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.     

Electron microscopy in structural studies of Photosystem II

Various techniques of electron microscopy (EM) such as ultrathin sectioning, freeze-fracturing, freeze-etching, negative staining and (cryo-)electron crystallography of two-dimensional crystals have been employed, since now, to obtain much of the structural information of the Photosystem II (PS II) pigment–protein complex at both low and high resolution. This review summarizes information about the structure of this membrane complex as well as its arrangement and interactions with the antenna proteins in thylakoid membranes of higher plants and cyanobacteria obtained by means of EM. Results on subunit organization, with the emphasis on the proteins of the oxygen-evolving complex (OEC), are compared with the data obtained by X-ray crystallography of cyanobacterial PS II. This revised version was published online in August 2006 with corrections to the Cover Date.     

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

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

Computer simulation study of pH-dependent regulation of electron transport in chloroplasts

A mathematical model is presented that describes the key steps of photosynthetic electron transport and transmembrane proton transfer in chloroplasts. Numerical modeling has been performed with due regard for regulatory processes at the donor and acceptor parts of photosystem (PS) I. The influence of pH-dependent activation of the Calvin cycle enzymes and energy dissipation in PS II (nonphotochemical quenching of chlorophyll fluorescence) on the light-induced redox transients of P₇₀₀, plastoquinone, and NADP as well as on the changes in intrathylakoid pH and ATP level is examined. It is demonstrated that pH-dependent regulatory processes alter the distribution of electron fluxes on the acceptor side of PS I and the total rate of electron flow between PS II and PS I. The light-induced activation of the Calvin cycle leads to significant enhancement of the electron flow from PS I to NADP⁺ and attenuation of the electron flow to molecular oxygen.     

Progress towards structural elucidation of Photosystem II

In recent years Photosystem II, and in particular the oxygen evolving component of the enzyme, have been the subject of intense biochemical and biophysical analysis. To date no high resolution structural model of the complex has been produced. As a consequence unambiguous interpretation of much experimental data has proven difficult, leading to a lack of consensus over many basic questions regarding the mechanisms involved, the oligomerization state of the enzyme in vivo and even the exact biochemical composition.This review is a summary of the progress towards the production of a structural model of PS II-derived from either X-ray crystallography or electron microscopy based techniques-and the current opinions, which have arisen from these structural analyses, on the structural topology and assemblage of the various subunits that constitute the complex.Abbreviations C₁₂-M dodecyl maltoside - CP chlorophyll protein - cyt b-559 cytochrome b-559 - DMPC dimyristoyl phosphatidyl choline - EC electron crystallography - EM electron microscopy - LHC II light harvesting complex II - OEC oxygen evolving complex - OG octyl--glucopyranoside - PS I Photosystem I - PS II Photosystem II - Tris N-tris (hydroxymethyl) amino ethane     

Assignment of the low-temperature fluorescence in oxygen-evolving Photosystem II

Low-temperature absorption and fluorescence spectra of fully active cores and membrane-bound PS II preparations are compared. Detailed temperature dependence of fluorescence spectra between 5 and 70 K are presented as well as 1.7-K fluorescence line-narrowed (FLN) spectra of cores, confirming that PS II emission is composite. Spectra are compared to those reported for LHCII, CP43, CP47 and D1/D2/cytit b₅₅₉ subunits of PS II. A combination of subunit spectra cannot account for emission of active PS II. The complex temperature dependence of PS II fluorescence is interpretable by noting that excitation transfer from CP43 and CP47 to the reaction centre is slow, and strongly dependent on the precise energy at which a ‘slow-transfer’ pigment in CP43 or CP47 is located within its inhomogeneous distribution. PS II fluorescence arises from CP43 and CP47 ‘slow-transfer’ states, convolved by this dependence. At higher temperatures, thermally activated excitation transfer to the PS II charge-separating system bypasses such bottlenecks. As the charge-separating state of active PS II absorbs at >700 nm, PS II emission in the 680–700 nm region is unlikely to arise from reaction centre pigments. PS II emission at physiological temperatures is discussed in terms of these results.     

Analysis of the slow phases of the in vivo chlorophyll fluorescence induction curve. Changes in the redox state of Photosystem II electron acceptors and fluorescence emission from Photosystems I and II

An analysis of the photo-induced decline in the in vivo chlorophyll a fluorescence emission (Kautsky phenomenon) from the bean leaf is presented. The redox state of PS II electron acceptors and the fluorescence emission from PS I and PS II were monitored during quenching of fluorescence from the maximum level at P to the steady state level at T. Simultaneous measurement of the kinetics of fluorescence emission associated with PS I and PS II indicated that the ratio of PS I/PS II emission changed in an antiparallel fashion to PS II emission throughout the induction curve. Estimation of the redox state of PS II electron acceptors at given points during P to T quenching was made by exposing the leaf to additional excitation irradiation and determining the amount of variable PS II fluorescence generated. An inverse relationship was found between the proportion of PS II electron acceptors in the oxidised state and PS II fluorescence emission. The interrelationships between the redox state of PS II electron acceptors and fluorescence emission from PS I and PS II remained similar when the shape of the induction curve from P to T was modified by increasing the excitation photon flux density. The contributions of photochemical and non-photochemical quenching to the in vivo fluorescence decline from P to T are discussed.     

State 1-State 2 transitions in the cyanobacterium Synechococcus 6301 are controlled by the redox state of electron carriers between Photosystems I and II

The mechanism by which state 1-state 2 transitions in the cyanobacterium Synechococcus 6301 are controlled was investigated by examining the effects of a variety of chemical and illumination treatments which modify the redox state of the plastoquinone pool. The extent to which these treatments modify excitation energy distribution was determined by 77K fluorescence emission spectroscopy. It was found that treatment which lead to the oxidation of the plastoquinone pool induce a shift towards state 1 whereas treatments which lead to the reduction of the plastoquinone pool induce a shift towards state 2. We therefore propose that state transitions in cyanobacteria are triggered by changes in the redox state of plastoquinone or a closely associated electron carrier. Alternative proposals have included control by the extent of cyclic electron transport around PS I and control by localised electrochemical gradients around PS I and PS II. Neither of these proposals is consistent with the results reported here.Abbreviations DBMIB 2,5-dibromo-3methyl-6-isopropyl-p-benzoquinone - Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DQH₂ duroquinol (tetramethyl-p-hydroquinone) - LHC II light-harvesting chlorophyll a/b-binding protein of PS II - Light 1 light predominantly exciting PS I - Light 2 light predominantly exciting PS II - M.V. methyl viologen - PS photosystem     

When X-rays modify the protein structure: radiation damage at work

The majority of 3D structures of macromolecules are currently determined by macromolecular crystallography, which employs the diffraction of X-rays on single crystals. However, during diffraction experiments, the X-rays can damage the protein crystals by ionization processes, especially when powerful X-ray sources at synchrotron facilities are used. This process of radiation damage generates photo-electrons that can get trapped in protein moieties. The 3D structure derived from such experiments can differ remarkably from the structure of the native molecule. Recently, the crystal structures of different oxidation states of horseradish peroxidase and nickel-containing superoxide dismutase were determined using crystallographic redox titration performed during the exposure of the crystals to the incident X-ray beam. Previous crystallographic analyses have not shown the distinct structures of the active sites associated with the redox state of the structural features of these enzymes. These new studies show that, for protein moieties that are susceptible to radiation damage and prone to reduction by photo-electrons, care is required in both the design of the diffraction experiment and the analysis and interpretation.     

Influence of thylakoid protein phosphorylation on photosynthetic electron transport and photophosphorylation

Data are reported which show that thylakoid protein phosphorylation decreases photosystem II fluorescence yield and enhances the photosystem I dependent photophosphorylation catalyzed by phenazinemethosulphate in the presence of DCMU. The stimulation is larger at low light intensity, but is still observed at high intensity. These observations are interpreted to demonstrate that thylakoid protein phosphorylation causes a transfer of excitation energy from PS II to PS I, but may also have an independent stimulatory effect on PS I dependent photophosphorylation.     

E. Antonini Plenary lecture. A structural basis of light energy and electron transfer in biology

Aspects of intramolecular light energy and electron transfer will be discussed for three protein cofactor complexes, whose three-dimensional structures have been elucidated by X-ray crystallography: components of light-harvesting cyanobacterial phycobilisomes, the purple bacterial reaction centre and the blue multi-copper oxidases. A wealth of functional data is available for these systems which allow specific correlations between structure and function, and general conclusions about light energy and electron transfer in biological materials to be made.     

Lipid-assisted protein-protein interactions that support photosynthetic and other cellular activities

Glycoglycerolipids are dominant lipids of photosynthetic organisms, i.e. higher plants and cyanobacteria. X-ray crystallographic localization of glycerolipids revealed that they are present at functionally and structurally important sites of both the PS I and PS II reaction centres. Phosphatidylglycerol (PG) is an indispensible member of glycerolipids, including the formation of functionally active oligomers of the reaction centres both PS I and PS II. Lipids assist in the assembly of protein subunits of the photosynthetic machinery by pasting the individual protein components together. PG is needed to glue CP43 to the reaction centre core. PG and digalactosyldiacylglycerol (DGDG) interact in photosynthetic processes: PG alone controls electron transport at the acceptor site of PS II, and together with DGDG is involved in electron transport at the donor site of PS II. PG is crucial for the formation of division rings and is implicated in the fission of cyanobacteria.     

Influence of protein phosphorylation on the electron-transport properties of Photosystem II

Many of the core proteins in Photosystem II (PS II) undergo reversible phosphorylation. It is known that protein phosphorylation controls the repair cycle of Photosystem II. However, it is not known how protein phosphorylation affects the partial electron transport reactions in PS II. Here we have applied variable fluorescence measurements and EPR spectroscopy to probe the status of the quinone acceptors, the Mn cluster and other electron transfer components in PS II with controlled levels of protein phosphorylation. Protein phosphorylation was induced in vivo by varying illumination regimes. The phosphorylation level of the D1 protein varied from 10 to 58% in PS II membranes isolated from pre-illuminated spinach leaves. The oxygen evolution and Q_A ⁻ to Q_B(Q_B ⁻) electron transfer measured by flash-induced fluorescence decay remained similar in all samples studied. Similar measurements in the presence of DCMU, which reports on the status of the donor side in PS II, also indicated that the integrity of the oxygen-evolving complex was preserved in PS II with different levels of D1 protein phosphorylation. With EPR spectroscopy we examined individual redox cofactors in PS II. Both the maximal amplitude of the charge separation reaction (measured as photo-accumulated pheophytin⁻) and the EPR signal from the Q_A ⁻ Fe²⁺ complex were unaffected by the phosphorylation of the D1 protein, indicating that the acceptor side of PS II was not modified. Also the shape of the S₂ state multiline signal was similar, suggesting that the structure of the Mn-cluster in Photosystem II did not change. However, the amplitude of the S₂ multiline signal was reduced by 35% in PS II, where 58% of the D1 protein was phosphorylated, as compared to the S₂ multiline in PS II, where only 10% of the D1 protein was phosphorylated. In addition, the fraction of low potential Cyt b ₅₅₉ was twice as high in phosphorylated PS II. Implications from these findings, were precise quantification of D1 protein phosphorylation is, for the first time, combined with high-resolution biophysical measurements, are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Senescence-induced alterations in the photosystem II functions of Cucumis sativus cotyledons: probing of senescence driven alterations of photosystem II by chlorophyll a fluorescence induction O-J-I-P transients

Senescence-induced alterations in photosystem II (PS II) structure and photofunctions were probed in cucumber (Cucumis sativus) cotyledons, using fast O-J-I-P Chlorophyll a (Chl a) fluorescence transients. Analysis of measured and derived parameters of the fast fluorescence O-J-I-P transient revealed senescence-induced alterations in (i), PS II acceptor side electron transfer equilibrium between QA and QB, the primary stable and secondary acceptors of PS II; (ii), intersystem PQ pool size and (iii), affected electron transfer from PS II to PS I. Also, senescence of cotyledons triggered conversion of QA-reducing (fully active) to non- QA-reducing PS II (heat sink) centres. Further, some of the remaining active PS II centres showed a high apparent trapping efficiency due to clustering and energetic connectivity (grouping) between the antennae of active and inactive centers. The overall density of active PS II reaction centers showed a temporal decrease due to the onset of foliar senescence. Thus, the fast Chl a fluorescence transients, with a time resolution of at least 50 mircosec and use of the equations of JIP-test, provide a valuable, non-invasive rapid biophysical probe to study the ageing in plants in terms of detecting photosynthetic activities and the heterogeneity of different types of photosynthetic units. Further, these results were found to be in agreement with the earlier in vitro studies using thylakoids isolated from senescing cotyledons where it was shown that senescence induced heterogeneity in PS II centers affected acceptor side QA<-->QB equilibrium.     

Electron transfer through the acceptor side of photosystem I: Interaction with exogenous acceptors and molecular oxygen

This review considers the state-of-the-art on mechanisms and alternative pathways of electron transfer in photosynthetic electron transport chains of chloroplasts and cyanobacteria. The mechanisms of electron transport control between photosystems (PS) I and II and the Calvin–Benson cycle are considered. The redistribution of electron fluxes between the noncyclic, cyclic, and pseudocyclic pathways plays an important role in the regulation of photosynthesis. Mathematical modeling of light-induced electron transport processes is considered. Particular attention is given to the electron transfer reactions on the acceptor side of PS I and to interactions of PS I with exogenous acceptors, including molecular oxygen. A kinetic model of PS I and its interaction with exogenous electron acceptors has been developed. This model is based on experimental kinetics of charge recombination in isolated PS I. Kinetic and thermodynamic parameters of the electron transfer reactions in PS I are scrutinized. The free energies of electron transfer between quinone acceptors A_1A/A_1B in the symmetric redox cofactor branches of PS I and iron–sulfur clusters F_X, F_A, and F_B have been estimated. The second-order rate constants of electron transfer from PS I to external acceptors have been determined. The data suggest that byproduct formation of superoxide radical in PS I due to the reduction of molecular oxygen in the A1 site (Mehler reaction) can exceed 0.3% of the total electron flux in PS I.     

The nature of the photosystem II reaction centre in the chlorophyll d-containing prokaryote, Acaryochloris marina.

Pigment-protein complexes enriched in photosystem II (PS II) have been isolated from the chlorophyll (Chl) d containing cyanobacterium, Acaryochloris marina. A small PS II-enriched particle, we call 'crude reaction centre', contained 20 Chl d, 0.5 Chl a and 1 redox active cytochrome b-559 per 2 pheophytin a, plus the D1 and D2 proteins. A larger PS II-enriched particle, we call 'core', additionally bound the antenna complexes, CP47 and CP43, and had a higher chlorophyll per pheophytin ratio. Pheophytin a could be photoreduced in the presence of a strong reductant, indicating that it is the primary electron acceptor in photosystem II of A. marina. A substoichiometric amount of Chl a (less than one chlorophyll a per 2 pheophytin a) strongly suggests that Chl a does not have an essential role in the photochemistry of PS II in this organism. We conclude that PS II, in A. marina, utilizes Chl d and not Chl a as primary electron donor and that the primary electron acceptor is one of two molecules of pheophytin a.     

Effect of exogenous histidine on restoration of electron transfer on the donor side of Photosystem II depleted of Mn

It is shown that restoration of photoinduced electron flow with added Mn²⁺ (measured by photoreduction of DCPIP and photoinduced change of chlorophyll fluorescence yield) in Mn-depleted Photosystem II (PS II) membrane fragments isolated from spinach chloroplasts, is considerably increased by exogenous histidine (His). The stimulating effect of His is not observed if other electron donors (NH₂OH or diphenylcarbazide) are used instead of Mn²⁺. His added alone does not induce electron transfer in Mn-depleted PS II preparations. Investigation of pH dependence of the stimulating effect of 2 mM His shows that the effect is observed only at pH > 5.0, it gives a 50% activation around pH 6.0 and saturates at pH 7.0–7.5. Nearly 200 μM His is required for a 50 effect at pH 7.0. It is suggested that the added His can be involved in stimulation of electron transfer on the donor side of PS II through direct interaction of Mn²⁺ with deprotonated form(s) of His resulting in formation of Mn–His complexes capable of efficient electron donation to PS II (though it is not excluded that His serves as a base that takes part in proton exchange coupled with redox reactions on the donor side of PS II or as an electron donor to the oxidized Mn).     

Protein-cofactor interactions in bioenergetic complexes: The role of the A1A and A1B phylloquinones in Photosystem I

This review focuses on phylloquinone as an indispensable link between light-induced charge separation and subsequent charge stabilization in Photosystem I (PS I). Here, the role of the polypeptide in conferring the necessary kinetic and thermodynamic properties to phylloquinone so as to specify its functional role in PS I electron transfer is discussed. Photosynthetic electron transfer and the role of quinones in Type I and Type II reaction centers are introduced at the outset with particular emphasis on the determination of redox potentials of the cofactors. Currently used methodologies, particularly time-resolved optical spectroscopy and varieties of magnetic resonance spectroscopy that have become invaluable in uncovering the details of phylloquinone function are described in depth. Recent studies on the selective alteration of the protein environment and on the incorporation of foreign quinones either by chemical or genetic means are explored to assess how these studies have improved our understanding of protein-quinone interactions. Particular attention is paid to the function of the H-bond, methyl group and phytyl tail of the phylloquinone in interacting with the protein environment.