Secondary stabilization reactions and proton-coupled electron transport in photosystem II investigated by electroluminescence and fluorescence spectroscopy

The oxidized primary electron donor in photosystem II, P(680)(+), is reduced in several phases, extending over 4 orders of magnitude in time. Especially the slower phases may reflect the back-pressure exerted by water oxidation and provide information on the reactions involved. The kinetics of secondary electron-transfer reactions in the microseconds time range after charge separation were investigated in oxygen-evolving thylakoids suspended in H2O or D2O. Flash-induced changes of chlorophyll fluorescence yield and electric field-induced recombination luminescence were decomposed into contributions from oxidation states S(0), S(1), S(2), and S(3) of the oxygen-evolving complex and interpreted in terms of stabilization kinetics of the initial charge-separated state S(j)Y(Z)P(680)(+)Q(A)(-)Q(B). In approximately 10% of the centers, only charge recombination took place. Otherwise, no static heterogeneity was involved in the microsecond reduction of P(680)(+) by Y(Z) (stabilization) or Q(A)(-) (recombination). The recombination component in active centers occurs mainly upon charge separation in S(3), and, in the presence of D2O, in S(2) as well and is tentatively attributed to the presence of Y(Z)(ox)S(j-1) in equilibrium with Y(Z)S(j). A 20-30 micros stabilization occurs in all S-states, but to different extents. Possible mechanisms for this component are discussed. D2O was found to decrease: (i) the rate of the reaction Y(Z)(ox)S(1) to Y(Z)S(2), (ii) the equilibrium constant between P680(+)Y(Z)S(2) and P(680)Y(Z)(ox)S(2), (iii) the rate of the slow phase of P(680)(+) reduction for the S(3) --> S(0) transition, and (iv) the rate of electron transfer from Q(A)(-) to Q(B) /Q(B)(-). The increased 'miss probability' in D2O is due to (iii).     

Electrogenic Protonation of the Secondary Quinone Acceptor Q<Subscript>B</Subscript> in Spinach Photosystem II Complexes Incorporated into Lipid Vesicles

The generation of transmembrane electric potential difference (delta psi) in quinone acceptor complex of proteoliposomes containing core complexes of photosystem II from spinach was studied using for the measurements a direct electrometric technique. Besides the fast increase in the membrane potential associated with the electron transfer between the redox-active tyrosine 161 residue (Y(Z)) in D1 polypeptide and the primary quinone acceptor Q(A), an additional electrogenic phase with tau approximately 0.85 msec at pH 7.3 and the maximal relative amplitude of approximately 11% of the Y(Z)ox Q(A)- phase was observed after the second light flash. The sensitivity of this phase to diuron (an inhibitor of electron transfer between Q(A) and the secondary quinone acceptor Q(B)), the dependence of its amplitude on the light flash parity, and also a decrease in its rate constant with increase in pH indicated that it was due to dismutation of Q(A)- and Q(B)- with the subsequent protonation of a doubly reduced plastoquinone molecule: Q(A)- Q(B)- + 2H+ --> Q(A)Q(B)H2.     

Inhibition of photosystem II of spinach by the respiration inhibitors piericidin A and thenoyltrifluoroacetone

The effects of several respiration inhibitors on photosystem II (PS II) were investigated. Among the agents tested, piericidin A and thenoyltrifluoroacetone (TTFA) inhibited the photosynthetic electron transport of spinach as measured from chlorophyll (Chl) fluorescence parameters (Fm'-F)/Fm' and Fv/Fm. Using specific donors and acceptors of electrons, we identified the sites of inhibition in and around the PS II complex; the site of inhibition by TTFA was between QA, primary quinone acceptor in PS II, and QB, secondary quinone acceptor, in the acceptor side of P680, the reaction center Chl of PS II, while inhibition by piericidin A of the acceptor side was downstream of Q(B), out of the PS II complex. Both agents also inhibited the donor side of P680, probably between tyrosine-161 of the reaction center protein of PS II and P680.     

The rate of charge recombination in Photosystem II

Loss by recombination of the charge separated state P(680+)Q(A-) limits the performance of Photosystem II (PS II) as a photochemical energy converter. Time constants reported in literature for this process are mostly either near 0.17 ms or near 1.4 ms. The shorter time is found in plant PS II when reduction of P(680+) by the secondary electron donor Tyrosine Z cannot occur because Y(Z) is already oxidized. The 1.4 ms recombination is seen in Y(Z)-less mutants of the cyanobacterium Synechocystis. However, the rate of P(680+)Q(A-) recombination that actually competes with the stabilization of the charge separation has not been previously reported. We have measured the kinetics of the flash-induced fluorescence yield changes in the microsecond time domain in Tris-washed spinach chloroplasts. In this way the kinetics and yield of P(680+) reduction by Y(Z) were obtained, and the rate of the competing P(680+)Q(A-) recombination could be evaluated. The recombination time was less than 0.5 ms; the best-fitting time constant was 0.1 ms. The presence of Y(Z)(ox) slightly decreased the efficiency of excitation trapping but did not seem to accelerate P(680+)Q(A-) recombination. The two P(680+)Q(A-) lifetimes in the literature probably reflect a significant difference between plant and cyanobacterial PS II.     

Photosynthetic water oxidation in Synechocystis sp. PCC6803: mutations D1-E189K, R and Q are without influence on electron transfer at the donor side of photosystem II.

The oxygen-evolving manganese cluster (OEC) of photosynthesis is oxidised by the photochemically generated primary oxidant (P(+*)(680)) of photosystem II via a tyrosine residue (Y(Z), Tyr161 on the D1 subunit of Synechocystis sp. PCC6803). The redox span between these components is rather small and probably tuned by protonic equilibria. The very efficient electron transfer from Y(Z) to P(+*)(680) in nanoseconds requires the intactness of a hydrogen bonded network involving Y(Z), D1-His190, and presumably D1-Glu189. We studied photosystem II core particles from photoautotrophic mutants where the residue D1-E189 was replaced by glutamine, arginine and lysine which were expected to electrostatically differ from the glutamate in the wild-type (WT). Surprisingly, the rates of electron transfer from Y(Z) to P(+*)(680) as well as from the OEC to Y(ox)(Z) were the same as in the WT. With the generally assumed proximity between D1-His190 (and thus D1-Glu189) and Y(Z), the lack of any influence on the electron transfer around Y(Z) straightforwardly implies a strongly hydrophobic environment forcing Glu (acid) and Lys, Arg (basic) at position D1-189 into electro-neutrality. As one alternative, D1-Glu189 could be located at such a large distance from the OEC, Y(Z) and P(+*)(680) that a charge on D1-189X does not influence the electron transfer. This seems less likely in the light of the drastic influence of its direct neighbour, D1-His190, on Y(Z) function. Another alternative is that D1-Glu189 is negatively charged, but is located in a cluster of acid/base groups that compensates for an alteration of charge at position 189, leaving the overall net charge unchanged in the Gln, Lys, and Arg mutants.     

Electrostatics and proton transfer in photosynthetic water oxidation

Photosystem II (PSII) oxidizes two water molecules to yield dioxygen plus four protons. Dioxygen is released during the last out of four sequential oxidation steps of the catalytic centre (S(0) --> S(1), S(1) --> S(2), S(2) --> S(3), S(3) --> S(4) --> S(0)). The release of the chemically produced protons is blurred by transient, highly variable and electrostatically triggered proton transfer at the periphery (Bohr effect). The extent of the latter transiently amounts to more than one H(+)/e(-) under certain conditions and this is understood in terms of electrostatics. By kinetic analyses of electron-proton transfer and electrochromism, we discriminated between Bohr-effect and chemically produced protons and arrived at a distribution of the latter over the oxidation steps of 1 : 0 : 1 : 2. During the oxidation of tyr-161 on subunit D1 (Y(Z)), its phenolic proton is not normally released into the bulk. Instead, it is shared with and confined in a hydrogen-bonded cluster. This notion is difficult to reconcile with proposed mechanisms where Y(Z) acts as a hydrogen acceptor for bound water. Only in manganese (Mn) depleted PSII is the proton released into the bulk and this changes the rate of electron transfer between Y(Z) and the primary donor of PSII P(+)(680) from electron to proton controlled. D1-His190, the proposed centre of the hydrogen-bonded cluster around Y(Z), is probably further remote from Y(Z) than previously thought, because substitution of D1-Glu189, its direct neighbour, by Gln, Arg or Lys is without effect on the electron transfer from Y(Z) to P(+)(680) (in nanoseconds) and from the Mn cluster to Y(ox)(Z).     

Coupling of electron and proton transfer in oxidative water cleavage in photosynthesis

This minireview addresses questions on the mechanism of oxidative water cleavage with special emphasis on the coupling of electron (ET) and proton transfer (PT) of each individual redox step of the reaction sequence and on the mode of O-O bond formation. The following topics are discussed: (1) the multiphasic kinetics of Y(Z)(ox) formation by P680(+*) originate from three different types of rate limitations: (i) nonadiabatic electron transfer for the "fast" ns reaction, (ii) local "dielectric" relaxation for the "slow" ns reaction, and (iii) "large-scale" proton shift for the micros kinetics; (2) the ET/PT-coupling mode of the individual redox transitions within the water oxidizing complex (WOC) driven by Y(Z)(ox) is assumed to depend on the redox state S(i): the oxidation steps of S(0) and S(1) comprise separate ET and PT pathways while those of S(2) and S(3) take place via proton-coupled electron transfer (PCET) analogous to Jerry Babcock's hydrogen atom abstractor model [Biochim. Biophys. Acta, 1458 (2000) 199]; (3) S(3) is postulated to be a multistate redox level of the WOC with fast dynamic equilibria of both redox isomerism and proton tautomerism. The primary event in the essential O-O bond formation is the population of a state S(3)(P) characterized by an electronic configuration and nuclear geometry that corresponds with a complexed hydrogen peroxide; (4) the peroxidic type S(3)(P) is the entatic state for formation of complexed molecular oxygen through S(3) oxidation by Y(Z)(ox); and (5) the protein matrix itself is proposed to exert catalytic activity by functioning as "PCET director". The WOC is envisaged as a supermolecule that is especially tailored for oxidative water cleavage and acts as a molecular machine.     

Possible states of photosystem II reaction center and thermoluminescence of the higher plants. I. Analysis of thermoluminescence models and their applicability to the description of experimental data

A method for the deconvolution of experimental glow curves into overlapping bands and determination of the activation energies for these bands is proposed. The model includes the S-states of the water-splitting complex, tyrozine Z and P680 on the donor side, pheophytine, primary and secondary quinone acceptors on the acceptor side, and takes into account the connection between different states of the reaction center complex. The rate constants of forward electron transport and the activation energies of backward reactions of electron transfer in photosystem II reaction center, included into the model, are estimated from the known experimental data using the proposed approach.     

Thermodynamics of electron transfer in oxygenic photosynthetic reaction centers: volume change, enthalpy, and entropy of electron-transfer reactions in manganese-depleted photosystem II core complexes

We have previously reported the thermodynamic data of electron transfer in photosystem I using pulsed time-resolved photoacoustics [Hou et al. (2001) Biochemistry 40, 7109-7116]. In the present work, using preparations of purified manganese-depleted photosystem II (PS II) core complexes from Synechocystis sp. PCC 6803, we have measured the DeltaV, DeltaH, and estimated TDeltaS of electron transfer on the time scale of 1 micros. At pH 6.0, the volume contraction of PS II was determined to be -9 +/- 1 A3. The thermal efficiency was found to be 52 +/- 5%, which corresponds to an enthalpy change of -0.9 +/- 0.1 eV for the formation of the state P680+Q(A-) from P680*. An unexpected volume expansion on pulse saturation of PS II was observed, which is reversible in the dark. At pH 9.0, the volume contraction, the thermal efficiency, and the enthalpy change were -3.4 +/- 0.5 A3, 37 +/- 7%, and -1.15 +/- 0.13 eV, respectively. The DeltaV of PS II, smaller than that of PS I and bacterial centers, is assigned to electrostriction and analyzed using the Drude-Nernst equation. To explain the small DeltaV for the formation of P680+Q(A-) or Y(Z*)Q(A-), we propose that fast proton transfer into a polar region is involved in this reaction. Taking the free energy of charge separation of PS II as the difference between the energy of the excited-state P680* and the difference in the redox potentials of the donor and acceptor, the apparent entropy change (TDeltaS) for charge separation of PS II is calculated to be negative, -0.1 +/- 0.1 eV at pH 6.0 (P680+Q(A-)) and -0.2 +/- 0.15 eV at pH 9.0 (Y(Z*)Q(A-)). The thermodynamic properties of electron transfer in PS II core reaction centers thus differ considerably from those of bacterial and PS I reaction centers, which have DeltaV of approximately -27 A3, DeltaH of approximately -0.4 eV, and TDeltaS of approximately +0.4 eV.     

Proton and hydrogen currents in photosynthetic water oxidation

The photosynthetic processes that lead to water oxidation involve an evolution in time from photon dynamics to photochemically-driven electron transfer to coupled electron/proton chemistry. The redox-active tyrosine, Y(Z), is the component at which the proton currents necessary for water oxidation are switched on. The thermodynamic and kinetic implications of this function for Y(Z) are discussed. These considerations also provide insight into the related roles of Y(Z) in preserving the high photochemical quantum efficiency in Photosystem II (PSII) and of conserving the highly oxidizing conditions generated by the photochemistry in the PSII reaction center. The oxidation of Y(Z) by P(680)(+) can be described well by a treatment that invokes proton coupling within the context of non-adiabatic electron transfer. The reduction of Y(.)(Z), however, appears to proceed by an adiabatic process that may have hydrogen-atom transfer character.     

Formate-induced inhibition of the water-oxidizing complex of photosystem II studied by EPR

The effects of various formate concentrations on both the donor and the acceptor sides in oxygen-evolving PS II membranes (BBY particles) were examined. EPR, oxygen evolution and variable chlorophyll fluorescence have been observed. It was found that formate inhibits the formation of the S(2) state multiline signal concomitant with stimulation of the Q(A)(-)Fe(2+) signal at g = 1.82. The decrease and the increase in intensities of the multiline and Q(A)(-)Fe(2+) signals, respectively, had a linear relation for formate concentrations between 5 and 500 mM. The g = 4.1 signal formation measured in the absence of methanol was not inhibited by formate up to 250 mM in the buffer. In the presence of 3% methanol the g = 4.1 signal evolved as formate concentration increased. The evolved signal could be ascribed to the inhibited centers. Oxygen evolution measured in the presence of an electron acceptor, phenyl-p-benzoquinone, was also inhibited by formate proportionally to the decrease in the multiline signal intensity. The inhibition seemed to be due to a retarded electron transfer from the water-oxidizing complex to Y(Z)(+), which was observed in the decay kinetics of the Y(Z)(+) signal induced by illumination above 250 K. These results show that formate induces inhibition of water oxidation reactions as well as electron transfer on the PS II acceptor side. The inhibition effects of formate in PS II were found to be reversible, indicating no destructive effect on the reaction center induced by formate.     

The mechanism of photosynthetic water oxidation

Photosynthetie water oxidation is unique to plants and cyanobacteria, it occurs in thylakoid membranes. The components associated with this process include: a reaction center polypeptide, having a molecular weight (Mr) of 47–50 kilodaltons (kDa), containing a reaction center chlorophyll a labeled as P680, a plastoquinol(?)-electron donor Z, a primary electron acceptor pheophytin, and a quinone electron acceptor Q_A; three ‘extrinsic’ polypeptides having Mr of approximately 17 kDa, 23 kDa, and 33 kDa; and, in all likelihood, an approximately 34 kDa ‘intrinsic’ polypeptide associated with manganese (Mn) atoms. In addition, chloride and calcium ions appear to be essential components for water oxidation. Photons, absorbed by the so-called photosystem II, provide the necessary energy for the chemical oxidation-reduction at P680; the oxidized P680 (P680⁺), then, oxidizes Z, which then oxidizes the water-manganese system contained, perhaps, in a protein matrix. The oxidation of water, leading to O₂ evolution and H⁺ release, requires four such independent acts, i.e., there is a charge accumulating device (the so-called S-states). In this minireview, we have presented our current understanding of the reaction center P680, the chemical nature of Z, a possible working model for water oxidation, and the possible roles of manganese atoms, chloride ions, and the various polypeptides, mentioned above. A comparison with cytochrome c oxidase, which is involved in the opposite process of the reduction of O₂ to H₂O, is stressed. This minireview is a prelude to the several minireviews, scheduled to be published in the forthcoming issues of Photosynthesis Research, including those on photosystem II (by H.J. van Gorkom); polypeptides of the O₂-evolving system (by D.F. Ghanotakis and C.F. Yocum); and the role of chloride in O₂ evolution (by S. Izawa).     

Recruitment of a foreign quinone into the A(1) site of photosystem I. II. Structural and functional characterization of phylloquinone biosynthetic pathway mutants by electron paramagnetic resonance and electron-nuclear double resonance spectroscopy

Electron paramagnetic resonance (EPR) and electron-nuclear double resonance studies of the photosystem (PS) I quinone acceptor, A(1), in phylloquinone biosynthetic pathway mutants are described. Room temperature continuous wave EPR measurements at X-band of whole cells of menA and menB interruption mutants show a transient reduction and oxidation of an organic radical with a g-value and anisotropy characteristic of a quinone. In PS I complexes, the continuous wave EPR spectrum of the photoaccumulated Q(-) radical, measured at Q-band, and the electron spin-polarized transient EPR spectra of the radical pair P700(+) Q(-), measured at X-, Q-, and W-bands, show three prominent features: (i) Q(-) has a larger g-anisotropy than native phylloquinone, (ii) Q(-) does not display the prominent methyl hyperfine couplings attributed to the 2-methyl group of phylloquinone, and (iii) the orientation of Q(-) in the A(1) site as derived from the spin polarization is that of native phylloquinone in the wild type. Electron spin echo modulation experiments on P700(+) Q(-) show that the dipolar coupling in the radical pair is the same as in native PS I, i.e. the distance between P700(+) and Q(-) (25.3 +/- 0.3 A) is the same as between P700(+) and A(1)(-) in the wild type. Pulsed electron-nuclear double resonance studies show two sets of resolved spectral features with nearly axially symmetric hyperfine couplings. They are tentatively assigned to the two methyl groups of the recruited plastoquinone-9, and their difference indicates a strong inequivalence among the two groups when in the A(1) site. These results show that Q (i) functions in accepting an electron from A(0)(-) and in passing the electron forward to the iron-sulfur clusters, (ii) occupies the A(1) site with an orientation similar to that of phylloquinone in the wild type, and (iii) has spectroscopic properties consistent with its identity as plastoquinone-9.     

Quantum efficiency distributions of photo-induced side-pathway donor oxidation at cryogenic temperature in photosystem II

We monitored illuminated-minus-dark absorption difference spectra in the range of 450-1100 nm induced by continuous illumination at 8 K of photosystem II (PSII) core complexes from Thermosynechococcus elongatus. The photo-induced oxidation of the side-path donors Cytb(559), beta-carotene and chlorophyll Z, as well as the concomitant stable (t(1/2) > 1 s) reduction of the first plastoquinone electron acceptor, Q(A) (monitored by the well-known 'C550' shift), were quantified as a function of the absorbed photons per PSII. The Q(A) photo-induced reduction data can be described by three distinct quantum efficiency distributions: (i) a very high efficiency of approximately 0.5-1, (ii) a middle efficiency with a very large range of approximately 0.014-0.2, and (iii) a low efficiency of approximately 0.002. Each of the observed side-path donors exhibited similar quantum efficiency distributions, which supports a branched pathway model for side-path oxidation where beta-carotene is the immediate electron donor to the photo-oxidized chlorophyll (P680(+)). The yields of the observed side-path donors account quantitatively for the wide middle efficiency range of photo-induced Q(A) reduction, but not for the PSII fractions that exhibit the highest and lowest efficiencies. The high-efficiency component may be due to Tyr(Z) oxidation. A donor that does not exhibit an identified absorption in the visible-near-IR region is mainly responsible for the lowest efficiency component.     

Mechanism of light induced water splitting in Photosystem II of oxygen evolving photosynthetic organisms

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. Gl?ckner, 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 1.9?. 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.     

Acceptor side effects on the electron transfer at cryogenic temperatures in intact photosystem II

In intact PSII, both the secondary electron donor (Tyr(Z)) and side-path electron donors (Car/Chl(Z)/Cyt(b)(559)) can be oxidized by P(680)(+) at cryogenic temperatures. In this paper, the effects of acceptor side, especially the redox state of the non-heme iron, on the donor side electron transfer induced by visible light at cryogenic temperatures were studied by EPR spectroscopy. We found that the formation and decay of the S(1)Tyr(Z) EPR signal were independent of the treatment of K(3)Fe(CN)(6), whereas formation and decay of the Car(+)/Chl(Z)(+) EPR signal correlated with the reduction and recovery of the Fe(3+) EPR signal of the non-heme iron in K(3)Fe(CN)(6) pre-treated PSII, respectively. Based on the observed correlation between Car/Chl(Z) oxidation and Fe(3+) reduction, the oxidation of non-heme iron by K(3)Fe(CN)(6) at 0 degrees C was quantified, which showed that around 50-60% fractions of the reaction centers gave rise to the Fe(3+) EPR signal. In addition, we found that the presence of phenyl-p-benzoquinone significantly enhanced the yield of Tyr(Z) oxidation. These results indicate that the electron transfer at the donor side can be significantly modified by changes at the acceptor side, and indicate that two types of reaction centers are present in intact PSII, namely, one contains unoxidizable non-heme iron and another one contains oxidizable non-heme iron. Tyr(Z) oxidation and side-path reaction occur separately in these two types of reaction centers, instead of competition with each other in the same reaction centers. In addition, our results show that the non-heme iron has different properties in active and inactive PSII. The oxidation of non-heme iron by K(3)Fe(CN)(6) takes place only in inactive PSII, which implies that the Fe(3+) state is probably not the intermediate species for the turnover of quinone reduction.     

Phosphatidylglycerol requirement for the function of electron acceptor plastoquinone Q(B) in the photosystem II reaction center

Phosphatidylglycerol (PG), a ubiquitous constituent of thylakoid membranes of chloroplasts and cyanobacteria, is demonstrated to be essential for the functionality of plastoquinone electron acceptor Q(B) in the photosystem II reaction center of oxygenic photosynthesis. Growth of the pgsA mutant cells of Synechocystis sp. PCC6803 that are defective in phosphatidylglycerolphosphate synthase and are incapable of synthesizing PG, in a medium without PG, resulted in a 90% decrease in PG content and a 50% loss of photosynthetic oxygen-evolving activity as reported [Hagio, M., Gombos, Z., Várkonyi, Z., Masamoto, K., Sato, N., Tsuzuki, M., and Wada, H. (2000) Plant Physiol. 124, 795-804]. We have studied each step of the electron transport in photosystem II of the pgsA mutant to clarify the functional site of PG. Accumulation of Q(A)(-) was indicated by the fast rise of chlorophyll fluorescence yield under continuous and flash illumination. Oxidation of Q(A)(-) by Q(B) plastoquinone was shown to become slow, and Q(A)(-) reoxidation required a few seconds when measured by double flash fluorescence measurements. Thermoluminescence measurements further indicated the accumulation of the S(2)Q(A)(-) state but not of the S(2)Q(B)(-) state following the PG deprivation. These results suggest that the function of Q(B) plastoquinone was inactivated by the PG deprivation. We assume that PG is an indispensable component of the photosystem II reaction center complex to maintain the structural integrity of the Q(B)-binding site. These findings provide the first clear identification of a specific functional site of PG in the photosynthetic reaction center.     

Fast structural changes (200-900ns) may prepare the photosynthetic manganese complex for oxidation by the adjacent tyrosine radical

The Mn complex of photosystem II (PSII) cycles through 4 semi-stable states (S(0) to S(3)). Laser-flash excitation of PSII in the S(2) or S(3) state induces processes with time constants around 350ns, which have been assigned previously to energetic relaxation of the oxidized tyrosine (Y(Z)(ox)). Herein we report monitoring of these processes in the time domain of hundreds of nanoseconds by photoacoustic (or 'optoacoustic') experiments involving pressure-wave detection after excitation of PSII membrane particles by ns-laser flashes. We find that specifically for excitation of PSII in the S(2) state, nuclear rearrangements are induced which amount to a contraction of PSII by at least 30?(3) (time constant of 350ns at 25°C; activation energy of 285+/-50meV). In the S(3) state, the 350-ns-contraction is about 5 times smaller whereas in S(0) and S(1), no volume changes are detectable in this time domain. It is proposed that the classical S(2)=>S(3) transition of the Mn complex is a multi-step process. The first step after Y(Z)(ox) formation involves a fast nuclear rearrangement of the Mn complex and its protein-water environment (~350ns), which may serve a dual role: (1) The Mn- complex entity is prepared for the subsequent proton removal and electron transfer by formation of an intermediate state of specific (but still unknown) atomic structure. (2) Formation of the structural intermediate is associated (necessarily) with energetic relaxation and thus stabilization of Y(Z)(ox) so that energy losses by charge recombination with the Q(A)(-) anion radical are minimized. The intermediate formed within about 350ns after Y(Z)(ox) formation in the S(2)-state is discussed in the context of two recent models of the S(2)=>S(3) transition of the water oxidation cycle. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: From Natural to Artificial.     

Studies on thylakoid phosphorylation and noncyclic electron transport

The effect of thylakoid phosphorylation on noncyclic electron transport in spinach chloroplasts was investigated by measuring both the reduction of nicotinamide adenine dinucleotide phosphate (NADP) and the steady-state redox level of the primary electron acceptor quinone of photosystem II (Q) during electron flow to NADP. These data are compared with the theoretical predictions for an electron transport model which relates both the redox levels of Q and the photosystem II optical cross section to the overall velocity of noncyclic electron flow. It is demonstrated that transfer of 15-20% of the photosystem II antenna to photosystem I may stimulate electron flow to NADP only if Q is less than 60-70% oxidized (this condition exists with our thylakoids, even at extremely low absorption fluxes, when the illumination is not specifically enriched in photosystem I absorbed wavelengths); in phosphorylated thylakoids the steady-state redox level Q is substantially shifted to a more oxidized one (measurements of this parameter using light of different wavelengths quantitatively support the idea that thylakoid phosphorylation leads to increased photosystem I and decreased photosystem II cross sections); thylakoid phosphorylation leads to stimulated noncyclic electron flow to NADP only when the increased photosystem I antenna is able to bring about large increases in the steady-state level of oxidized Q.     

Amino acid residues involved in the coordination and assembly of the manganese cluster of photosystem II. Proton-coupled electron transport of the redox-active tyrosines and its relationship to water oxidation

The combination of site-directed mutagenesis, isotopic labeling, new magnetic resonance techniques and optical spectroscopic methods have provided new insights into cofactor coordination and into the mechanism of electron transport and proton-coupled electron transport in photosystem II. Site-directed mutations in the D1 polypeptide of this photosystem have implicated a number of histidine and carboxylate residues in the coordination and assembly of the manganese cluster, responsible for photosynthetic water oxidation. Many of these are located in the carboxy-terminal region of this polypeptide close to the processing site involved in its maturation. This maturation is a required precondition for cluster assembly. Recent proposals for the mechanism of water oxidation have directly implicated redox-active tyrosine Y(Z) in this mechanism and have emphasized the importance of the coupling of proton and electron transfer in the reduction of Y(Z)(radical) by the Mn cluster. The interaction of both homologous redox-active tyrosines Y(Z) and Y(D) with their respective homologous proton acceptors is discussed in an effort to better understand the significance of such coupling.