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This review summarizes our current state of knowledge on the structural organization and functional pattern of photosynthetic water splitting in the multimeric Photosystem II (PS II) complex, which acts as a light-driven water: plastoquinone-oxidoreductase. The overall process comprises three types of reaction sequences: (1) photon absorption and excited singlet state trapping by charge separation leading to the ion radical pair \( {\text{P}}680^{ + \bullet } {\text{Q}}_{\text{A}}^{ - \bullet } \left( { \overset{\wedge}{=}{\text{P}}_{\text{D1}}^{ + \bullet } {\text{Q}}_{\text{A}}^{ - \bullet } } \right) \) formation, (2) oxidative water splitting into four protons and molecular dioxygen at the water oxidizing complex (WOC) with \( {\text{P}}680^{ + \bullet } \) as driving force and tyrosine YZ as intermediary redox carrier, and (3) reduction of plastoquinone to plastoquinol at the special QB binding site with \( {\text{Q}}_{\text{A}}^{ - \bullet } \) acting as reductant. Based on recent progress in structure analysis and using new theoretical approaches the mechanism of reaction sequence (1) is discussed with special emphasis on the excited energy transfer pathways and the sequence of charge transfer steps: \( ^{1} \left( {\text{RC-PC}} \right)^{ *} {\text{Q}}_{\text{A}} \to {\text{P}}_{{{\text{D}}2}} {\text{P}}_{{{\text{D}}1}} {\text{Chl}}_{{{\text{D}}1}}^{ + \bullet } {\text{Pheo}}_{{{\text{D}}1}}^{ - \bullet } {\text{Q}}_{\text{A}} \to {\text{P}}_{{{\text{D}}2}} {\text{P}}_{{{\text{D}}1}}^{ + \bullet } {\text{Chl}}_{{{\text{D}}1}} {\text{Pheo}}_{{{\text{D}}1}}^{ - \bullet } {\text{Q}}_{\text{A}} \to {\text{P}}_{{{\text{D}}2}} {\text{P}}_{{{\text{D}}1}}^{ + \bullet } {\text{Chl}}_{\text{D1}} {\text{Pheo}}_{\text{D1}} {\text{Q}}_{\text{A}}^{ - \bullet } , \) where 1(RC-PC)* denotes the excited singlet state 1P680* of the reaction centre pigment complex. The structure of the catalytic Mn4OXCa cluster of the WOC and the four step reaction sequence leading to oxidative water splitting are described and problems arising for the electronic configuration, in particular for the nature of redox state S3, are discussed. The unravelling of the mode of O–O bond formation is of key relevance for understanding the mechanism of the process. This problem is not yet solved. A multistate model is proposed for S3 and the functional role of proton shifts and hydrogen bond network(s) is emphasized. Analogously, the structure of the QB site for PQ reduction to PQH2 and the energetic and kinetics of the two step redox reaction sequence are described. Furthermore, the relevance of the protein dynamics and the role of water molecules for its flexibility are briefly outlined. We end this review by presenting future perspectives on the water oxidation process.

This short communication addresses three topics of photosynthetic water cleavage in Photosystem II (PS II): (a) effect of protonation in the acidic range on the extent of the ‘fast’ ns kinetics of P680+· reduction by Y Z , (b) mechanism of O–O bond formation and (c) role of protein flexibility in the functional integrity of PS II. Based on measurements of light-induced absorption changes and quasielastic neutron scattering in combination with mechanistic considerations, evidence is presented for the protein acting as a functionally active constituent of the water cleavage machinery, in particular, for directed local proton transfer. A specific flexibility emerging above a threshold of about 230 K is an indispensable prerequisite for oxygen evolution and plastoquinol formation.

This historical minireview describes basic lines of progress in our understanding of the functional pattern of photosynthetic water oxidation and the structure of the Photosystem II core complex. After a short introduction into the state of the art about 35 years ago, results are reviewed that led to identification of the essential cofactors of this process and the kinetics of their reactions. Special emphasis is paid on the flash induced oxygen measurements performed by Pierre Joliot (in Paris, France) and Bessel Kok (Baltimore, MD) and their coworkers that led to the scheme, known as the Kok-cycle. These findings not only unraveled the reaction pattern of oxidation steps leading from water to molecular oxygen but also provided the essential fingerprint as prerequisite for studying individual redox reactions. Starting with the S. Singer and G. Nicolson model of membrane organization, attempts were made to gain information on the structure of the Photsystem II complex that eventually led to the current stage of knowledge based on the recently published X-ray crystal structure of 3.8 A resolution in Berlin (Germany).With respect to the mechanism of water oxidation, the impact of Gerald T. Babcock's hydrogen abstractor model and all the considerations of electron/proton transfer coupling are outlined. According to my own model cosiderations, the protein matrix is not only a `cofactor holder' but actively participates by fine tuning via hydrogen bond networks, playing most likely an essential role in water substrate coordination and in oxygen-oxygen bond formation as the key step of the overall process.

The adverse effect of low intensity, small band UV-B irradiation (λ = 305 ± 5 nm, I = 300 mW m−2) on PS II has been studied by comparative measurements of laser flash-induced changes of the absorption at 325 nm, ΔA325(t), as an indicator of redox changes in QA, and of the relative fluorescence quantum yield, F(t)/Fo, in PS II membrane fragments. The properties of untreated control were compared with those of samples where the oxygen evolution rate under illumination with continuous saturating light was inhibited by up to 95%. The following results were obtained: a) the detectable initial amplitude (at a time resolution of 30 μs) of the 325 nm absorption changes, ΔA325, remained virtually invariant whereas the relaxation kinetics exhibit significant changes, b) the 300 μs kinetics of ΔA325 dominating the relaxation in UV-B treated samples was largely replaced by a 1.3 ms kinetics after addition of MnCl2, c) the extent of the flash induced rise of the relative fluorescence quantum yield was severely diminished in UV-B treated PS II membrane fragments but the relaxation kinetics remain virtually unaffected. Based on these results the water oxidizing complex (WOC) is inferred to be the primary target of UV-B impairment of PS II while the formation of the ‘stable’ radical pair P680+·QA−● is almost invariant to this UV-B treatment.

The efficiency of oxidized endogenous plastoquinone-9 (PQ-9) as a non-photochemical quencher of chlorophyll fluorescence has been analyzed in spinach thylakoids and PS II membrane fragments isolated by Triton X-100 fractionation of grana stacks. The following results were obtained: (a) After subjection of PS II membrane fragments to ultrasonic treatment in the presence of PQ-9, the area over the induction curve of chlorophyll fluorescence owing to actinic cw light increases linearly with the PQ-9/PS II ratio in the reconstitution assay medium; (b) the difference of the maximum fluorescence levels, Fmax, of the induction curves, measured in the absence and presence of DCMU, is much more pronounced in PS II membrane fragments than in thylakoids; (c) the ratio Fmax(-DCMU)/Fmax(+DCMU) increases linearly with the content of oxidized PQ-9 that is varied in the thylakoids by reoxidation of the pool after preillumination and in PS II membrane fragments by the PQ-9/PS II ratio in the reconstitution assay; (d) the reconstitution procedure leads to tight binding of PQ-9 to PS II membrane fragments, and PQ-9 cannot be replaced by other quinones; (e) the fluorescence quenching by oxidized PQ-9 persists at low temperatures, and (f) oxidized PQ-9 preferentially affects the F695 of the fluorescence emission spectrum at 77 K. Based on the results of this study the oxidized PQ-9 is inferred to act as a non-photochemical quencher via a static mechanism. Possible implications for the nature of the quenching complex are discussed.

The mode of photoinhibition as a function of the ambient redox potential (Eambient) in suspensions of Tris-washed PS II membrane fragments has been analyzed by monitoring flash-induced absorption changes at 830 nm. It was found: (a) the detectable initial amplitude, ΔAtotal830, as a measure of the capacity to form the `stable' radical pair, P680+ċ Q−ċA, drastically decreases during a 10 min photoinhibition at Eambient values below +350 mV; (b) conversely, the normalized extent of the 18 μs relaxation kinetics, ΔA18 μ s830 as a measure of the electron transfer from YZ to P680+ċ becomes highly susceptible to light stress when Eambient exceeds values of about +350 mV; (c) effects of the ambient redox potentials are highly pronounced during light exposure under anaerobic conditions, while much smaller differences arise under aerobic conditions; (d) the extent of damage does not correlate with the total concentration of K3[Fe(CN)6] and K4[Fe(CN)6] in the suspension during photoinhibition but rather depends on the Em-values; (e) qualitatively similar features are observed when the redox buffer system K3[Fe(CN)6]/Na2S2O4 is replaced by K2[IrCl6]/Na2S2O4; (f) the characteristic Eambient-dependence of photoinhibition is observed only under anaerobic conditions. The results are discussed with respect to different redox components that might be involved, including brief comments on a possible role of Cyt b559.

The present study describes a novel fluorometer system which permits the simultaneous monitoring of the time and wavelength dependence of chlorophyll fluorescence in the picosecond time domain. The key element of this equipment is a microchannel-plate photomultiplier with delay-line anode. The comparatively short acquisition times in combination with full spectral and temporal resolution of this device are of high advantage, especially for measurements on photosynthetic samples at cryogenic temperatures. For a convenient numerical data evaluation of complex overlapping spectra a decay-associated gaussian deconvolution technique was installed by developing a fitting program with graphical user-interface. In order to illustrate the potential of the new set-up, surface plots and decay spectra gathered from measurements with anaerobically photoinhibited Photosystem II particles at 277 K and 10 K are presented.

Photoinhibition under aerobic and anaerobic conditions was analyzed in O2-evolving and in Tris-treated PS II-membrane fragments from spinach by measuring laser-flash-induced absorption changes at 826 nm reflecting the transient P680+ formation and the chlorophyll fluorescence lifetime. It was found that anaerobic photoinhibitory treatment leads in both types of samples to the appearence of two long-lived fluorescence components with lifetimes of 7 ns and 16 ns, respectively. The extent of these fluorescence kinetics depends on the state of the reaction center (open/closed) during the fluorescence measurements: it is drastically higher in the closed state. It is concluded that this long-lived fluorescence is mainly emitted from modified reaction centers with singly reduced QA(QA -). This suggests that the observation of long-lived fluorescence components cannot necessarily be taken as an indicator for reaction centers with missing or doubly reduced and protonated QA (QAH2). Time-resolved measurements of 826 nm absorption changes show that the rate of photoinhibition of the stable charge separation (P680*QA → P680+QA -), is nearly the same in O2-evolving and in Tris-treated PS II-membrane fragments. This finding is difficult to understand within the framework of the QAH2-mechanism for photoinhibition of stable charge separation because in that case the rate of photoinhibition should strongly depend on the functional integrity of the donor side of PS II. Based on the results of this study it is inferred, that several processes contribute to photoinhibition within the PS II reaction center and that a mechanism which comprises double reduction and protonation of QA leading to QAH2 formation is only of marginal – if any – relevance for photoinhibition of PS II under both, aerobic and anaerobic, conditions.

Newly developed equipment is described that permits the monitoring of laser flash induced transients of the normalised chlorophyll-a fluorescence quantum yield in isolated PS II preparations and whole leaves with a high time resolution. The essential operational unit of the set-up is a rapidly gated photomultiplier. In this way, the fluorescence artefact, due to the high intensity excitation laser flash, is sufficiently suppressed and the dead time of the signal response is reduced to about 500 ns. It is shown that the fluorescence rise kinetics in the μs time-domain, after flash excitation is strongly dependent on the redox state of the primary electron donor of PS II (P680). At high excitation energies, the decay of carotenoid triplets, which are very efficient quenchers of chlorophyll singlet states, dominates the rise kinetics of the flash induced fluorescence yield in the μs time domain.

In the present study the light induced formation of superoxide and intrinsic superoxide dismutase (SOD) activity in PS II membrane fragments and D1/D2/Cytb559-complexes from spinach have been analyzed by the use of ferricytochrome c (cyt c(III)) reduction and xanthine/xanthine oxidase as assay systems. The following results were obtained: 1.) Photoreduction of Cyt c (III) by PS II membrane fragments is induced by addition of sodium azide, tetracyane ethylene (TCNE) or carbonylcyanide-p-trifluoromethoxy-phenylhydrazone (FCCP) and after removal of the extrinsic polypeptides by a 1M CaCl2-treatment. This activity which is absent in control samples becomes completely inhibited by the addition of exogenous SOD. 2.) The TCNE induced cyt c(III) photoreduction by PS II membrane fragments was found to be characterized by a half maximal concentration of c1/2=10 μM TCNE. Simultaneously, TCNE inhibits the oxygen evolution rate of PS II membrane fragments with c1/2≈ 3 μM. 3.) The photoproduction of O2 (-) is coupled with H(+)-uptake. This effect is diminished by the addition of the O2 (-)-trap cyt c(III). 4.) D1/D2/Cytb559-complexes and PS II membrane fragments deprived of the extrinsic proteins and manganese exhibit no SOD-activity but are capable of producing O2 (-) in the light if a PS II electron donor is added.Based on these results the site(s) of light induced superoxide formation in PS II is (are) inferred to be located at the acceptor side. A part of the PS II donor side and Cyt b559 in its HP-form are proposed to provide an intrinsic superoxide dismutase (SOD) activity.

The functional properties of a purified homogeneous spinach PS II-core complex with high oxygen evolution capacity (Haag et al. 1990a) were investigated in detail by measuring thermoluminescence and oscillation patterns of flash induced oxygen evolution and fluorescence quantum yield changes. The following results were obtained: a) Depending on the illumination conditions the PS II-core complexes exhibit several thermoluminescence bands corresponding to the A band, Q band and Zv band in PS II membrane fragments. The lifetime of the Q band (Tmax=10°C) was determined to be 8s at T=10°C. No B band corresponding to S2QB (-) or S3QB (-) recombination could be detected. b) The flash induced transient fluorescence quantum yield changes exhibit a multiphasi relaxation kinetics shich reflect the reoxidation of Q A (-) . In control samples without exogenous acceptors this process is markedly slower than in PS II membrane fragments. The reaction becomes significantly retarded by addition of 10 μM DCMU. After dark incubation in the presence of K3[Fe(CN)6 c) Excitation of dark-adapted samples with a train of short saturating flashes gives rise to a typical pattern dominated by a high O2 yield due to the third flash and a highly damped period four oscillation. The decay of redox states S2 and S3 are dominated by short life times of 4.3 s and 1.5 s, respectively, at 20°C. The results of the present study reveal that in purified homogeneous PS II-core complexes with high oxygen evolution isolated from higher plants by β-dodecylmaltoside solubilization the thermodynamic properties and the kinetic parameters of the redox groups leading to electron transfer from water to QA are well preserved. The most obvious phenomenon is a severe modification of the QB binding site. The implications of this finding are discussed.

Effects of photoinhibition at 0 °C on the PS II acceptor side have been analyzed by comparative studies in isolated thylakoids, PS II membrane fragments and PS II core complexes from spinach under conditions where degradation of polypeptide(s) D1(D2) is highly retarded. The following results were obtained by measurements of the transient fluorescence quantum and oxygen yield, respectively, induced by a train of short flashes in dark-adapted samples: (a) in the control the decay of the fluorescence quantum yield is very rapid after the first flash, if the dark incubation was performed in the presence of 300 μM K3[Fe(CN)6]; whereas, a characteristic binary oscillation was observed in the presence of 100 μM phenyl-p-benzoquinone with a very fast relaxation after the even flashes (2nd, 4th. . . ) of the sequence; (b) illumination of the samples in the presence of K3[Fe(CN)6] for only 5 min with white light (180 W m(-2)) largely eliminates the very fast fluorescence decay after the first flash due to QA (-) reoxidation by preoxidized endogenous non-heme Fe(3+), while a smaller effect arises on the relaxation kinetics of the fluorescence transients induced by the subsequent flashes; (c) the extent of the normalized variable fluorescence due to the second (and subsequent) flash(es) declines in all sample types with a biphasic time dependence at longer illumination. The decay times of the fast (6-9 min) and the slow degradation component (60-75 min) are practically independent of the absence or presence of K3[Fe(CN)6] and of anaerobic and aerobic conditions during the photo-inhibitory treatment, while the relative extent of the fast decay component is higher under anaerobic conditions. (d) The relaxation kinetics of the variable fluorescence induced by the second (and subsequent) flash(es) become retarded due to photoinhibition, and (e) the oscillation pattern of the oxygen yield caused by a flash train is not drastically changed due to photoinhibition.Based on these findings, it is concluded that photoinhibition modifies the reaction pattern of the PS II acceptor side prior to protein degradation. The endogenous high spin Fe(2+) located between QA and QB is shown to become highly susceptible to modification by photoinhibition in the presence of K3[Fe(CN)6] (and other exogenous acceptors), while the rate constant of QA (-) reoxidation by QB(QB (-)) and other acceptors (except the special reaction via Fe(3+)) is markedly less affected by a short photoinhibition. The equilibrium constant between QA (-) and QB(QB (-)) is not drastically changed as reflected by the damping parameters of the oscillation pattern of oxygen evolution.

This contribution describes incoherent quasielastic neutron scattering (QENS) as a suitable tool for investigations of protein dynamics with special emphasis on applications in photosynthesis research. QENS characterizes protein dynamics via the measurement of energy and momentum exchange between sample system and incident low-energy neutrons (1 meV < E < 20 meV). This method is especially sensitive for picosecond motions of hydrogen atoms because it makes use of the exceptionally large incoherent neutron scattering cross section of protons and their almost homogeneous distribution in proteins. After a short introduction into the basic principles of neutron scattering, a more detailed description of QENS will be presented including a short overview on instrumentation and theory. Recent QENS results will be discussed for the antenna complex LHC II and PS II membrane fragments. It is shown that diffusive protein dynamics is indispensable for enabling \( {\text{Q}}_{\text{A}}^{ - \bullet } \) reoxidation by QB at temperatures above 240 K, which explains the strong dependence of this electron transfer step on temperature and hydration level of the sample. Finally, a new laser-QENS pump-probe technique will be introduced which permits in situ monitoring of protein dynamics correlated with a change of the functional state of the sample, i.e. a direct observation of structure-dynamics-function relationships in real time.

From a chemical point of view methanol is one of the closest analogues of water. Consistent with this idea EPR spectroscopy studies have shown that methanol binds at-or at least very close to-the Mn(4)O(x)Ca cluster of photosystem II (PSII). In contrast, Clark-type oxygen rate measurements demonstrate that the O(2) evolving activity of PSII is surprisingly unaffected by methanol concentrations of up to 10%. Here we study for the first time in detail the effect of methanol on photosynthetic water-splitting by employing a Joliot-type bare platinum electrode. We demonstrate a linear dependence of the miss parameter for S( i ) state advancement on the methanol concentrations in the range of 0-10% (v/v). This finding is consistent with the idea that methanol binds in PSII with similar affinity as water to one or both substrate binding sites at the Mn(4)O(x)Ca cluster. The possibility is discussed that the two substrate water molecules bind at different stages of the cycle, one during the S(4) --S(0) and the other during the S(2) --S(3) transition.

The dark incubation at room temperature of photosystem II (PS II) membrane fragments in a chloride-free medium at pH 6.3 slowly leads to large chloride-restorable and non-restorable O2 evolution activity losses with time as compared with control samples incubated in the presence of 10 mM NaCl. The chloride requirement in O2 evolution generated under these conditions reveals a complex interplay among various experimental parameters, including the source of the plant material, the times of incubation, the sample concentration, the chloride concentration, as well as those treatments which are believed to specifically displace chloride from PS II such as alkaline pH pretreatment and Na2SO4 addition. The results indicate that secondary, structural changes within the PS II complex are an important factor in determining the influence of chloride on the O2 evolution activity and raise the question whether or not chloride ions actually play a direct cofactor role in the water-oxidizing reactions leading to O2 evolution.

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+*QA(-*) ; (b) reduction of plastoquinone to plastoquinol at the QB site via a two-step reaction sequence with QA(-*) 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 YZ 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 (QB-site) and oxidative water splitting (Mn4OxCa 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.

This minireview is an attempt to summarize our current knowledge on oxidative water splitting in photosynthesis. Based on the extended Kok model (Kok, Forbush, McGloin (1970) Photochem Photobiol 11:457-476) as a framework, the energetics and kinetics of two different types of reactions comprising the overall process are discussed: (i) P680+* reduction by the redox active tyrosine YZ of polypeptide D1 and (ii) Yz (ox) induced oxidation of the four step sequence in the water oxidizing complex (WOC) leading to the formation of molecular oxygen. The mode of coupling between electron transport (ET) and proton transfer (PT) is of key mechanistic relevance for the redox turnover of YZ and the reactions within the WOC. The peculiar energetics of the oxidation steps in the WOC assure that redox state S1 is thermodynamically most stable. This is a general feature in all oxygen evolving photosynthetic organisms and assumed to be of physiological relevance. The reaction coordinate of oxidative water splitting is discussed on the basis of the available information about the Gibbs energy differences between the individual redox states Si+1 and Si and the data reported for the activation energies of the individual oxidation steps in the WOC. Finally, an attempt is made 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 on the active role of the protein in tuning the local proton activity that depends on time and redox state Si. The O-O linkage is assumed to take place at the level of a complexed peroxide.

This historical minireview describes basic lines of progress in our understanding of the functional pattern of photosynthetic water oxidation and the structure of the Photosystem II core complex. After a short introduction into the state of the art about 35 years ago, results are reviewed that led to identification of the essential cofactors of this process and the kinetics of their reactions. Special emphasis is paid on the flash induced oxygen measurements performed by Pierre Joliot (in Paris, France) and Bessel Kok (Baltimore, MD) and their coworkers that led to the scheme, known as the Kok-cycle. These findings not only unraveled the reaction pattern of oxidation steps leading from water to molecular oxygen but also provided the essential fingerprint as prerequisite for studying individual redox reactions. Starting with the S. Singer and G. Nicolson model of membrane organization, attempts were made to gain information on the structure of the Photsystem II complex that eventually led to the current stage of knowledge based on the recently published X-ray crystal structure of 3.8 A resolution in Berlin (Germany).With respect to the mechanism of water oxidation, the impact of Gerald T. Babcock's hydrogen abstractor model and all the considerations of electron/proton transfer coupling are outlined. According to my own model cosiderations, the protein matrix is not only a 'cofactor holder' but actively participates by fine tuning via hydrogen bond networks, playing most likely an essential role in water substrate coordination and in oxygen-oxygen bond formation as the key step of the overall process.

This issue of Photosynthesis Research is dedicated to the memory of Bessel Kok, the discoverer of the photoactive reaction center pigment of Photosystem I, P700, and a pioneer in the field of biophysics of photosynthesis. We particularly salute his formulation of the ‘5-step S-state O2 clock’, based on the elegant experiments of Pierre Joliot. Pierre observed a characteristic period four oscillation of the O2 yield when dark-adapted photosynthetic samples were illuminated with a train of single turn-over flashes. We honor Pierre by inviting him to write his personal perspective in which he discusses the events that led to this seminal discovery.

A comparative study of X-band EPR and ENDOR of the S2 state of photosystem II membrane fragments and core complexes in the frozen state is presented. The S2 state was generated either by continuous illumination at T=200 K or by a single turn-over light flash at T=273 K yielding entirely the same S2 state EPR signals at 10 K. In membrane fragments and core complex preparations both the multiline and the g=4.1 signals were detected with comparable relative intensity. The absence of the 17 and 23 kDa proteins in the core complex preparation has no effect on the appearance of the EPR signals. (1)H-ENDOR experiments performed at two different field positions of the S2 state multiline signal of core complexes permitted the resolution of four hyperfine (hf) splittings. The hf coupling constants obtained are 4.0, 2.3, 1.1 and 0.6 MHz, in good agreement with results that were previously reported (Tang et al. (1993) J Am Chem Soc 115: 2382-2389). The intensities of all four line pairs belonging to these hf couplings are diminished in D2O. A novel model is presented and on the basis of the two largest hfc's distances between the manganese ions and the exchangeable protons are deduced. The interpretation of the ENDOR data indicates that these hf couplings might arise from water which is directly ligated to the manganese of the water oxidizing complex in redox state S2.

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 Si (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 Si, 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 YZ (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.

The functional size of Photosystem II (PS II) was investigated by radiation inactivation. The technique provides an estimate of the functional mass required for a specific reaction and depends on irradiating samples with high energy γ-rays and assaying the remaining activity. The analysis is based on target theory that has been modified to take into account the temperature dependence of radiation inactivation of proteins. Using PS II enriched membranes isolated from spinach we determined the functional size of primary charge separation coupled to water oxidation and quinone reduction at the QB site: H2O → (Mn)4 → Yz → P680 → Pheophytin → Q → phenyl-p-benzoquinone. Radiation inactivation analysis indicates a functional mass of 88 ± 12 kDa for electron transfer from water to phenyl-p-benzoquinone. It is likely that the reaction center heterodimer polypeptides, D1 and D2, contribute approximately 70 kDa to the functional mass, in which case polypeptides adding up to approximately 20 kDa remain to be identified. Likely candidates are the α and β subunits of cytochrome b 559and the 4.5 kDa psbI gene product.

The present study describes the formation of different forms of peroxide in Photosystem II (PS II) by using a chemiluminescence detection technique. Four chemiluminescence signals (A, B, C and D) of the luminolperoxidase (Lu-Per) system, which detects peroxide, are found in illuminated O2-evolving Photosystem II (PS II) membrane fragments isolated from spinach. Signal A ('free peroxide') peaking around 0.2-0.3 s after mixing PS II membrane fragments with Lu-Per is eliminated by catalase or removal of oxygen from the suspension and ascribed to O2 interaction with reduced PS II electron acceptors. In contrast, signal B peaking around 1.5 min remains largely unaffected under anaerobic conditions, as well as in the presence of catalase (20 μg/ml). Under flash illumination the extent of this signal exhibits a weak period four oscillation (maximum at third and 7th flash). Its yield increases up to the third flash, but is close to zero in the fourth flash. An analogous behaviour is observed in flashes 5 to 8. Signal B is ascribed to Lu-Per interaction with the water-oxidizing system being in S2 and/or S3-state. Signal C ('bound peroxide') detected as free peroxide after acid decomposition of illuminated PS II particles is observed on the 1 st flash and oscillates with period 2 with superposition of period 4. It is evidently related to peroxide either released from S2 or formed at S2 upon acid shock treatment. Signal D ('slowly released peroxide') peaking around 2-3 s after mixing is observed in samples after various treatments (LCC-incubation, washing with 1 M NaCl at pH 8 or with 1 M CaCl2, Cl(-)-depletion) that lead to at least partial removal of the extrinsic proteins of 18, 24 and 33 kDa without Mn extraction. The average amplitude of this signal corresponds with a yield of about 0.2 H2O2 molecules per RC and flash. In a flash train, the extent of signal D exhibits an oscillation pattern with a minimum at the 3rd flash. We assume that these treatments increase the release of 'bound' peroxide (upon injection into the Lu-Per assay) either formed in the normal oxidative pathway of the water oxidase in the S2 or the S3-state or give rise to peroxide formation due to higher accessibility of the Mn-cluster to water molecules.

Utilizing a two-beam technique in the frequency domain, the pumped absorption of PS II membrane fragments from spinach and of acetonic chlorophyll-a solutions was measured at room temperature. In a very narrow wavelength region (0.2 nm around the pump pulse wavelength) the relative test beam transmission exhibited either a decrease or an increase, respectively, dependent on the intensity of a strong pump beam. In contrast, the transmission changes of chl-a solutions were not affected by the wavelength mistuning between pump and test beam. The data obtained for PS II membrane fragments were interpreted in terms of excited state absorption of pigment-protein clusters within the light-harvesting complex of PS II. The interpretation of the small absorption band as a homogeneously broadened line led to a transversal relaxation time for chlorophyll in vivo of about 1 ps.

Photoinhibition was analyzed in O2-evolving and in Tris-treated PS II membrane fragments by measuring flash-induced absorption changes at 830 nm reflecting the transient P680(+) formation and oxygen evolution. Irradiation by visible light affects the PS II electron transfer at two different sites: a) photoinhibition of site I eliminates the capability to perform a 'stable' charge separation between P680(+) and QA (-) within the reaction center (RC) and b) photoinhibition of site II blocks the electron transfer from YZ to P680(+). The quantum yield of site I photoinhibition (2-3×10(-7) inhibited RC/quantum) is independent of the functional integrity of the water oxidizing system. In contrast, the quantum yield of photoinhibition at site II depends strongly on the oxygen evolution capacity. In O2-evolving samples, the quantum yield of site II photoinhibition is about 10(-7) inhibited RC/quantum. After selective elimination of the O2-evolving capacity by Tris-treatment, the quantum yield of photoinhibition at site II depends on the light intensity. At low intensity (3 W/m(2)), the quantum yield is 10(-4) inhibited RC/quantum (about 1000 times higher than in oxygen evolving samples). Based on these results it is inferred that the dominating deleterious effect of photoinhibition cannot be ascribed to an unique target site or a single mechanism because it depends on different experimental conditions (e.g., light intensity) and the functional status of the PS II complex.

The possibility to determine the difference spectra Δεi+1jλ of each univalent redox step Si→Si+1(i=0,...3) of the water-oxidizing enzyme system was analyzed by theoretical calculations and by measurements of 320 nm absorption changes induced by a train of saturating laser flashes (FWHM:7 ns) in PS II membrane fragments. It was found: a) Lipophilic quinones complicate the experimental determination of optical changes due the Si-state transitions because they lead to an additional binary oscillation probably caused by a reductant-induced oxidation of the Fe(2+) at the PS II acceptor side. b) In principle, a proper separation can be achieved at sufficiently high K3[Fe(CN)6] concentrations. c) An unequivocal deconvolution into the difference spectra Δεi+1jλ of flash train-induced optical changes which are exclusively due to Si-state transitions is impossible unless the Kok parameters α, β and [Si]0 can be determined by an independent method.Measurements of the oxygen yield induced by a flash train reveals, that in thylakoids and PS II membrane fragments Si is the stable state of dark adapted samples even at alkaline pH (up to pH=9). However, in PS II membrane fragments at pH7.7 the misses probability α markedly increases, in contrast to the properties of intact thylakoids. Based on these data the possibility is discussed that an equilibrium exists of two types of S2-states with different properties.

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 QB-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.

The protolytic reactions of PSII membrane fragments were analyzed by measurements of absorption changes of the water soluble indicator dye bromocresol purple induced by a train of 10 μs flashes in dark-adapted samples. It was found that: a) in the first flash a rapid H(+)-release takes place followed by a slower H(+)-uptake. The deprotonation is insensitive to DCMU but is completely eliminated by linolenic acid treatment of the samples; b) the extent of the H(+)-uptake in the first flash depends on the redox potential of the suspension. In this time domain no H(+)-uptake is observed in the subsequent flashes; c) the extent of the H(+)-release as a function of the flash number in the sequence exhibits a characteristic oscillation pattern. Multiphasic release kinetics are observed. The oscillation pattern can be satisfactorily described by a 1, 0, 1, 2 stoichiometry for the redox transitions Si → Si+1 (i=0, 1, 2, 3) in the water oxidizing enzyme system Y. The H(+)-uptake after the first flash is assumed to be a consequence of the very fast reduction of oxidized Q400(Fe(3+)) formed due to dark incubation with K3[Fe(CN)6]. The possible participation of component Z in the deprotonation reactions at the PSII donor side is discussed.