Comparative properties of hydroquinone and hydroxylamine reduction of the Ca(2+)-stabilized O2-evolving complex of photosystem II: reductant-dependent Mn2+ formation and activity inhibition.

Calcium binding to photosystem II slows NH2OH inhibition of O2 evolution; Mn2+ is retained by the O2-evolving complex [Mei, R., & Yocum, C. F. (1991) Biochemistry 30, 7836-7842]. This Ca(2+)-induced stability has been further characterized using the large reductant hydroquinone. Salt-washed photosystem II membranes reduced by hydroquinone in the presence of Ca2+ retain 80% of steady-state O2 evolution activity and contain about 2 Mn2+/reaction center that can be detected at room temperature by electron paramagnetic resonance. This Mn2+ produces a weak enhancement of H2O proton spin-lattice relaxation rates, cannot be easily extracted by a chelator, and is reincorporated into the O2-evolving complex upon illumination. A comparison of the properties of Ca(2+)-supplemented photosystem II samples reduced by hydroquinone or NH2OH alone or in sequence reveals the presence of a subpopulation of manganese atoms at the active site of H2O oxidation that is not accessible to facile hydroquinone reduction. At least one of these manganese atoms can be readily reduced by NH2OH following a noninhibitory hydroquinone reduction step. Under these conditions, about 3 Mn2+/reaction center are lost and O2 evolution activity is irreversibly inhibited. We interpret the existence of distinct sites of reductant action on manganese as further evidence that the Ca(2+)-binding site in photosystem II participates in regulation of the organization of manganese-binding ligands and the overall structure of the O2-evolving complex.     

Mechanism of photosynthetic water oxidation in the dimeric oxygen-evolving complex of chloroplast photosystem II

Based on the analysis of the molecular organization and properties of an isolated oxygen-evolving complex of photosystem II of plant chloroplasts, a mechanism of water oxidation and oxygen release during photosynthesis was proposed. It is suggested that the photolysis of water occurs in a dimeric oxygen-evolving complex consisting of two core complexes. In the region of contact of these complexes, a hydrophobic "boiler" is formed where the conditions for screening and stabilization of Z-linanded manganese cations accumulating positive charges for the oxidation of water molecules are created. A prerequisite to the photolysis of water is the formation of a binuclear [Mn(3+)-OH ... HO-Mn3+] hydroxyl-manganese associate, which appears in the dimeric oxygen-evolving complex after the first two light flashes as a result of photohydrolysis of photochemically oxidized Z-liganded manganese cations. The process is accompanied by the release of the first water protons to the medium. The photosynthetic oxidation of water hydroxyls occurs at the next stage and is considered as synchronous detachment of four electrons from two bound OH-groups of the associate upon photooxidation of Mn3+ cations to Mn4+ cations after two subsequent light flashes. This process is accompanied by the disproportionation of electron density and the formation of a bond between oxygen atoms of hydroxyls followed by the evolution of molecular oxygen and protons, and regeneration of two starting Mn2+ cations and the primary state of the system.     

Reduction-induced inhibition and Mn(II) release from the photosystem II oxygen-evolving complex by hydroquinone or NH2OH are consistent with a Mn(III)/Mn(III)/Mn(IV)/Mn(IV) oxidation state for the dark-adapted enzyme

Hydroxylamine and hydroquinone were used to probe the oxidation states of Mn in the oxygen-evolving complex of dark-adapted intact (hydroxylamine) and salt-washed (hydroquinone) photosystem II. These preparations were incubated in the dark for 24 h in the presence of increasing reductant/photosystem II ratios, and the loss of oxygen evolution activity and of Mn(II) was determined for each incubation mixture. Monte Carlo simulations of these data yielded models that provide insight into the structure, reactivity, and oxidation states of the manganese in the oxygen-evolving complex. Specifically, the data support oxidation states of Mn(III)(2)/Mn(IV)(2) for the dark stable S(1) state of the O(2)-evolving complex. Activity and Mn(II) loss data were best modeled by assuming an S(1) --> S(-)(1) conversion of intermediate probability, a S(-)(1) --> S(-)(3) reaction of high probability, and subsequent step(s) of low probability. This model predicts that photosystem II Mn clusters that have undergone an initial reduction step become more reactive toward a second reduction, followed by a slower third reduction step. Analysis of the Mn(II) release parameters used to model the data suggests that the photosystem II manganese cluster consists of three Mn atoms that exhibit a facile reactivity with both reductants, and a single Mn that is reducible but sterically trapped at or near its binding site. Activity assays indicate that intact photosystem II centers reduced to S(-)(1) can evolve oxygen upon illumination, but that these centers are inactive in preparations depleted of the extrinsic 23 and 17 kDa polypeptides. Finally, it was found that a substantial population of the tyrosine D radical is reduced by hydroxylamine, but a smaller population reacts with hydroquinone over the course of a 24 h exposure to the reductant.     

What Are the Oxidation States of Manganese Required To Catalyze Photosynthetic Water Oxidation?

Photosynthetic O(2) production from water is catalyzed by a cluster of four manganese ions and a tyrosine residue that comprise the redox-active components of the water-oxidizing complex (WOC) of photosystem II (PSII) in all known oxygenic phototrophs. Knowledge of the oxidation states is indispensable for understanding the fundamental principles of catalysis by PSII and the catalytic mechanism of the WOC. Previous spectroscopic studies and redox titrations predicted the net oxidation state of the S(0) state to be (Mn(III))(3)Mn(IV). We have refined a previously developed photoassembly procedure that directly determines the number of oxidizing equivalents needed to assemble the Mn(4)Ca core of WOC during photoassembly, starting from free Mn(II) and the Mn-depleted apo-WOC complex. This experiment entails counting the number of light flashes required to produce the first O(2) molecules during photoassembly. Unlike spectroscopic methods, this process does not require reference to synthetic model complexes. We find the number of photoassembly intermediates required to reach the lowest oxidation state of the WOC, S(0), to be three, indicating a net oxidation state three equivalents above four Mn(II), formally (Mn(III))(3)Mn(II), whereas the O(2) releasing state, S(4), corresponds formally to (Mn(IV))(3)Mn(III). The results from this study have major implications for proposed mechanisms of photosynthetic water oxidation.     

Reactions of hydroxylamine with the electron-donor side of photosystem II

The reaction of hydroxylamine with the O2-evolving center of photosystem II (PSII) in the S1 state delays the advance of the H2O-oxidation cycle by two charge separations. In this paper, we compare and contrast the reactions of hydroxylamine and N-methyl-substituted analogues with the electron-donor side of PSII in both O2-evolving and inactivated [tris(hydroxymethyl)aminomethane- (Tris-) washed] spinach PSII membrane preparations. We have employed low-temperature electron paramagnetic resonance (EPR) spectroscopy in order to follow the oxidation state of the Mn complex in the O2-evolving center and to detect radical oxidation products of hydroxylamine. When the reaction of hydroxylamine with the S1 state in O2-evolving membranes is allowed to proceed to completion, the S2-state multiline EPR signal is suppressed until after three charge separations have occurred. Chemical removal of hydroxylamine from treated PSII membrane samples prior to illumination fails to reverse the effects of the dark reaction, which argues against an equilibrium coordination of hydroxylamine to a site in the O2-evolving center. Instead, the results indicate that the Mn complex is reduced by two electrons by hydroxylamine, forming the S-1 state. An additional two-electron reduction of the Mn complex to a labile "S-3" state probably occurs by a similar mechanism, accounting for the release of Mn(II) ions upon prolonged dark incubation of O2-evolving membranes with high concentrations of hydroxylamine. In N,N-dimethylhydroxylamine-treated, Tris-washed PSII membranes, which lack O2 evolution activity owing to loss of the Mn complex, a large yield of dimethyl nitroxide radical is produced immediately upon illumination at temperatures above 0 degrees C. The dimethyl nitroxide radical is not observed upon illumination under similar conditions in O2-evolving PSII membranes, suggesting that one-electron photooxidations of hydroxylamine do not occur in centers that retain a functional Mn complex. We suggest that the flash-induced N2 evolution observed in hydroxylamine-treated spinach thylakoid membrane preparations arises from recombination of hydroxylamine radicals formed in inactivated O2-evolving centers.     

Water oxidation chemistry of photosystem II

The O(2)-evolving complex of photosystem II catalyses the light-driven four-electron oxidation of water to dioxygen in photosynthesis. In this article, the steps leading to photosynthetic O(2) evolution are discussed. Emphasis is given to the proton-coupled electron-transfer steps involved in oxidation of the manganese cluster by oxidized tyrosine Z (Y(*)(Z)), the function of Ca(2+) and the mechanism by which water is activated for formation of an O-O bond. Based on a consideration of the biophysical studies of photosystem II and inorganic manganese model chemistry, a mechanism for photosynthetic O(2) evolution is presented in which the O-O bond-forming step occurs via nucleophilic attack on an electron-deficient Mn(V)=O species by a calcium-bound water molecule. The proposed mechanism includes specific roles for the tetranuclear manganese cluster, calcium, chloride, Y(Z) and His190 of the D1 polypeptide. Recent studies of the ion selectivity of the calcium site in the O(2)-evolving complex and of a functional inorganic manganese model system that test key aspects of this mechanism are also discussed.     

Manganese proteins isolated from spinach thylakoid membranes and their role in O2 evolution. II. A binuclear manganese-containing 34 kilodalton protein, a probable component of the water dehydrogenase enzyme

Extraction conditions have been found which result in the retention of manganese to the 33-34 kDa protein, first isolated as an apoprotein by Kuwabara and Murata (Kuwabara, T. and Murata, N. (1979) Biochim. Biophys Acta 581, 228-236). By maintaining an oxidizing-solution potential, with hydrophilic and lipophilic redox buffers during protein extraction of spinach grana-thylakoid membranes, the 33-34 kDa protein is observed to bind a maximum of 2 Mn/protein which are not released by extended dialysis versus buffer. This manganese is a part of the pool of 4 Mn/Photosystem II normally associated with the oxygen-evolving complex. The mechanism for retention of Mn to the protein during isolation appears to be by suppression of chemical reduction of natively bound, high-valent Mn to the labile Mn(II) oxidation state. This protein is also present in stoichiometric levels in highly active, O2-evolving, detergent-extracted PS-II particles which contain 4-5 Mn/PS II. Conditions which result in the loss of Mn and O2 evolution activity from functional membranes, such as incubation in 1.5 mM NH2OH or in ascorbate plus dithionite, also release Mn from the protein. The protein exists as a monomer of 33 kDa by gel filtration and 34 kDa by gel electrophoresis, with an isoelectric point of 5.1 +/- 0.1. The protein exhibits an EPR spectrum only below 12 K which extends over at least 2000 G centered at g = 2 consisting of non-uniformly separated hyperfine transitions with average splitting of 45-55 G. The magnitude of this splitting is nominally one-half the splitting observed in monomeric manganese complexes having O or N donor ligands. This is apparently due to electronic coupling of the two 55Mn nuclei in a presumed binuclear site. Either a ferromagnetically coupled binuclear Mn2(III,III) site or an antiferromagnetically coupled mixed-valence Mn2(II,III) site are considered as possible oxidation states to account for the EPR spectrum. Qualitatively similar hyperfine structure splittings are observed in ferromagnetically coupled binuclear Mn complexes having even-spin ground states. The extreme temperature dependence suggests the population of low-lying excited states such as are present in weakly coupled dimers and higher clusters of Mn ions, or, possibly, from efficient spin relaxation such as occurs in the Mn(III) oxidation state. Either 1.5 mM NH2OH or incubation with reducing agents abolishes the low temperature EPR signal and releases two Mn(II) ions to solution. This is consistent with the presence of Mn(III) in the isolated protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Improvement by benzoquinones of the quantum yield of photoactivation of photosynthetic oxygen evolution: direct evidence for the two-quantum mechanism.

Effects of eight differently substituted 1,4-benzoquinones (BQs) on the quantum yield of photoactivation of oxygen evolution (reconstitution of the Mn cluster) were examined with wheat photosystem II (PSII) membranes depleted of the Mn cluster by treatment with 1.0 mM NH2OH. Illumination with 10 flashes at 0.25-s intervals of the PSII membranes in the presence of 2.0 mM Mn2+, 20 mM Ca2+, and 1.2 M Cl- restored 14% of oxygen-evolving activity destroyed by the NH2OH treatment. Among the benzoquinones tested, DBMIB (2,5-dibromo-3-methyl-6-isopropyl-BQ) and tetramethyl-BQ did not enhance the activity recovery, but all the others doubled the recovery when present at their optimal concentrations during illumination. The order of effectiveness was tetrabromo-, phenyl-, and 2,6-dichloro-BQs greater than or equal to 2,5-dichloro-BQ greater than tetrachloro-BQ greater than 2,5-dimethyl-BQ, though the differences were small. This order reflects their efficiencies as electron acceptors of PSII. This finding, together with others, suggests that the enhancement of activity recovery results from rapid oxidation by the benzoquinones of the reduced form of the quinone acceptors in PSII, QA- and QB-, which cause loss of an oxidized intermediate through charge-recombination reaction with Mn3+. The flash-number dependence of the recovery of oxygen-evolving activity indicated that the activity was not restored after one flash but recovered significantly after illumination with two flashes and then further increased upon additional flashes. This provides direct evidence that the minimum quantum requirement for photoactivation is two.     

Ethanol inhibits leptin-induced STAT3 activation in Huh7 cells

The manganese (Mn) complex of photosystem II catalyzes water oxidation. For the first time, its advancement through the reaction cycle was monitored by time-resolved X-ray absorption measurements at the Mn K-edge at room temperature. The complex was stepped through its four oxidation states by nano-second-laser flashes applied to samples exposed to the X-ray beam. Time courses of the X-ray fluorescence intensity were recorded during a flash sequence. Extended X-ray absorption fine-structure spectra were recorded with the S(1), S(2), and S(3) oxidation states highly populated. The room temperature data is compatible with the formation of a third di-mu-oxo bridge between two Mn atoms upon the S(2)-->S(3) transition.     

Mechanism of photosynthetic water oxidation: combining biophysical studies of photosystem II with inorganic model chemistry

A mechanism for photosynthetic water oxidation is proposed based on a structural model of the oxygen-evolving complex (OEC) and its placement into the modeled structure of the D1/D2 core of photosystem II. The structural model of the OEC satisfies many of the geometrical constraints imposed by spectroscopic and biophysical results. The model includes the tetranuclear manganese cluster, calcium, chloride, tyrosine Z, H190, D170, H332 and H337 of the D1 polypeptide and is patterned after the reversible O2-binding diferric site in oxyhemerythrin. The mechanism for water oxidation readily follows from the structural model. Concerted proton-coupled electron transfer in the S2-->S3 and S3-->S4 transitions forms a terminal Mn(V)=O moiety. Nucleophilic attack on this electron-deficient Mn(V)=O by a calcium-bound water molecule results in a Mn(III)-OOH species, similar to the ferric hydroperoxide in oxyhemerythrin. Dioxygen is released in a manner analogous to that in oxyhemerythrin, concomitant with reduction of manganese and protonation of a mu-oxo bridge.     

Topology of NH2OH-induced Mn(II) release from chloroplast thylakoid membranes

Permeant and impermeant metal ion chelators have been used in conjunction with NMR relaxation time measurements (T₁) of solvent protons to probe the membrane topology of the Mn(II) released from the water-oxidizing center of chloroplast thylakoid membranes by NH₂OH. Chelex, a tightly binding divalent metal ion exchanger, quantitatively removes Mn²⁺ (added as MnCl₂) from the external thylakoid membrane without significantly affecting oxygen evolution activity or photophosphorylation efficiency. Because of its obvious impermeance (the resin is supplied as 0.2 mm beads), chelex selectively removes only manganese that is in equilibrium with the external aqueous phase. Both internal and external manganese pools are removed by chelex in the presence of A23187, a divalent cation-specific ionophore. Topological experiments using these reagents have shown that NH₂OH releases Mn(II) predominantly to the loculus in freshly prepared, dark-adapted thylakoid membranes at 0–3°C. This topology changes radically as a result of three pretreatments: (1) incubation of thylakoid membranes in the dark at 25°C, which redirects Mn(II) release toward the external medium with a half-time of 10–15 min; (2) illumination with saturating white light, which decreases the half-time of reorientation to about 1 min; (3) freeze-thawing in 0.4 M sucrose, which results in the appearance of 40–60% of the NH₂OH-liberated Mn(II) in the external medium. None of these treatments substantially degrades O₂ evolution activity or osmotic integrity as judged from measurements of photophosphorylation efficiency. It is concluded that the topology of the manganese site associated with photosystem II is not static but changes dramatically in response to external stimuli, possibly reflecting a regulatory mechanism of photophosphorylation.     

Reactivation of oxygen accumulation function after complete removal of manganese from the photosystem II particles

Reactivation of 02 evolution function has been studied in PS-2 particles after complete removal of Mn and water soluble 10, 17, 24, 33 kDa proteins, It has been shown that 02 evolution function in such particles can be reactivated by adding 5 μM Mn2 and 20 mM Ca2(+). Preliminary illumination of the sample is necessary to exhibit the reactivation effect of 02 evolution. The maximum value of the reactivation of 02 evolution rate is about 60% of the control. Upon illumination of the reactivated particles with flashes of 1s duration and at a frequency of 0,1 Hz, 02 evolution occurs according to the mechanism analogous to that in the initial parties of PS 2. Thus the reactivation of water oxidation and 02 evolution after complete removal of Mn and water soluble 10, 17, 24, 33 kDa proteins resulting in the suppression of 02 evolution function has been shown for the first time and it can serve as a basic approach for profound investigation of the mechanism of photosynthetic water oxidation.     

Evidence that bicarbonate is not the substrate in photosynthetic oxygen evolution

It is widely accepted that the oxygen produced by photosystem II of cyanobacteria, algae, and plants is derived from water. Earlier proposals that bicarbonate may serve as substrate or catalytic intermediate are almost forgotten, though not rigorously disproved. These latter proposals imply that CO2 is an intermediate product of oxygen production in addition to O2. In this work, we investigated this possible role of exchangeable HCO3- in oxygen evolution in two independent ways. (1) We studied a possible product inhibition of the electron transfer into the catalytic Mn4Ca complex during the oxygen-evolving reaction by greatly increasing the pressure of CO2. This was monitored by absorption transients in the near UV. We found that a 3,000-fold increase of the CO2 pressure over ambient conditions did not affect the UV transient, whereas the S(3) --> S(4) --> S(0) transition was half-inhibited by raising the O2 pressure only 10-fold over ambient, as previously established. (2) The flash-induced O2 and CO2 production by photosystem II was followed simultaneously with membrane inlet mass spectrometry under approximately 15% H2(18)O enrichment. Light flashes that revealed the known oscillatory O2 release failed to produce any oscillatory CO2 signal. Both types of results exclude that exchangeable bicarbonate is the substrate for (and CO2 an intermediate product of) oxygen evolution by photosynthesis. The possibility that a tightly bound carbonate or bicarbonate is a cofactor of photosynthetic water oxidation has remained.     

The photosystem II-associated Cah3 in Chlamydomonas enhances the O2 evolution rate by proton removal

Water oxidation in photosystem II (PSII) is still insufficiently understood and is assumed to involve HCO(3)(-). A Chlamydomonas mutant lacking a carbonic anhydrase associated with the PSII donor side shows impaired O(2) evolution in the absence of HCO(3)(-). The O(2) evolution for saturating, continuous illumination (R(O2)) was slower than in the wild type, but was elevated by HCO(3)(-) and increased further by Cah3. The R(O2) limitation in the absence of Cah3/HCO(3)(-) was amplified by H(2)O/D(2)O exchange, but relieved by an amphiphilic proton carrier, suggesting a role of Cah3/HCO(3)(-) in proton translocation. Chlorophyll fluorescence indicates a Cah3/HCO(3)(-) effect at the donor side of PSII. Time-resolved delayed fluorescence and O(2)-release measurements suggest specific effects on proton-release steps but not on electron transfer. We propose that Cah3 promotes proton removal from the Mn complex by locally providing HCO(3)(-), which may function as proton carrier. Without Cah3, proton removal could become rate limiting during O(2) formation and thus, limit water oxidation under high light. Our results underlie the general importance of proton release at the donor side of PSII during water oxidation.     

pH dependence of the multiline, manganese EPR signal for the 'S2' state in PS II particles. Absence of proton release during the S1----S2 electron transfer step of the oxygen evolving system

The pH dependence of oxygen evolution rates, 2,6-dichlorophenolindophenol (DCIP) reduction rates and the intensity of the multiline manganese EPR signal associated with the S2K ok state has been studied using oxygen-evolving spinach (PS) II particles. The oxygen evolution and DCIP reduction rates are found to be very sensitive to pH, with the maximal rates occurring at pH 6.5-7.0. Both the rate and yield of the S2 multiline manganese EPR signal intensity, produced by single flash excitation at room temperature or by continuous illumination at 200 K, are found to be independent of pH, indicating that no proton is released from this manganese site during the S1----S2 electron transfer. These results agree with those from other laboratories showing no proton release on this transition, but using techniques monitoring other species.     

Mechanism of photoinhibition of photosynthetic water oxidation by Cl- depletion and F- substitution: oxidation of a protein residue

New evidence on the chloride requirement for photosynthetic O2 evolution has indicated that Cl- facilitates oxidation of the manganese cluster by the photosystem II (PSII) Tyr-Z+ radical. Illumination above 250 K of spinach PSII centers which are inhibited in O2 evolution by either Cl- depletion or F- substitution produces a new EPR signal which has magnetic characteristics similar to one recently discovered in samples inhibited by depletion of Ca2+ only [Boussac et al. (1989) Biochemistry 28, 8984; Sivaraja et al. (1989) Biochemistry 28, 9459]. The physiological roles of Cl- and Ca2+ in water oxidation are thus linked. The characteristics include a nearly isotropic g = 2.00 +/- 0.005, a symmetric line shape with line width = 16 +/- 2 mT, almost stoichiometric spin concentration relative to Try-D+ = 0.6 +/- 0.3 spin/PSII, very rapid spin relaxation at all temperatures measured down to 6 K, and an undetectable change in magnetic susceptibility upon formation (less than 1 mu B2). The signal appears to originate from a spin doublet (radical) in magnetic dipolar contact with a transition-metal ion, most probably a photooxidized protein residue within 10 A of the Mn cluster (Mn-proximal radical). It is distinct from the three other protein-bound radical-type electron donors found in the PSII reaction center: Tyr-D+, Tyr-Z+, and C+. This signal photoaccumulates to a stable level under continuous illumination at 270 K and decays only after illumination stops.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heat inactivation of oxygen evolution in Photosystem II particles and its acceleration by chloride depletion and exogenous manganese

Heat inactivation of oxygen evolution by isolated Photosystem II particles was accelerated by Cl⁻ depletion and exogenous Mn²⁺. Weak red light also accelerated heat inactivation. Heat treatment released the 33, 24 and 18 kDa proteins and Mn from the Photosystem II particles. The protein release was stimulated by Cl⁻ depletion and exogenous Mn²⁺, and the Mn release was also stimulated by Cl⁻ depletion. A 50% loss of Mn corresponded to full inactivation of oxygen evolution, whereas no direct correlation seemed to exist between the loss of any one protein and inactivation of oxygen evolution. Removal of the 24 and 18 kDa proteins from photosystem II particles only slightly decreased the heat stability of oxygen evolution.     

NADH oxidation by manganese peroxidase with or without alpha-hydroxy acid

NADH oxidation by manganese peroxidase (MnP) was done in a reaction mixture including either alpha-hydroxy acid or acetate. The oxidation in the former reaction mixture was inhibited by a catalase and was accelerated by exogenous H2O2, while the oxidation in the latter reaction mixture was inhibited by a superoxide dismutase and was not accelerated by the exogenous H2O2. These results indicated that there are significant differences between the two reaction systems, particularly, in the active oxygen species involved in the reactions. Additionally, the experiment of MnP reduction with Mn(II) suggests that MnP has a separate catalytic activity other than an oxidation of Mn(II) to Mn(III) in the reaction mixture including acetate.     

Bridging-type changes facilitate successive oxidation steps at about 1 V in two binuclear manganese complexes--implications for photosynthetic water-oxidation

The redox behavior of two synthetic manganese complexes illustrates a mechanistic aspect of importance for light-driven water oxidation in Photosystem II (PSII) and design of biomimetic systems (artificial photosynthesis). The coupling between changes in oxidation state and structural changes was investigated for two binuclear manganese complexes (1 and 2), which differ in the set of first sphere ligands to Mn (N(3)O(3) in 1, N(2)O(4) in 2). Both complexes were studied by electron paramagnetic resonance (EPR) and X-ray absorption spectroscopy (XAS) in three oxidation states which had been previously prepared either electro- or photochemically. The following bridging-type changes are suggested. In 1: Mn(II)-(mu-OR)(mu-OCO)(2)-Mn(II)<-->Mn(II)-(mu-OR)(mu-OCO)(2)-Mn(III)-->Mn(III)-(mu-OR)(mu-OCO)(mu-O)-Mn(III). In 2: Mn(II)-(mu-OR)(mu-OCO)(2)-Mn(III)<-->Mn(III)-(mu-OR)(mu-OCO)(2)-Mn(III)-->Mn(III)-(mu-OR)(mu-OCO)(mu-O)-Mn(IV). In both complexes, the first one-electron oxidation proceeds without bridging-type change, but involves a redox-potential increase by 0.5-1V. The second one-electron oxidation likely is coupled to mu-oxo-bridge (or mu-OH) formation which seems to counteract a further potential increase. In both complexes, mu-O(H) bridge formation is associated with a redox transition proceeding at approximately 1V, but the mu-O(H) bridge is observed at the Mn(2)(III,III) level in 1 and at the Mn(III,IV) level in 2, demonstrating modulation of the redox behavior by the terminal ligands. It is proposed that also in PSII bridging-type changes facilitate successive oxidation steps at approximately the same potential.     

Proton evolution from photosystem II. Stoichiometry and mechanistic considerations

1. In a sequence of flashes given to dark-adapted chloroplasts, the flash yield of proton release oscillates with a period of 4, which is similar but not identical to the oscillation of the O2 flash yield. 2. Using the proton release associated with ferricyanide reduction as a calibration, we computed that two protons are released in the terminal O2-liberating reaction; the other two protons are released in precursor conversion steps. 3. Analysis of the effect of preflashes on the oscillation pattern showed that the S1 leads to S2 transition releases no proton, the S0 leads to S1 transition somewhat less than one (0.75), and the S2 leads to S3 transition somewhat more than one (1.25). 4. The precision of the data was sufficient to exclude the possibility that in the four-step water oxidation, proton release follows a simple 1, 0, 1, 2 pattern. A possible model to interpret the observed flash yield patterns is discussed.