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Chlorophylls and bacteriochlorophylls, together with carotenoids, serve, noncovalently bound to specific apoproteins, as principal light-harvesting and energy-transforming pigments in photosynthetic organisms. In recent years, enormous progress has been achieved in the elucidation of structures and functions of light-harvesting (antenna) complexes, photosynthetic reaction centers and even entire photosystems. It is becoming increasingly clear that light-harvesting complexes not only serve to enlarge the absorption cross sections of the respective reaction centers but are vitally important in short- and long-term adaptation of the photosynthetic apparatus and regulation of the energy-transforming processes in response to external and internal conditions. Thus, the wide variety of structural diversity in photosynthetic antenna “designs” becomes conceivable. It is, however, common for LHCs to form trimeric (or multiples thereof) structures. We propose a simple, tentative explanation of the trimer issue, based on the 2D world created by photosynthetic membrane systems.

All cyanobacteria, algae, and plants use a similar water-oxidizing catalyst for water oxidation. This catalyst is housed in Photosystem II, a membrane-protein complex that functions as a light-driven water oxidase in oxygenic photosynthesis. Water oxidation is also an important reaction in artificial photosynthesis because it has the potential to provide cheap electrons from water for hydrogen production or for the reduction of carbon dioxide on an industrial scale. The water-oxidizing complex of Photosystem II is a Mn-Ca cluster that oxidizes water with a low overpotential and high turnover frequency number of up to 25-90 molecules of O2 released per second. In this Review, we discuss the atomic structure of the Mn-Ca cluster of the Photosystem II water-oxidizing complex from the viewpoint that the underlying mechanism can be informative when designing artificial water-oxidizing catalysts. This is followed by consideration of functional Mn-based model complexes for water oxidation and the issue of Mn complexes decomposing to Mn oxide. We then provide a detailed assessment of the chemistry of Mn oxides by considering how their bulk and nanoscale properties contribute to their effectiveness as water-oxidizing catalysts.

Transient absorption changes induced by excitation of isolated reaction centers (RCs) from Rhodobacter sphaeroides with 600nm laser pulses of 20fs (full width at half maximum) were monitored in the wavelength region of 420-560nm. The spectral features of the spectrum obtained are characteristic for an electrochromic band shift of the single carotenoid (Car) molecule spheroidene, which is an integral constituent of these RCs. This effect is assigned to an electrochromic bandshift of Car due to the local electric field of the dipole moment formed by electronic excitation of bacteriochlorophyll (BChl) molecule(s) in the neighborhood of Car. Based on the known distances between the pigments, the monomeric BChl (B(B)) in the inactive B-branch is inferred to dominate this effect. The excitation of B(B) at 600nm leads to a transition into the S(2) state (Q(x) band), which is followed by rapid internal conversion to the S(1) state (Q(y) band), thus leading to a change of strength and orientation of the dipole moment, i.e., of the electric field acting on the Car molecule. Therefore, the time course of the electrochromic bandshift reflects the rate of the internal conversion from S(2) to S(1) of B(B). The evaluation of the kinetics leads to a value of 30fs for this relaxation process. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

The reactions of light induced oxidative water splitting were analyzed within the framework of the empirical rate constant-distance relationship of non-adiabatic electron transfer in biological systems (C. C. Page, C. C. Moser, X. Chen , P. L. Dutton, Nature 402 (1999) 47-52) on the basis of structure information on Photosystem II (PS II) (A. Guskov, A. Gabdulkhakov, M. Broser, C. Glöckner, J. Hellmich, J. Kern, J. Frank, W. Saenger, A. Zouni, Chem. Phys. Chem. 11 (2010) 1160-1171, Y. Umena, K. Kawakami, J-R Shen, N. Kamiya, Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9Å. Nature 47 (2011) 55-60). Comparison of these results with experimental data leads to the following conclusions: 1) The oxidation of tyrosine Y(z) by the cation radical P680(+·) in systems with an intact water oxidizing complex (WOC) is kinetically limited by the non-adiabatic electron transfer step and the extent of this reaction is thermodynamically determined by relaxation processes in the environment including rearrangements of hydrogen bond network(s). In marked contrast, all Y(z)(ox) induced oxidation steps in the WOC up to redox state S(3) are kinetically limited by trigger reactions which are slower by orders of magnitude than the rates calculated for non-adiabatic electron transfer. 3) The overall rate of the triggered reaction sequence of Y(z)(ox) reduction by the WOC in redox state S(3) eventually leading to formation and release of O(2) is kinetically limited by an uphill electron transfer step. Alternative models are discussed for this reaction. The protein matrix of the WOC and bound water molecules provide an optimized dynamic landscape of hydrogen bonded protons for catalyzing oxidative water splitting energetically driven by light induced formation of the cation radical P680(+·). In this way the PS II core acts as a molecular machine formed during a long evolutionary process. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

The present work describes results obtained on hybrid systems formed in aqueous buffer solution by self-assembly of different CdSe quantum dots (QDs) surrounded by a ZnS shell and functionalized by covering the surface with anionic and cationic groups and various isolated pigment–protein complexes from the light-harvesting antennae of photosynthetic organisms (light-harvesting complexes 1 and 2 (LH1 and LH2, respectively) from purple bacteria, phycobiliproteins (PBPs) from cyanobacteria and the rod-shaped PBP from the cyanobacterium Acaryochloris marina). Excitation energy transfer (EET) from QDs to PBP rods was found to take place with varying and highly temperature-dependent efficiencies of up to 90%. Experiments performed at room temperature on hybrid systems with different QDs show that no straightforward correlation exists between the efficiency of EET and the parameter J / (R126) given by the theory of Forster resonance energy transfer (FRET), where J is the overlap integral of the normalized QD emission and PBP absorption and R12 the distance between the transition dipole moments of donor and acceptor. The results show that the hybrid systems cannot be described as randomly orientated aggregates consisting of QDs and photosynthetic pigment–protein complexes. Specific structural parameters are inferred to play an essential role. The mode of binding and coupling seems to change with the size of QDs and with temperature. Efficient EET and fluorescence enhancement of the acceptor was observed at particular stoichiometric ratios between QDs and trimeric phycoerythrin (PE). At higher concentrations of PE, a quenching of its fluorescence is observed in the presence of QDs. This effect is explained by the existence of additional quenching channels in aggregates formed within hybrid systems. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Vibrational and conformational protein dynamics of photosystem II (PS II) membrane fragments from spinach were investigated by elastic and inelastic incoherent neutron scattering (EINS and IINS). As to the EINS experiments, the average atomic mean square displacement values of PS II membrane fragments hydrated at a relative humidity of 57% exhibit a dynamical transition at ~230K. In contrast, the dynamical transition was absent at a relative humidity of 44%. These findings are in agreement with previous studies which reported a “freezing” of protein mobility due to dehydration (Pieper et al. (2008) Eur. Biophys. J. 37: 657–663) and its correlation with an inhibition of electron transfer from QA− to QB (Kaminskaya et al. (2003) Biochemistry 42, 8119–8132). IINS spectra of a sample hydrated at a relative humidity of 57% show a distinct Boson peak at ~7.5meV at 20K, which shifts towards lower energy values upon temperature increase to 250K. This unexpected effect is interpreted in terms of a “softening” of the protein matrix along with the onset of conformational protein dynamics as revealed by the EINS experiments. Information on the density of vibrational states of pigment–protein complexes is important for a realistic calculation of excitation energy transfer kinetics and spectral lineshapes and is often routinely obtained by optical line-narrowing spectroscopy at liquid helium temperature. The data presented here demonstrate that IINS is a valuable experimental tool in determining the density of vibrational states not only at cryogenic, but also at nearly physiological temperatures up to 250K. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

It has been shown by the use of steady-state and time-resolved fluorimetry and kinetic phosphorescent spectroscopy that a polycationic fullerene derivative forms complexes with eosine Y in solution due to electrostatic interactions. It has been found that singlet excited states of eosine Y are effectively quenched due to either the excitation energy transfer or electron transfer from the dye to the fullerene core. This leads to a substantial increase in the photodynamic activity of the fullerene derivative and the dye in the structure of the complex when it is excited by light in the absorption band of the dye.

The functional domain size for efficient excited singlet state quenching was studied in artificial aggregates of the main light-harvesting complex II (LHCIIb) from spinach and in native thylakoid membranes by picosecond time-resolved fluorescence spectroscopy and quantum yield measurements. The domain size was estimated from the efficiency of added exogenous singlet excitation quenchers—phenyl- p -benzoquinone (PPQ) and dinitrobenzene (DNB). The mean fluorescence lifetimes τ av were quantified for a range of quencher concentrations. Applying the Stern–Volmer formalism, apparent quenching rate constants k q were determined from the dependencies on quencher concentration of the ratio τ 0 av / τ av , where τ 0 av is the average fluorescence lifetime of the sample without addition of an exogenous quencher. The functional domain size was gathered from the ratio k q ′/ k q , i.e., the apparent quenching rate constants determined in aggregates (or membranes), k q ′, and in detergent-solubilised LHCII trimers, k q , respectively. In LHCII macroaggregates, the resulting values for the domain size were 15–30 LHCII trimers. In native thylakoid membranes the domain size was equivalent to 12–24 LHCII trimers, corresponding to 500–1000 chlorophylls. Virtually the same results were obtained when membranes were suspended in buffers promoting either membrane stacking or destacking. These domain sizes are orders of magnitude smaller than the number of physically connected pigment–protein complexes. Therefore our results imply that the physical size of an antenna system beyond the numbers of a functional domain size has little or no effect on improving the light-harvesting efficiency.

This short review paper describes spectroscopic studies on pigment–pigment and pigment–protein interactions of chlorophyll (Chl) a and b bound to the recombinant protein of class IIa water soluble chlorophyll protein (WSCP) from cauliflower. Two Chls form a strongly excitonically coupled open sandwich dimer within the tetrameric protein matrix. In marked contrast to the mode of excitonic coupling of Chl and bacterio-Chl molecules in light harvesting complexes and reaction centers of all photosynthetic organisms, the unique structural pigment array in the Chl dimer of WSCP gives rise to an upper excitonic state with a large oscillator strength. This property opens the way for thorough investigations on exciton relaxation processes in Chl–protein complexes. Lifetime measurements of excited singlet states show that the unusual stability towards photodamage of Chls bound to WSCP, which lack any protective carotenoid molecule, originates from a high diffusion barrier to interaction of molecular dioxygen with Chl triplets. Site selective spectroscopic methods provide a wealth of information on the interactions of the Chls with the protein matrix and on the vibronic structure of the pigments. The presented data and discussions illustrate the great potential of WSCP as a model system for systematic experimental and theoretical studies on the functionalizing of Chls by the protein matrix. It opens the way for further detailed analyses and a deeper understanding of the properties of pigment protein complexes.

The essential steps of photosynthetic water splitting take place in Photosystem II (PS II) and comprise three different reaction sequences: (i) light induced formation of the radical pair P680+ Q A - , (ii) P680+ driven oxidative water splitting into O2 and four protons, and (iii) two step plastoquinone reduction to plastoquinol by Q A - . This mini-review briefly summarizes our state of knowledge on energetics, kinetics and mechanism of oxidative water splitting. Essential features of the two types of reactions involved are described: (a) P680+ reduction by the redox active tyrosine Yz and (b) sequence of oxidation steps induced by Y z ox in the water-oxidizing complex (WOC). The rate of the former reaction is limited by the non-adiabatic electron transfer (NET) step and the multi-phase kinetics shown to originate from a sequence of relaxation processes. In marked contrast, the rate of the stepwise oxidation by Y z ox of the WOC up to the redox level S3 is not limited by NET but by trigger reactions which probably comprise proton shifts and/or conformational changes. The overall rate of the final reaction sequence leading to formation and release of O2 is assumed to be limited by the electron transfer step from the S3 state of WOC to Y z ox due to involvement of an endergonic redox equilibrium. Currently discussed controversial ideas on possible pathways are briefly outlined. Several crucial points of the mechanism of oxidative water splitting, like O–O bond formation, role of local proton shift(s), details of hydrogen bonding, are still not clarified and remain a challenging topic of future research.

Difference fluorescence line-narrowing spectroscopy at 4.5 K was employed to investigate electron-phonon and electron-vibrational coupling strengths of the lower exciton level of water-soluble chlorophyll-binding protein (WSCP) from cauliflower reconstituted with chlorophyll a or chlorophyll b, respectively. The electron-phonon coupling is found to be moderate with integral Huang-Rhys factors S in the order of 0.81-0.85. A weak dependence of S on excitation wavelength within the inhomogeneously broadened fluorescence origin band is attributed to a sizable contribution of nonresonant excitation that varies with excitation wavelength. The strongly asymmetric and highly structured one-phonon profile is characterized by a peak phonon frequency (ω(m)) of ~24 cm(-1) and further discernible peaks at 48 and 88 cm(-1), respectively. A structural assignment of this unusual one-phonon profile is proposed. As will be shown in the accompanying paper (part II) (DOI 10.1021/jp111457t), the parameters of electron-phonon coupling readily account for shape and position of the fluorescence origin bands at 666.1 and 683.8 nm for chlorophyll b- and chlorophyll a-WSCP, respectively. A rich structure of S(1)→S(0) vibrational frequencies was resolved in the wavenumber range between 180 and 1665 cm(-1) for both chlorophyll a- and chlorophyll b-WSCP. The corresponding individual Huang-Rhys factors fall in the range between 0.0011 and 0.0500. To the best of our knowledge, this is the first report of S-factors for vibrational modes of chlorophyll b. Most remarkable is the presence of two additional modes at 228 and 327 cm(-1) compared with the vibrational spectrum of chlorophyll in solution. The additional modes can most likely be attributed to H-bond formation in the vicinity of the chlorophyll molecule bound by WSCP.

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.

Protein dynamics in hydrated and vacuum-dried photosystem II (PS II) membrane fragments from spinach has been investigated by quasielastic neutron scattering (QENS) in the temperature range between 5 and 300 K. Three distinct temperature ranges can be clearly distinguished by active type(s) of protein dynamics: (A) At low temperatures (T120 K), the protein dynamics of both dry and hydrated PS II is characterized by harmonic vibrational motions. (B) In the intermediate temperature range (120T240 K), the total mean square displacementu2total slightly deviates from the predicted linear behavior. The QENS data indicate that this deviation, which is virtually independent of the extent of hydration, is due to a partial onset of diffusive protein motions. (C) At temperatures above 240 K, the protein flexibility drastically changes because of the onset of diffusive (large-amplitude) protein motions. This dynamical transition is clearly hydration-dependent since it is strongly suppressed in dry PS II. The thermally activated onset of protein flexibility as monitored by QENS is found to be strictly correlated with the temperature-dependent increase of the electron transport efficiency from Q(A)(-) to QB (Garbers et al. (1998) Biochemistry 37, 11399-11404). Analogously, the freezing of protein mobility by dehydration in dry PS II appears to be responsible for the blockage of Q(A)(-) reoxidation by Q(B) at hydration values lower than 45% r.h. (Kaminskaya et al. (2003) Biochemistry 42, 8119-8132). Similar effects were observed for reactions of the water-oxidizing complex as outlined in the Discussion section.

Acaroychloris (A.) marina is a unique oxygen evolving organism that contains a large amount of chlorophyll d (Chl d) and only very few Chl a molecules. This feature raises questions on the nature of the photoactive pigment, which supports light-induced oxidative water splitting in Photosystem II (PS II). In this study, flash-induced oxygen evolution patterns (FIOPs) were measured to address the question whether the S(i) state transition probabilities and/or the redox-potentials of the water oxidizing complex (WOC) in its different S(i) states are altered in A. marina cells compared to that of spinach thylakoids. The analysis of the obtained data within the framework of different versions of the Kok model reveals that in light activated A. marina cells the miss probability is similar compared to spinach thylakoids. This finding indicates that the redox-potentials and kinetics within the WOC, of the reaction center (P680) and of Y(Z) are virtually the same for both organisms. This conclusion is strongly supported by lifetime measurements of the S(2) and S(3) states. Virtually identical time constants were obtained for the slow phase of deactivation. Kinetic differences in the fast phase of S(2) and S(3) decay between A. marina cells and spinach thylakoids reflect a shift of the E(m) of Y(D)/Y(D)(ox) to lower values in the former compared to the latter organisms, as revealed by the observation of an opposite change in the kinetics of S(0) oxidation to S(1) by Y(D)(ox). A slightly increased double hit probability in A. marina cells is indicative of a faster Q(A)(-) to Q(B) electron transfer in these cells compared to spinach thylakoids.

"Reduced minus oxidized" difference extinction coefficients Deltavarepsilon in the alpha-bands of Cyt b559 and Cyt c550 were determined by using functionally and structurally well-characterized PS II core complexes from the thermophilic cyanobacterium Thermosynechococcus elongatus. Values of 25.1+/-1.0 mM(-1) cm(-1) and 27.0+/-1.0 mM(-1) cm(-1) were obtained for Cyt b559 and Cyt c550, respectively. Anaerobic redox titrations covering the wide range from -250 up to +450 mV revealed that the heme groups of both Cyt b559 and Cyt c550 exhibit homogenous redox properties in the sample preparation used, with E(m) values at pH 6.5 of 244+/-11 mV and -94+/-21 mV, respectively. No HP form of Cyt b559 could be detected. Experiments performed on PS II membrane fragments of higher plants where the content of the high potential form of Cyt b559 was varied by special treatments (pH, heat) have shown that the alpha-band extinction of Cyt b559 does not depend on the redox form of the heme group. Based on the results of this study the Cyt b559/PSII stoichiometry is inferred to be 1:1 not only in thermophilic cyanobacteria as known from the crystal structure but also in PSII of plants. Possible interrelationships between the structure of the Q(B) site and the microenvironment of the heme group of Cyt b559 are discussed.

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.

The decay kinetics of chlorophyll (Chl) fluorescence of solubilized pigment protein complexes of the distal, proximal, and core antenna of photosystem II from higher plants have been analyzed in th...

The multiphasic P680+˙ reduction kinetics by YZ and their temperature dependence were investigated in PS II core complexes with high oxygen evolution capacity, isolated from a thermophilic cyanobacterium (Thermosynechococcus elongatus) and a higher plant (Spinacea oleracea). Measurements and kinetic analyses of laser flash induced 820 nm absorption changes (reflecting the turnover of P680) led to the following results: (a) the pattern of multiphasic P680+˙ reduction is basically the same in both species, (b) the activation energy of the “fast” nanosecond kinetics is 20 ± 5 kJ mol−1 and 14 ± 5 kJ mol−1 for the samples from T. elongatus and S. oleracea, respectively, (c) the activation energies of this reaction are nearly the same in complexes with water oxidizing complex (WOC) in redox states S1 and S2, (d) the activation energy of the “slow” nanosecond kinetics ascribed to “local” relaxation processes is larger by almost a factor of two compared to that of the “fast” nanosecond kinetics, and (e) the normalized amplitudes of the “fast” and “slow” nanosecond kinetics are virtually independent of temperature in the physiological range for PS II core complexes from both organisms. Based on these findings the energetics and kinetics of P680+˙ reduction in fully competent PS II is briefly discussed within the framework of a dynamic model of sequential relaxation processes. The protein dynamics are inferred to provide the major contribution to the driving force of the redox reaction.

The present study describes a comparative analysis on the fluorescence properties of the manganese-stabilizing protein (MSP), a synthetic peptide corresponding to its C terminus and a 7:1 (molar ratio) mixture of N-acetyl-tyrosine and N-acetyl-tryptophan, respectively, together with reconstitution experiments of oxygen evolution in MSP-depleted photosystem II (PS II) membrane fragments. It is found: (i) at neutral pH, the fluorescence from Trp241 is strongly diminished in MSP solutions, whereas it highly dominates the overall emission from the C-terminus peptide; (ii) at alkaline pH, the emission of Tyr and Trp is quenched in both, MSP and C-terminus peptide, with increasing pH but the decline curve is shifted by about two pH units towards the alkaline region in MSP; (iii) a drastically different pattern emerges in the 7:1 mixture where the Trp emission even slightly increases at high pH; (iv) the anisotropy of the fluorescence emission is wavelength-independent (310–395 nm) and indicative of one emitter type (Trp) in the C-terminus peptide and of two emitter types (Tyr, Trp) in MSP; and (v) in MSP-depleted PS II membrane fragments the oxygen evolution is restored (up to 85% of untreated control) by rebinding of MSP but not by the C-terminus peptide, however, the presence of the latter diminishes the restoration effect of MSP. A quenching mechanism of Trp fluorescence by a next neighbored tyrosinate in the peptide chain is proposed and the relevance of the C terminus of MSP briefly discussed.

In spinach photosystem II (PSII) membranes, the tetranuclear manganese cluster of the oxygen-evolving complex (OEC) can be reduced by incubation with nitric oxide at -30 degrees C to a state which is characterized by an Mn(2)(II, III) EPR multiline signal [Sarrou, J., Ioannidis, N., Deligiannakis, Y., and Petrouleas, V. (1998) Biochemistry 37, 3581-3587]. This state was recently assigned to the S(-)(2) state of the OEC [Schansker, G., Goussias, C., Petrouleas, V., and Rutherford, A. W. (2002) Biochemistry 41, 3057-3064]. On the basis of EPR spectroscopy and flash-induced oxygen evolution patterns, we show that a similar reduction process takes place in PSII samples of the thermophilic cyanobacterium Synechococcus elongatus at both -30 and 0 degrees C. An EPR multiline signal, very similar but not identical to that of the S(-)(2) state in spinach, was obtained with monomeric and dimeric PSII core complexes from S. elongatus only after incubation at -30 degrees C. The assignment of this EPR multiline signal to the S(-)(2) state is corroborated by measurements of flash-induced oxygen evolution patterns and detailed fits using extended Kok models. The small reproducible shifts of several low-field peak positions of the S(-)(2) EPR multiline signal in S. elongatus compared to spinach suggest that slight differences in the coordination geometry and/or the ligands of the manganese cluster exist between thermophilic cyanobacteria and higher plants.

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.

Stepwise two-photon excitation of chlorophyll a and b in the higher plant main light-harvesting complex (LHC II) and the minor complex CP29 (as well as in organic solution) with 100-fs pulses in the Q(y) region results in a weak blue fluorescence. The dependence of the spectral shape of the blue fluorescence on excitation wavelength offers a new approach to elucidate the long-standing problem of the origin of spectral "chlorophyll forms" in pigment-protein complexes, in particular the characterization of chlorophyll a/b-heterodimers. As a first result we present evidence for the existence of strong chlorophyll a/b-interactions (excitonically coupled transitions at 650 and 680 nm) in LHC II at ambient temperature. In comparison with LHC II, the experiments with CP29 provide further evidence that the lowest energy chlorophyll a transition (at approximately 680 nm) is not excitonically coupled to chlorophyll b.

Mössbauer spectroscopy was applied to study the properties of cytochrome b559 (Cyt b559) in isolated D1/D2/Cyt b559 preparations (from spinach) that are completely deprived of non-heme iron. In these samples, all Cyt b559 exists as low-potential form(s) with the iron center attaining the low-spin ferric state in the absence of a strong reductant. The Mössbauer spectra were analyzed using isomer shift and quadrupole splitting parameters below 100 K, gathered from an extrapolation of the temperature dependence of experimental data of photosystem II membrane fragments from spinach. The calculations, based on the Griffith model, lead to the conclusion that the crystal field around the heme iron of Cyt b559 is characterized by a strong rhombic distortion. The g-values obtained are in agreement with recently published EPR results. The use of an extended theoretical approach permits the description of the relaxation changes of the Mössbauer spectra in the temperature region from 5 K to 60 K. It shows that the environment of the heme iron in D1/D2/Cyt b559 is not homogeneous but most likely reflects the existence of two different forms. We assume that factors other than changes of the first coordination sphere are responsible for the drastic negative shift in the redox potential of Cyt b559 that takes place during the isolation procedure of D1/D2/Cyt b559 complexes. Possible implications of these findings are discussed.

Time-resolved and integrated two-pulse photon echo signals were measured at 5 K in the wavelength range from 640 to 690 nm (with an increment of 5 nm) in samples of solubilized light-harvesting complex II (LHC II) from spinach. Furthermore, the dephasing time was determined at 680 nm in the temperature range 5 ≤ T ≤ 300 K. The following results and conclusions were gathered from these experiments: (a) the photon echo intensity as a function of the temporal distance between both excitation pulses exhibits a nonexponential decay; (b) data analysis on the basis of a superposition of several dephasing processes leads to three characteristic dephasing time domains (A, B, and C) with markedly different wavelength dependencies of dephasing time and relative amplitude, T2A ≤ 1.7 ps from 640 to 675 nm, T2B = 4−13 ps over the whole wavelength region of 640 to 690 nm, and T2C ≥ 40 ps from 675 to 685 nm; (c) the dephasing times T2A and T2B are attributed to the kinetics of excitation energy transfer and to multiexci...

The fluorescence properties of the isolated extrinsic 33 kDa subunit acting as ‘manganese stabilizing protein’ (MSP) of the water oxidizing complex in photosynthesis was analyzed in buffer solution. Measurements of the emission spectra as a function of excitation wavelength, pH and temperature led to the following results: (a) under all experimental conditions the spectra monitored were found to be the composite of two contributions referred to as ‘306 nm band’ and ‘long-wavelength band’, (b) the excitation spectra of these two bands closely resemble those of tyrosine and tryptophan in solution, respectively, (c) the spectral shape of the ‘306 nm band’ is virtually independent on pH but its amplitude drastically decreases in the alkaline with a p K of 11.7, (d) the amplitude of the ‘long-wavelength’ emission band at alkaline pH slightly increases when the pH rises from 7.2 to about 11.3 followed by a sharp decline at higher pH, and (e) the shape of the overall spectrum at pH 7.2 is only slightly changed upon heating to 90°C whereas the amplitude significantly declines. Based on these findings the two distinct fluorescence bands are ascribed to tyrosine(s) (‘306 nm band’) and the only tryptophan residue W241 of MSP from higher plants (‘long-wavelength band’) as emitters which are both embedded into a rather hydrophobic environment.

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.

A detailed analysis of the properties of cytochrome b(559) (Cyt b(559)) in photosystem II (PS II) preparations with different degrees of structural complexity is presented. It reveals that (i) D1-D2-Cyt b(559) complexes either in solubilized form or incorporated into liposomes contain only one type of Cyt b(559) with E(m) values of 60 +/- 5 and 100 +/- 10 mV, respectively, at pH 6.8; (ii) in oxygen-evolving solubilized PS II core complexes Cyt b(559) exists predominantly (85%) as an LP form with an E(m,7) of 125 +/- 10 mV and a minor fraction with an E(m,7) of -150 +/- 15 mV; (iii) in oxygen-evolving PS II membrane fragments three different redox forms are discernible with E(m) values of 390 +/- 15 mV (HP form), 230 +/- 20 mV (IP form), and 105 +/- 25 mV (LP form) and relative amplitudes of 58, 24, and 18%, respectively, at pH 7.3; (iv) the E(m) values are almost pH-independent between pH 6 and 9.5 in all sample types except D1-D2-Cyt b(559) complexes incorporated into liposomes with a slope of -29 mV/pH unit, when the pH increases from 6 to 9.5 (IP and LP form in PS II membrane fragments possibly within a restricted range from pH 6.5 to 8); (v) at pH8 the HP Cyt b(559) progressively converts to the IP form with increasing pH; (vi) the reduced-minus-oxidized optical difference spectra of Cyt b(559) are very similar in the lambda range of 360-700 nm for all types except for the HP form which exhibits pronounced differences in the Soret band; and (vii) PS II membrane fragments and core complexes are inferred to contain about two Cyt b(559) hemes per PS II. Possible implications of conformational changes near the heme group and spin state transitions of the iron are discussed.

The complexities involved in interpretation of the phonon structure in hole burned (HB) and fluorescence line narrowed (FLN) spectra are discussed by analyzing the four contributions to the spectra, one of which is the zero-phonon hole (line) and the remaining three due to multi-phonon transitions. It is concluded that HB spectra allow for a more reliable qualitative assessment of the electron–phonon coupling. In addition, related HB experiments provide good first estimates of the Huang–Rhys factor S, the mean phonon frequency ωm and Γinh. An application is given for the LHC II chlorophyll a/b antenna complex of photosystem II.

In this study, the reversible conversion between the high- (HP) and low-potential (LP) forms of Cytb(559) has been analyzed in Tris-washed photosystem II (PSII) enriched membranes. These samples are deprived of the Mn cluster of the water-oxidizing complex (WOC) and the extrinsic regulatory proteins. The results obtained by application of optical and EPR spectroscopy reveal that (i) under aerobic conditions, the vast majority of Cytb(559) exhibits a low midpoint potential, (ii) after removal of O(2) in the dark, a fraction of Cytb(559) is converted to the high-potential form which reaches level of about 25% of the total Cytb(559), (iii) a similar dark transformation of LP --HP Cytb(559) occurs under reducing conditions (8 mM hydroquinone), (iv) under anaerobic conditions and in the presence of 8 mM hydroquinone, about 60% of the Cytb(559) attains the HP form, (v) the interconversion is reversible with the re-establishment of aerobic conditions, and (vi) aerobic and oxidizing conditions (2 mM ferricyanide or 0.5 mM potassium iridate) induce a decrease of the amount of the HP form, also showing that the conversion is reversible. This reversible interconversion between LP and HP Cytb(559) is not observed in PSII membrane fragments with an intact WOC. On the basis of these findings, the possibility is discussed that the O(2)-dependent conversion of Cytb(559) in PSII complexes lacking a functionally competent WOC is related to a protective role of Cytb(559) in photoinhibition and/or that it is involved in the regulation of the assembly of a competent water-oxidizing complex in PSII.

The rise of fluorescence as an indicator for P680(+)* reduction by YZ and the period-four oscillation of oxygen yield induced by a train of saturating flashes were measured in dark-adapted thylakoids as a function of pH in the absence of exogenous electron acceptors. The results reveal that: (i) the average amplitude of the nanosecond kinetics and the average of the maximum fluorescence attained at 100 micros after the flash in the acidic range decrease with decreasing pH; (ii) the oxygen yield exhibits a pronounced period-four oscillation at pH 6.5 and higher damping at both pH 5.0 and pH 8.0; (iii) the probability of misses in the Si-state transitions of the water oxidizing complex is affected characteristically when exchangeable protons are replaced by deuterons [at pH6.5, the ratio alpha(D)/alpha(H) is larger than 1 whereas at pH7.0 values of1 are observed]. The results are discussed within the framework of a combined mechanism for P680(+)* reduction where the nanosecond kinetics reflect an electron transfer coupled with a "rocket-type" proton shift within a hydrogen bridge from YZ to a nearby basic group, X [Eckert, H.-J., and Renger, G. (1988) FEBS Lett. 236, 425-431], and subsequent relaxations within a network of hydrogen bonds. It is concluded that in the acidic region the hydrogen bond between YZ and X (most likely His 190 of polypeptide D1) is interrupted either by direct protonation of X or by conformational changes due to acid-induced Ca2+ release. This gives rise to a decreased P680(+)* reduction by nanosecond kinetics and an increase of dissipative P680(+)* recombination at low pH. A different mechanism is responsible for the almost invariant amplitude of nanosecond kinetics and increase of alpha in the alkaline region.

The quenching of chlorophyll fluorescence by triplets in solubilized trimeric light harvesting complexes was analyzed by comparative pump-probe experiments that monitor with weak 2-ns probe pulses the fluorescence yield and changes of optical density, Δ OD, induced by 2-ns pump pulses. By using a special array for the measurement of the probe fluorescence (Schodel R., F. Hillman, T. Schrotter, K.-D. Irrgang, J. Voight, and G. Renger, 1996. Biophys. J. 71:3370–3380) the emission caused by the pump pulses could be drastically reduced so that even at highest pump pulse intensities, I P , no significant interference with the signal due to the probe pulse was observed. The data obtained reveal: a) at a fixed time delay of 50ns between pump and probe pulse the fluorescence yield of the latter drastically decreased with increasing I P , b) the recovery of the fluorescence yield in the μ s time domain exhibits kinetics which are dependent on I P , c) Δ OD at 507nm induced by the pump pulse and monitored by the probe pulse with a delay of 50ns (reflecting carotenoid triplets) increases with I P without reaching a saturation level at highest I P values, d) an analogous feature is observed for the bleaching at 675nm but it becomes significant only at very high I P values, e) the relaxation of Δ OD at 507nm occurs via a monophasic kinetics at all I P values whereas Δ OD at 675nm measured under the same conditions is characterized by a biphasic kinetics with τ values of about 1 μ s and 7–9 μ s. The latter corresponds with the monoexponential decay kinetics of Δ OD at 507nm. Based on a Stern-Volmer plot, the time-dependent fluorescence quenching is compared with the relaxation kinetics of triplets. It is shown that the fluorescence data can be consistently described by a quenching due to triplets.

In the present study the rate of triplet transfer from chlorophyll to carotenoids in solubilized LHCII was investigated by flash spectroscopy using laser pulses of approximately 2 ns for both pump and probe. Special attention has been paid to calibration of the experimental setup and to avoid saturation effects. Carotenoid triplets were identified by the pronounced positive peak at approximately 507 nm in the triplet-singlet difference spectra. DeltaOD (507 nm) exhibits a monoexponential relaxation kinetics with characteristic lifetimes of 2-9 micros (depending on the oxygen content) that was found to be independent of the pump pulse intensity. The rise of DeltaOD (507 nm) was resolved via a pump probe technique where an optical delay of up to 20 ns was used. A thorough analysis of these experimental data leads to the conclusion that the kinetics of carotenoid triplet formation in solubilized LHCII is almost entirely limited by the lifetime of the excited singlet state of chlorophyll but neither by the pulse width nor by the rate constant of triplet-triplet transfer. Within the experimental error the rate constant of triplet-triplet transfer from chlorophyll to carotenoids was estimated to be kTT > (0.5 ns)-1. This value exceeds all data reported so far by at least one order of magnitude. The implications of this finding are briefly discussed.

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 YZ 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 Si with values of about 0.6 eV for the reactions YZOXS0→YZS1 and YZOXS1→YZS2, 1.6 eV for the reaction YZOXS2→YZS3 and 1.1 eV (above a characteristic temperature uc of about 6 °C) for the reaction YZOXS3→→YZS0+O2. Using an empirical rate constant-distance relationship, the van der Waals distance between YZ and P680 was found to be about 10 A, independent of the presence or absence of the WOC, whereas the distance between YZ and the manganese cluster in the WOC was ≥15 A. Based on the calculated activation energies the environment of YZ 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 S2→S3 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.

To analyze a possible correlation between the extent of QA-* reoxidation and protein dynamics, fluorometric and Mossbauer spectroscopic measurements were performed in photosystem II membrane fragments from spinach. Numerical evaluation of the flash-induced change of the normalized fluorescence quantum yield revealed that the extent of reoxidation starts to decrease below 275 K and is almost completely suppressed at 230 K. Detailed analyses of Mossbauer spectra measured at different temperatures in 57Fe-enriched material indicate that the onset of fluctuations between conformational substates of the protein matrix occurs also at around 230 K. Based on this correspondence, protein flexibility is inferred to play a key role for QA-* reoxidation in photosystem II. Taking into account the striking similarities with purple bacteria and the latest structural information on these reaction centers [Stowell, M. H. B., McPhillips, T. M., Rees, D. C., Soltis, S. M., Abresch, E., and Feher, G. (1997) Science 276, 812-816], it appears most plausible that also the headgroup of plastoquinone-9 bound to the QB-site in PSII requires a structural reorientation for its reduction to the semiquinone.

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.

Flash-induced absorption changes at 355 nm were measured at different temperatures within the range of 2 degrees C/= theta/= 25 degrees C in dark-adapted PS II core complexes from spinach [O2 evolution rate: 1500 +/- 100 micromol of O2 (mg of Chl)-1 h-1] that were dissolved either in H2O- or in D2O-containing buffer. Comparative measurements were performed at 20 degrees C in H2O- or D2O-containing suspensions of PS II membrane fragments [O2 evolution rate: 600 +/- 40 micromol of O2 (mg of Chl)-1 h-1]. The results obtained reveal the following: (a) The activation energies of the individual redox steps in the water oxidizing complex (WOC) are dependent on the redox state Si with EA(S1--S2) = 14 kJ/mol, EA(S2--S3) = 35 kJ/mol, and EA(S3----S0 + O2) = 21 kJ/mol for theta11 degrees C, 67 kJ/mol for theta11 degrees C in PS II core complexes dissolved in H2O; (b) replacement of exchangeable protons by deuterons causes only minor changes (/=15%) of the activation energies; and (c) the rate constants of these reactions in PS II core complexes are characterized by H/D isotope ratios, ki(H)/ki(D), of 1.6, 2.3, and 1.5 for the transitions S1 --S2, S2 --S3, and S3 ----S0 + O2, respectively. The corresponding values of PS II membrane fragments are 1.3, 1.3, and 1. 4. Based on these results and corresponding EA data reported in the literature for PS II membrane fragments from spinach [Renger, G.,Hanssum, B. (1992) FEBS Lett. 299, 28-32] and PS II particles from the thermophilic cyanobacterium Synechococcus vulcanus Copeland [Koike, H., Hanssum, B., Inoue, Y.,Renger, G. (1987) Biochim. Biophys. Acta 893, 524-533], the reaction coordinate of the redox sequence in the WOC is inferred to be almost invariant to the evolutionary development from cyanobacteria to higher plants. Furthermore, the rather high activation energy of the S2 --S3 transition provides evidence for a significant structural change coupled with this reaction. Implications for the mechanism of photosynthetic water oxidation are discussed.

The protein composition, steady state and time-resolved fluorescence emission spectra were studied in solubilized and aggregated LHCII complexes, that were prepared according to two different isolation protocols: (1) by fractionation of cation-depleted thylakoid membranes using the non-ionic detergent Triton X-100 according to the procedure of Burke et al. [(1978) Arch. Biochem. Biophys. 187, 252-263] or (2) by solubilization with N-beta-dodecyl maltoside (beta-DM) of photosystem II (PSII) membrane fragments in the presence of cations [Irrgang et al. (1988) Eur. J. Biochem. 178, 207-217]. Based on the analysis of the decay-associated emission spectra measured at 10 and 80 K five long-wavelength chlorophyll species were identified in aggregated LHCII complexes. These five forms are characterized by emission maxima at 681.5, 683, 687, 695, or 702 nm. All of these forms were found in both types of LHCII preparations but the relative amounts and temperature dependency of these species were markedly different in the aggregated LHCII complexes isolated by the two procedures. It was found that these differences cannot be simply explained by effects due to using a less mild detergent as beta-DM or by an ionic influence of Ca2+. Biochemical analysis of the protein composition showed that beta-DM type LHCII consists of all the chlorophyll (Chl)binding proteins belonging to the antenna system of PSII except the CP29 type II gene product (CP29). In contrast, the Triton X-100-solubilized LHCII is highly depleted in CP26 (CP 29 type I gene product) and is contaminated by a variety of unidentified polypeptides. It is proposed that the aggregates of LHCII prepared using Triton X-100 acquire specific spectral and kinetic features due to interaction between the bulk of LHCII subunits and minor protein(s).

The characteristic period four oscillation patterns of oxygen evolution induced by a train of single-turnover flashes were measured as a function of temperature in dark-adapted photosystem II (PS II) membrane fragments that were reconstituted with native plastoquinone-9 (PQ-9) by a recently developed procedure [Kurreck, J., Seeliger, A. G., Reifarth, F., Karge, M.,Renger, G. (1995) Biochemistry 34, 15721-15731]. The following results were obtained: (a) within the range 0-35 degrees C, the probabilities of misses (alpha) and double-hits (beta) and the dark population of redox state S1 exhibit similar dependencies on the temperature; (b) below a characteristic temperature theta(c) these parameters remain virtually independent of temperature, above theta(c) (theta(c) = 20 degrees C for alpha and beta; theta(c) = 30 degrees C for S1) the values of alpha and beta increase whereas S1 decreases; and (c) the dark decay of S2 and S3 via fast and slow kinetics owing to reduction of the water oxidase by Y(D) and other endogenous electron donor(s), respectively, exhibits comparatively strong temperature dependencies with the following activation energies: E(A)(S2(fast)) = 60 +/- 10 kJ/mol, EA(S3(fast)) = 55 +/- 10 kJ/mol, E(A)(S2(slow)) = 80 +/- 5 kJ/mol, and E(A)(S3(slow)) = 75 +/- 5 kJ/mol. These values of PQ-9 reconstituted PS II membrane fragments are very similar to those that were previously reported for thylakoids [Messinger, J., Schröder, W. P.,Renger, G. (1993) Biochemistry 32, 7658-7668]. These findings reveal that the reaction coordinates of feeding electrons by endogenous electron donors into the water oxidizing complex (WOC) that attains the redox states S2 and S3 is virtually invariant to Triton X-100 treatment used in the isolation procedure of PS II membrane fragments from thylakoids. Implications of these findings are discussed.

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.

The sulfolipid 6-sulfo-alpha-D-quinovosyldiacylglycerol is associated with the thylakoid membranes of many photosynthetic organisms. Previously, genes involved in sulfolipid biosynthesis have been characterized only in the purple bacterium Rhodobacter sphaeroides. Unlike plants and cyanobacteria, photosynthesis in this bacterium is anoxygenic due to the lack of a water splitting photosystem II. To test the function of sulfolipid in an organism with oxygenic photosynthesis, we isolated and inactivated a sulfolipid gene of the cyanobacterium Synechococcus sp. PCC7942. Extensive analysis of the sulfolipid-deficient null mutant revealed subtle changes in photosynthesis related biochemistry of O2. In addition, a slight increase in the variable room temperature chlorophyll fluorescence yield was observed. Regardless of these changes, it seems unlikely that sulfolipid is an essential constituent of a functional competent water oxidase or the core antenna complex of photosystem II. However, reduced growth of the mutant under phosphate-limiting conditions supports the hypothesis that sulfolipid acts as a surrogate for anionic phospholipids under phosphate-limiting growth conditions.

H/D isotope exchange effects on P680+· reduction by Yz and electron abstraction from the water oxidizing complex (WOC) in redox state S3 by YZOX were analyzed in PS II core complexes from spinach by measurements of laser flash induced absorption changes at 820 nm and 355 nm. The results obtained reveal: (1) the rate of YZ oxidation by P680+· is almost independent of the substitution of exchangeable protons by deuterons; and (2) the reaction between YZOX and the WOC in S3 exhibits a kinetic H/D isotope exchange effect of similar magnitude as that recently observed in PS II membrane fragments [Renger, G., Bittner, T. and Messinger, J. (1994) Biochem. Soc. Trans. 22, 318–322]. Based on these results it is inferred that photosynthetic dioxygen formation comprises the cleavage of at least one hydrogen bond.

To obtain a clearer understanding of the photosensitization process, we have investigated the effect of photosensitization on the photochemical and non-photochemical energy dissipation in green leaves of wheat ( Triticum aestivum L.) and lettuce ( Lactuca sativa L.), pretreated with 5-aminolaevulinic acid (ALA) for 2–24 h in the dark, using chlorophyll (Chl) fluorescence quenching analysis and measurement of the influence on CO 2 uptake and xanthophyll cycle pigments. In response to dark pretreatment, leaves accumulated high levels of protochlorophyllide (PChlide) and non-metabolized ALA. The dark pretreatment had no effect on the intrinsic photochemical efficiency of photosystem II (PS II). Although quantitative differences exist between wheat and lettuce, exposure to actinic light caused significant effects with a similar overall response pattern in both plant species. Changes in excitation energy dissipation were obtained already by a very low photon flux density of 85 μmol photons m −2 s −1 within a few minutes CO 2 uptake was almost completely suppressed in photosensitized leaves, but they were able to build up the ΔpH necessary to drive de-epoxidation of violaxanthin. Fluorescence quenching analysis revealed that a progressive increase in non-radiative energy dissipation (measured as the non-photochemical quenching of Chl fluorescence, q N ) was paralled by a stronger reduction in the primary quinone electron acceptor of PS II (Q A ) with increasing time of ALA pretreatment in the dark. In the early stages of photosensitization, high-energy state quenching mainly contributed to q N . In the later stages, q N was dominated by a photoinhibitory component together with the photodegradation of xanthophyll cycle pigments, in particular antheraxanthin and zeaxanthin. The latter phenomenon appeared to be promoted in response to a highly increased reduction state of Q A . If ALA was simultaneously applied to leaves with 4,6-dioxoheptanoic acid, which acts as a competitive inhibitor of the enzyme ALA dehydratase, photosensitization disappeared when PChlide synthesis was completely inhibited, but was enhanced when inhibition was incomplete. Electron paramagnetic resonance studies using the spin trapping technique revealed a specific ALA-mediated formation of hydroxyl and carbon-centred radicals in response to actinic light exposure of chloroplasts. On the basis of these findings, a possible role of ALA is proposed: ALA enhances the photosensitizing effect(s) triggered by PChlide.

The exciton equilibration in the major chlorophyll a/b light-harvesting complex from spinach thylakoid membranes was analysed by non-linear transmission spectroscopy and pump-probe spectroscopy under quasi-stationary conditions at room temperature. Investigations of the non-linear transmission in the Qy transitions of Chl a/b absorption indicate that excited state absorption dominates in the wavelength regions between 620 nm and 650 nm and between 685 nm and 690 nm, whereas bleaching and stimulated emission is predominant from 650 nm to 685 nm. The pump-probe transmission spectra suggest that in isolated light-harvesting complexes of Photosystem II (LHC II) the excitons are thermally equilibrated over all spectral chlorophyll forms nearly independent of the excitation wavelength. An analogous phenomenon is not observed in Photosystem II (PS II) membrane fragments. Regardless of differences between isolated LHC II and PS II membrane fragments in both samples, the maximum of the exciton density is located at around 680 nm independent of excitation intensity and wavelength. The maximum of exciton distribution in the Chl b absorption region is located at the absorption maximum of Chl b at 650 nm. The room-temperature exciton equilibration in the wavelength region of Chl a absorption can be described by a Boltzmann distribution. On the other hand, an inclusion of the Chl b excitons in the same distribution can be achieved only under the assumption of a strongly increased oscillator strength of Chl b states.

Oscillation patterns of the oxygen yield per flash induced by a train of single-turnover flashes were measured as a function of dark incubation and different pre-illumination conditions in several autotrophic mutant strains of Synechocystis sp. PCC 6803 carrying short deletions within the large, lumen-exposed hydrophilic region (loop E) of the chlorophyll a-binding photosystem II protein CP47. A physiological and biochemical characterization of these mutant strains has been presented previously [Eaton-Rye, J. J., & Vermaas, W. F. J. (1991) Plant Mol. Biol. 17, 1165-1177; Haag, E., Eaton-Rye, J. J., Renger, G., & Vermaas, W. F. J. (1993) Biochemistry 32, 4444-4454], and some functional properties were described recently [Gleiter, H. M., Haag, E., Shen, J.-R., Eaton-Rye, J. J., Inoue, Y., Vermaas, W. F. J., & Renger, G. (1994) Biochemistry 33, 12063-12071]. The present study shows that in several mutants the water-oxidizing complex (WOC) became inactivated during prolonged dark incubation, whereas the WOC of the wild-type strain remained active. The rate and extent of the inactivation in the mutants depend on the domain of loop E, where 3-8 amino acid residues were deleted. The most pronounced effects are observed in mutants delta(A373-D380) and delta(R384-V392). A competent WOC can be restored from the fully inactivated state by illumination with short saturating flashes. The number of flashes required for this process strongly depends on the site at which a deletion has been introduced into loop E. Again, the most prominent effects were found in mutants delta(A373-D380) and delta(R384-V392). Interestingly, the number of flashes required for activation was reduced by more than an order of magnitude in both mutants by the addition of 10 mM CaCl2 to the cell suspension. On the basis of a model for photoactivation proposed by Tamura and Cheniae (1987) [Biochim. Biophys. Acta 890, 179-194], a scheme is presented for the processes of dark inactivation and photoactivation in these mutants. The results presented here corroborate an important role of the large hydrophilic domain (loop E) of CP47 in a functional and stable WOC.