidation. Artificial electron acceptors withdraw electrons from this cycle thus keeping the primary quinone acceptor, Q_A, oxidized and thereby diminishing the reduction of P₆₈₀ ⁺ by cyclic PSII. This leads to the appearance of the Y_D Signal II_slow also in the photoheterotrophically grown algae.Abbreviations A-band- thermoluminescence band associated with S₂Q_A ^- charge recombination - DCQ- 2,5-dichlorobenzoquinone - D₂- structure protein of Photosystem II - EPR- electron paramagnetic resonance - OEC- oxygen evolving complex - PPQ- phenyl-p-benzoquinone - PS II- Photosystem II - P₆₈₀- reaction center of Photosystem II - Q-band- thermoluminescence band associated with S₂Q_A ^- charge recombination - S_i- oxidation levels of the OEC - Y_D- tyrosine-D accessory donor to P₆₈₀ - Y_Z- tyrosine-Z electron donor to P₆₈₀ Dedicated to Prof. Dr E. Schnepf/Heidelberg.     

Phosphate Sorption and Desorption on Pyrite in Primitive Aqueous Scenarios: Relevance of acidic → Alkaline Transitions

Phosphate (P _i) sorption assays onto pyrite in media simulating primeval aquatic scenarios affected by hydrothermal emissions, reveal that acidic conditions favour P _i sorption whereas mild alkaline media – as well as those simulating sulfur oxidation to SO²⁻ ₄ – revert this capture process. Several mechanisms relevant to P _i availability in prebiotic eras are implicated in the modulation of these processes. Those favouring sorption are: (a) hydrophobic coating of molecules, such as acetate that could be formed in the vicinity of hydrothermal vents; (b) water and Mg²⁺ bridging in the interface mineral-aqueous media; (c) surface charge neutralization by monovalent cations (Na⁺ and K⁺). The increase of both the medium pH and the SO²⁻ ₄ trapping by the mineral interface would provoke the release of sorbed P _i due to charge polarization. Moreover it is shown that P _i self-modulates its sorption, a mechanism that depends on the abundance of SO²⁻ ₄ in the interface. The relevance of the proposed mechanisms of P _i capture, release and trapping arises from the need of abundant presence of this molecule for primitive phosphorylations, since – similarly to contemporary aqueous media – inorganic phosphate concentrations in primitive seas should have been low. It is proposed that the presence of sulphide minerals with high affinity to P _i could have trapped this molecule in an efficient manner, allowing its concentration in specific niches. In these niches, the conditions studied in the present work would have been relevant for its availability in soluble form, specially in primitive insulated systems with pH gradients across the wall. R B-L and Y C-S contributed equally to this work; recipients of fellowships from the Brazilian National Research Council in the PIBIC and PINC-School of Medicine programs of the Universidade Federal de Rio de Janeiro     

Inhibition of the catalase reaction of Photosystem II by anions

A new binding site for anions which inhibit the water oxidizing complex (WOC) of Photosystem II in spinach has been identified. Anions which bind to this site inhibit the flash-induced S₂/S₀ catalase reaction (2H₂O₂2H₂O+O₂) of the WOC by displacing hydrogen peroxide. Using a mass spectrometer and gas permeable membrane to detect the ³²O₂ product, the yield and lifetime of the active state of the flash-induced catalase (to be referred to simply as flash-catalase) reaction were measured after forming the S₂ or S₀-states by a short flash. The increase in flash-catalase activity with H₂O₂ concentration exhibits a K_m=10–20 mM, and originates from an increase in the lifetime by 20-fold of the active state. The increased lifetime in the presence of peroxide is ascribed to formation of the long-lived S₀-state at the expense of the unstable S₂-state. The anion inhibition site differs from the chloride site involved in stimulating the photolytic water oxidation reaction (2H₂OO₂+4e^-+4H⁺). Whereas water oxidation requires Cl^- and is inhibited with increasing effectiveness by F^-CN^-N₃ ^-, the flash-catalase reaction is weakly inhibited by Cl^-, and with increasing effectiveness by F^-CN^-, N₃ ^-. Unlike water oxidation, chloride is unable to suppress or reverse inhibition of the flash-catalase reaction caused by these anions. The inhibitor effectiveness correlates with the pK_a of the conjugate acid, suggesting that the protonated species may be the active inhibitor. The reduced activity arises from a shortening of the lifetime of the flash-induced catalase active state by 3–10 fold owing to stronger anion binding in the flash-induced states, S₂ and S₀, than in the dark S-states, S₁ and S_-1. To account for the paradoxical result that higher anion concentrations are required to inhibit at lower H₂O₂ concentrations, where S₂ forms initially after the flash, than at higher H₂O₂ concentrations, where S₀ forms initially after the flash, stronger anion binding to the S₀-state than to the S₂-state is proposed. A kinetic model is given which accounts for these equilibria with anions and H₂O₂. The rate constant for the formation/release of O₂ by reduction of S₂ in the WOC is <0.4 s^-1.Abbreviations ADRY acceleration of the deactivation reactions of the water splitting enzyme system Y - BTP bis [tris(hydroxymethyl)methylamino]-propane - CCCP carbonylcyanide m-chlorophenylhyrazone - DCBQ 2,5-dichlorobenzoquinone - DMBQ 2,3-dimethylbenzoquinone - WOC water oxidizing complex