BBA - Bioenergetics (v.1767, #6)

Structure and Function of Photosystems by Suleyman I. Allakhverdiev; Vladimir A. Shuvalov; Vyacheslav V. Klimov (401-403).

Purification, crystallization and X-ray diffraction analyses of the T. elongatus PSII core dimer with strontium replacing calcium in the oxygen-evolving complex by Joanna Kargul; Karim Maghlaoui; James W. Murray; Zsuzsanna Deak; Alain Boussac; A. William Rutherford; Imre Vass; James Barber (404-413).
The core complex of photosystem II (PSII) was purified from thermophillic cyanobaterium Thermosynechococcus elongatus grown in Sr2+-containing and Ca2+-free medium. Functional in vivo incorporation of Sr2+ into the oxygen-evolving complex (OEC) was confirmed by EPR analysis of the isolated and highly purified SrPSII complex in agreement with the previous study of Boussac et al. [J. Biol. Chem. 279 (2004) 22809–22819]. Three-dimensional crystals of SrPSII complex were obtained which diffracted to 3.9 Å and belonged to the orthorhombic space group P212121 with unit cell dimensions of a  = 133.6 Å, b  = 236.6 Å, c  = 307.8 Å. Anomalous diffraction data collected at the Sr K-X-ray absorption edge identified a novel Sr2+-binding site which, within the resolution of these data (6.5 Å), is consistent with the positioning of Ca2+ in the recent crystallographic models of PSII [Ferreira et al. Science 303 (2004) 1831–1838, Loll et al. Nature 438 (2005) 1040–1044]. Fluorescence measurements on SrPSII crystals confirmed that crystallized SrPSII was active in transferring electrons from the OEC to the acceptor site of the reaction centre. However, SrPSII showed altered functional properties of its modified OEC in comparison with that of the CaPSII counterpart: slowdown of the QA-to-QB electron transfer and stabilized S2QA charge recombination.
Keywords: Photosynthesis; Photosystem II structure; Strontium replacement; X-ray crystallography; Fluorescence decay and imaging; Water splitting;

Photoinhibition of photosystem II under environmental stress by Norio Murata; Shunichi Takahashi; Yoshitaka Nishiyama; Suleyman I. Allakhverdiev (414-421).
Inhibition of the activity of photosystem II (PSII) under strong light is referred to as photoinhibition. This phenomenon is due to an imbalance between the rate of photodamage to PSII and the rate of the repair of damaged PSII. In the “classical” scheme for the mechanism of photoinhibition, strong light induces the production of reactive oxygen species (ROS), which directly inactivate the photochemical reaction center of PSII. By contrast, in a new scheme, we propose that photodamage is initiated by the direct effect of light on the oxygen-evolving complex and that ROS inhibit the repair of photodamaged PSII by suppressing primarily the synthesis of proteins de novo. The activity of PSII is restricted by a variety of environmental stresses. The effects of environmental stress on damage to and repair of PSII can be examined separately and it appears that environmental stresses, with the exception of strong light, act primarily by inhibiting the repair of PSII. Studies have demonstrated that repair-inhibitory stresses include CO2 limitation, moderate heat, high concentrations of NaCl, and low temperature, each of which suppresses the synthesis of proteins de novo, which is required for the repair of PSII. We postulate that most types of environmental stress inhibit the fixation of CO2 with the resultant generation of ROS, which, in turn, inhibit protein synthesis.
Keywords: Environmental stress; Photodamage; Photoinhibition; Photosystem II; Protein synthesis; Repair;

It has been shown [V.A. Shuvalov, Quantum dynamics of electrons in many-electron atoms of biologically important compounds, Biochemistry (Mosc.) 68 (2003) 1333-1354; V.A. Shuvalov, Quantum dynamics of electrons in atoms of biologically important molecules, Uspekhi biologicheskoi khimii, (Pushchino) 44 (2004) 79-108] that the orbit angular momentum L of each electron in many-electron atoms is L  =  mVr  =  nℏ and similar to L for one-electron atom suggested by N. Bohr. It has been found that for an atom with N electrons the total electron energy equation E  =  (Z eff)2 e 4 m/(2n 2 2 N) is more appropriate for energy calculation than standard quantum mechanical expressions. It means that the value of L of each electron is independent of the presence of other electrons in an atom and correlates well to the properties of virtual photons emitted by the nucleus and creating a trap for electrons. The energies for elements of the 1st up to the 5th rows and their ions (total amount 240) of Mendeleev' Periodical table were calculated consistent with the experimental data (deviations in average were 5 × 10− 3). The obtained equations can be used for electron dynamics calculations in molecules. For H2 and H2 + the interference of electron–photon orbits between the atoms determines the distances between the nuclei which are in agreement with the experimental values. The formation of resonance electron–photon orbit in molecules with the conjugated bonds, including chlorophyll-like molecules, appears to form a resonance trap for an electron with E values close to experimental data. Two mechanisms were suggested for non-barrier primary charge separation in reaction centers (RCs) of photosynthetic bacteria and green plants by using the idea of electron–photon orbit interference between the two molecules. Both mechanisms are connected to formation of the exciplexes of chlorophyll-like molecules. The first one includes some nuclear motion before exciplex formation, the second one is related to the optical transition to a charge transfer state.
Keywords: Many-electron atoms; Quantum dynamics of electrons in atoms and molecules; Femtosecond spectroscopy; Photosynthetic reaction centers; Non-barrier electron transfer;

A cluster of carboxylic groups in PsbO protein is involved in proton transfer from the water oxidizing complex of Photosystem II by Tatiana Shutova; Vyacheslav V. Klimov; Bertil Andersson; Göran Samuelsson (434-440).
The hypothesis presented here for proton transfer away from the water oxidation complex of Photosystem II (PSII) is supported by biochemical experiments on the isolated PsbO protein in solution, theoretical analyses of better understood proton transfer systems like bacteriorhodopsin and cytochrome oxidase, and the recently published 3D structure of PS II (Pdb entry 1S5L). We propose that a cluster of conserved glutamic and aspartic acid residues in the PsbO protein acts as a buffering network providing efficient acceptors of protons derived from substrate water molecules. The charge delocalization of the cluster ensures readiness to promptly accept the protons liberated from substrate water. Therefore protons generated at the catalytic centre of PSII need not be released into the thylakoid lumen as generally thought. The cluster is the beginning of a localized, fast proton transfer conduit on the lumenal side of the thylakoid membrane. Proton-dependent conformational changes of PsbO may play a role in the regulation of both supply of substrate water to the water oxidizing complex and the resultant proton transfer.
Keywords: Photosystem II; PsbO protein; Proton transfer; Cluster of carboxylic group;

Correlation of electron transfer rate in photosynthetic reaction centers with intraprotein dielectric properties by Sergey K. Chamorovsky; Dmitry A. Cherepanov; Constantin S. Chamorovsky; Alexey Yu. Semenov (441-448).
A number of the electrogenic reactions in photosystem I, photosystem II, and bacterial reaction centers (RC) were comparatively analyzed, and the variation of the dielectric permittivity (ε) in the vicinity of electron carriers along the membrane normal was calculated. The value of ε was minimal at the core of the complexes and gradually increased towards the periphery. We found that the rate of electron transfer (ET) correlated with the value of the dielectric permittivity: the fastest primary ET reactions occur in the low-polarity core of the complexes within the picosecond time range, whereas slower secondary reactions take place at the high-polarity periphery of the complexes within micro- to millisecond time range. The observed correlation was quantitatively interpreted in the framework of the Marcus theory. We calculated the reorganization energy of ET carriers using their van der Waals volumes and experimentally determined ε values. The electronic coupling was calculated by the empirical Moser–Dutton rule for the distance-dependent electron tunneling rate in nonadiabatic ET reactions. We concluded that the local dielectric permittivity inferred from the electrometric measurements could be quantitatively used to estimate the rate constant of ET reactions in membrane proteins with resolved atomic structure with the accuracy of less than one order of magnitude.
Keywords: Photosynthetic reaction center; Dielectric permittivity; Photoelectric activity; Rate constant; Marcus theory;

Recent advances in vectorial proteomics of protein domains exposed to the surface of photosynthetic thylakoid membranes of plants and the green alga Chlamydomonas reinhardtii allowed mapping of in vivo phosphorylation sites in integral and peripheral membrane proteins. In plants, significant changes of thylakoid protein phosphorylation are observed in response to stress, particularly in photosystem II under high light or high temperature stress. Thylakoid protein phosphorylation in the algae is much more responsive to the ambient redox and light conditions, as well as to CO2 availability. The light-dependent multiple and differential phosphorylation of CP29 linker protein in the green algae is suggested to control photosynthetic state transitions and uncoupling of light harvesting proteins from photosystem II under high light. The similar role for regulation of the dynamic distribution of light harvesting proteins in plants is proposed for the TSP9 protein, which together with other recently discovered peripheral proteins undergoes specific environment- and redox-dependent phosphorylation at the thylakoid surface. This review focuses on the environmentally modulated reversible phosphorylation of thylakoid proteins related to their membrane dynamics and affinity towards particular photosynthetic protein complexes.
Keywords: Light harvesting; Photosynthesis; Photosystem II; Protein phosphorylation; Stress responses; Thylakoid membrane;

This mini review is an attempt to briefly summarize our current knowledge on light driven oxidative water splitting in photosynthesis. The reaction leading to molecular oxygen and four protons via photosynthesis comprises thermodynamic and kinetic constraints that require a balanced fine tuning of the reaction coordinates. The mode of coupling between electron (ET) and proton transfer (PT) reactions is shown to be of key mechanistic relevance for the redox turnover of YZ and the reactions within the WOC. The WOC is characterized by peculiar energetics of its oxidation steps in the WOC. In all oxygen evolving photosynthetic organisms the redox state S1 is thermodynamically most stable and therefore this general feature is assumed to be of physiological relevance. Available information on the Gibbs energy differences between the individual redox states S i+1 and S i and on the activation energies of their oxidative transitions are used to construct a general reaction coordinate of oxidative water splitting in photosystem II (PS II). Finally, an attempt is presented to cast our current state of knowledge into a mechanism of oxidative water splitting with special emphasis on the formation of the essential O–O bond and the active role of the protein environment in tuning the local proton activity that depends on time and redox state S i . The O–O linkage is assumed to take place within a multistate equilibrium at the redox level of S3, comprising both redox isomerism and proton tautomerism. It is proposed that one state, S3(P), attains an electronic configuration and nuclear geometry that corresponds with a hydrogen bonded peroxide which acts as the entatic state for the generation of complexed molecular oxygen through S3(P) oxidation by YZ ox.
Keywords: Photosystem II; P680; Redox active tyrosine; Proton coupled electron transfer; Water splitting;

In oxygenic photosynthesis, water is split at a Mn4Ca complex bound to the proteins of photosystem II (PSII). Powered by four quanta of visible light, four electrons and four protons are removed from two water molecules before dioxygen is released. By this process, water becomes an inexhaustible source of the protons and electrons needed for primary biomass formation. On the basis of structural and spectroscopic data, we recently have introduced a basic reaction cycle of water oxidation which extends the classical S-state cycle [B. Kok, B. Forbush, M. McGloin, Cooperation of charges in photosynthetic O2 evolution- I. A linear four-step mechanism, Photochem. Photobiol. 11 (1970) 457–475] by taking into account also the role and sequence of deprotonation events [H. Dau, M. Haumann, Reaction cycle of photosynthetic water oxidation in plants and cyanobacteria, Science 312 (2006) 1471–1472]. We propose that the outwardly convoluted and irregular events of the classical S-state cycle are governed by a simple underlying principle: protons and electrons are removed strictly alternately from the Mn complex. Starting in I 0, eight successive steps of alternate proton and electron removal lead to I 8 and only then the O–O bond is formed. Thus not only four oxidizing equivalents, but also four bases are accumulated prior to the onset of dioxygen formation. After reviewing the kinetic properties of the individual S-state transition, we show that the proposed basic model explains a large body of experimental results straightforwardly. Furthermore we discuss how the I-cycle model addresses the redox-potential problem of PSII water oxidation and we propose that the accumulated bases facilitate dioxygen formation by acting as proton acceptors.
Keywords: Manganese complex; Oxygen evolution; Photosynthesis; Proton release; Kok cycle; Water oxidation;

The molecular mechanism of the water oxidation reaction in photosystem II (PSII) of green plants remains a great mystery in life science. This reaction is known to take place in the oxygen evolving complex (OEC) incorporating four manganese, one calcium and one chloride cofactors, that is light-driven to cycle four intermediates, designated S0 through S4, to produce four protons, five electrons and lastly one molecular oxygen, for indispensable resources in biosphere. Recent advancements of X-ray crystallography models established the existence of a catalytic Mn4Ca cluster ligated by seven protein amino acids, but its functional structure is not yet resolved. The 18O exchange rates of two substrate water molecules were recently measured for four Si-state samples (i  = 0–3) leading to 34O2 and 36O2 formations, revealing asymmetric substrate binding sites significantly depending on the Si-state. In this paper, we present a chemically complete model for the Mn4Ca cluster and its surrounding enzyme field, which we found out from some possible models by using the hybrid density functional theoretic geometry optimization method to confirm good agreements with the 3.0 Å resolution PSII model [B. Loll, J. Kern, W. Saenger, A. Zouni , J. Biesiadka, Nature 438 (2005) 1040–1044] and the S-state dependence of 18O exchange rates [W. Hillier and T. Wydrzynski, Phys. Chem. Chem. Phys. 6 (2004) 4882–4889]. Furthermore, we have verified that two substrate water molecules are bound to asymmetric cis-positions on the terminal Mn ion being triply bridged (μ-oxo, μ-carboxylato, and a hydrogen bond) to the Mn3CaO3(OH) core, by developing a generalized theory of 18O exchange kinetics in OEC to obtain an experimental evidence for the cross exchange pathway from the slow to the fast exchange process. Some important experimental data will be discussed in terms of this model and its possible tautomers, to suggest that a cofactor, Cl ion, may be bound to CP43-Arg357 nearby Ca2+ ion and that D1-His337 may be used to trap a released proton only in the S2-state.
Keywords: Photosynthesis; Photosystem II; Oxygen evolution; Water oxidation; Manganese cluster; DFT B3LYP method; Tautomerism; Manganese EPR; Water exchange kinetics;

The function and mechanism of TyrZ in active photosystem II (PSII) is one of the long-standing issues in the study of photosynthetic water oxidation. Based on recent investigations on active PSII and theoretical studies, a new model is proposed, in which D1-His190 acts as a bridge, to form a low-barrier hydrogen bond (LBHB) with TyrZ, and a coordination bond to Mn or Ca ion of the Mn-cluster. Accordingly, this new model differs from previous proposals concerning the mechanism of TyrZ function in two aspects. First, the LBHB plays a key role to decrease the activation energy for TyrZ oxidation and TyrZ · reduction during photosynthetic water oxidation. Upon the oxidation of TyrZ, the hydrogen bond between TyrZ and His190 changes from a LBHB to a weak hydrogen bond, and vice versa upon TyrZ · reduction. In both stages, the electron transfer and proton transfer are coupled. Second, the positive charge formed after TyrZ oxidation may play an important role for water oxidation. It can be delocalized on the Mn-cluster, thus helps to accelerate the proton release from substrate water on Mn-cluster. This model is well reconciled with observations of the S-state dependence of TyrZ oxidation and TyrZ · reduction, proton release, isotopic effect and recent EPR experiments. Moreover, the difference between TyrZ and TyrD in active PSII can also be readily rationalized. The His190 binding to the Mn-cluster predicted in this model is contradictious to the recent structure data, however, it has been aware that the crystal structure of the Mn-cluster and its environment are significantly modified by X-ray due to radiation damage and are different from that in active PSII. It is suggested that the His190 may be protonated during the radiation damage, which leads to the loss of its binding to Mn-cluster and the strong hydrogen bond with TyrZ. This type of change arising from radiation damage has been confirmed in other enzyme systems.
Keywords: Photosystem II; TyrosineZ; His190; Mn-cluster; Low-barrier hydrogen bond; Proton and electron transfer;

Subsequent events to GTP binding by the plant PsbO protein: Structural changes, GTP hydrolysis and dissociation from the photosystem II complex by Björn Lundin; Sophie Thuswaldner; Tatiana Shutova; Said Eshaghi; Göran Samuelsson; James Barber; Bertil Andersson; Cornelia Spetea (500-508).
Besides an essential role in optimizing water oxidation in photosystem II (PSII), it has been reported that the spinach PsbO protein binds GTP [C. Spetea, T. Hundal, B. Lundin, M. Heddad, I. Adamska, B. Andersson, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 1409–1414]. Here we predict four GTP-binding domains in the structure of spinach PsbO, all localized in the β-barrel domain of the protein, as judged from comparison with the 3D-structure of the cyanobacterial counterpart. These domains are not conserved in the sequences of the cyanobacterial or green algae PsbO proteins. MgGTP induces specific changes in the structure of the PsbO protein in solution, as detected by circular dichroism and intrinsic fluorescence spectroscopy. Spinach PsbO has a low intrinsic GTPase activity, which is enhanced fifteen-fold when the protein is associated with the PSII complex in its dimeric form. GTP stimulates the dissociation of PsbO from PSII under light conditions known to also release Mn2+ and Ca2+ ions from the oxygen-evolving complex and to induce degradation of the PSII reaction centre D1 protein. We propose the occurrence in higher plants of a PsbO-mediated GTPase activity associated with PSII, which has consequences for the function of the oxygen-evolving complex and D1 protein turnover.
Keywords: Photosystem II; PsbO protein; GTPase; Oxygen-evolving complex; D1 protein;

Lipids in photosystem II: Interactions with protein and cofactors by Bernhard Loll; Jan Kern; Wolfram Saenger; Athina Zouni; Jacek Biesiadka (509-519).
Photosystem II (PSII) is a homodimeric protein–cofactor complex embedded in the thylakoid membrane that catalyses light-driven charge separation accompanied by the oxidation of water during oxygenic photosynthesis. Biochemical analysis of the lipid content of PSII indicates a number of integral lipids, their composition being similar to the average lipid composition of the thylakoid membrane. The crystal structure of PSII at 3.0 Å resolution allowed for the first time the assignment of 14 integral lipids within the protein scaffold, all of them being located at the interface of different protein subunits. The reaction centre subunits D1 and D2 are encircled by a belt of 11 lipids providing a flexible environment for the exchange of D1. Three lipids are located in the dimerization interface and mediate interactions between the PSII monomers. Several lipids are located close to the binding pocket of the mobile plastoquinone QB, forming part of a postulated diffusion pathway for plastoquinone. Furthermore two lipids were found, each ligating one antenna chlorophyll a. A detailed analysis of lipid–protein and lipid–cofactor interactions allows to derive some general principles of lipid binding pockets in PSII and to suggest possible functional properties of the various identified lipid molecules.
Keywords: Photosystem II; Structure; Lipids; Dimerization; Thylakoid membrane; Plastoquinone diffusion;

Spare quinones in the QB cavity of crystallized photosystem II from Thermosynechococcus elongatus by Roland Krivanek; Jan Kern; Athina Zouni; Holger Dau; Michael Haumann (520-527).
The recent crystallographic structure at 3.0 Å resolution of PSII from Thermosynechococcus elongatus has revealed a cavity in the protein which connects the membrane phase to the binding pocket of the secondary plastoquinone QB. The cavity may serve as a quinone diffusion pathway. By fluorescence methods, electron transfer at the donor and acceptor sides was investigated in the same membrane-free PSII core particle preparation from T. elongatus prior to and after crystallization; PSII membrane fragments from spinach were studied as a reference. The data suggest selective enrichment of those PSII centers in the crystal that are intact with respect to O2 evolution at the manganese–calcium complex of water oxidation and with respect to the integrity of the quinone binding site. One and more functional quinone molecules (per PSII monomer) besides of QA and QB were found in the crystallized PSII. We propose that the extra quinones are located in the QB cavity and serve as a PSII intrinsic pool of electron acceptors.
Keywords: Chlorophyll fluorescence; Crystallization; Photosystem II; Quinone; Water oxidation;

Structural response of Photosystem 2 to iron deficiency: Characterization of a new Photosystem 2–IdiA complex from the cyanobacterium Thermosynechococcus elongatus BP-1 by Julia E.-M. Lax; Ana A. Arteni; Egbert J. Boekema; Elfriede K. Pistorius; Klaus-Peter Michel; Matthias Rögner (528-534).
Iron deficiency triggers various processes in cyanobacterial cells of which the synthesis of an additional antenna system (IsiA) around photosystem (PS) 1 is well documented [T.S. Bibby, J. Nield, J. Barber, Iron deficiency induces the formation of an antenna ring around trimeric photosystem I in cyanobacteria, Nature 412 (2001) 743–745, E.J. Boekema, A. Hifney, A.E. Yakushevska, M. Piotrowski, W. Keegstra, S. Berry, K.P. Michel, E.K. Pistorius, J. Kruip, A giant chlorophyll–protein complex induced by iron deficiency in cyanobacteria, Nature 412 (2001) 745–748]. Here we show that PS2 also undergoes prominent structural changes upon iron deficiency: Prerequisite is the isolation and purification of a PS2–IdiA complex which is exclusively synthesized under these conditions. Immunoblotting in combination with size exclusion chromatography shows that IdiA is only bound to dimeric PS2. Using single particle analysis of negatively stained specimens, IdiA can be localized in averaged electron micrographs on top of the CP43 subunit facing the cytoplasmic side in a model derived from the known 3D structure of PS2 [B. Loll, J. Kern, W. Saenger, A. Zouni, J. Biesiadka, Towards complete cofactor arrangement in the 3.0 Å resolution structure of photosystem II, Nature 438 (2005) 1040–4]. The presence of IdiA as integral part of PS2 is the first example of a new PS2 protein being expressed under stress conditions, which is missing in highly purified PS2 complexes isolated from iron-sufficient cells.
Keywords: Photosystem 2; Iron starvation; IdiA; Photosystem 2 structure; Thermosynechococcus elongatus BP-1;

A Fourier transform infrared (FTIR) difference spectrum of the oxygen-evolving Mn cluster upon the S1-to-S2 transition was obtained with Ca2+-depleted photosystem II (PSII) membranes to investigate the structural relevance of Ca2+ to the Mn cluster. Previously, Noguchi et al. [Biochim. Biophys. Acta 1228 (1995) 189] observed drastic changes in the carboxylate stretching region of the S2/S1 FTIR spectrum upon Ca2+ depletion, whereas Kimura and co-workers [Biochemistry 40 (2001) 14061; ibid. 41 (2002) 5844] later claimed that these changes were not ascribed to Ca2+ depletion itself but caused by the interaction of EDTA to the Mn cluster and/or binding of K+ at the Ca2+ site. In the present study, the preparation of the Ca2+-depleted PSII sample and its FTIR measurement were performed in the absence of EDTA and K+. The obtained S2/S1 spectrum exhibited the loss of carboxylate bands at 1587/1562 and 1364/1403 cm− 1 and diminished amide I intensities, which were identical to the previous observations in the presence of EDTA and K+. This result indicates that the drastic FTIR changes are a pure effect of Ca2+ depletion, and provides solid evidence for the general view that Ca2+ is strongly coupled with the Mn cluster.
Keywords: Ca2+; Carboxylate ligand; FTIR; Mn cluster; Oxygen evolution; Photosystem II;

The influence of hydrogen bonds on electron transfer rate in photosynthetic RCs by P.M. Krasilnikov; P.A. Mamonov; P.P. Knox; V.Z. Paschenko; A.B. Rubin (541-549).
Hydrogen bonds formed between photosynthetic reaction centers (RCs) and their cofactors were shown to affect the efficacy of electron transfer. The mechanism of such influence is determined by sensitivity of hydrogen bonds to electron density rearrangements, which alter hydrogen bonds potential energy surface. Quantum chemistry calculations were carried out on a system consisting of a primary quinone QA, non-heme Fe2+ ion and neighboring residues. The primary quinone forms two hydrogen bonds with its environment, one of which was shown to be highly sensitive to the QA state. In the case of the reduced primary quinone two stable hydrogen bond proton positions were shown to exist on [QA-HisM219] hydrogen bond line, while there is only one stable proton position in the case of the oxidized primary quinone. Taking into account this fact and also the ability of proton to transfer between potential energy wells along a hydrogen bond, theoretical study of temperature dependence of hydrogen bond polarization was carried out. Current theory was successfully applied to interpret dark P+/QA recombination rate temperature dependence.
Keywords: Photosynthetic reaction center; Electron transfer; Hydrogen bond; Proton tunneling; Dipole relaxation;

This study describes an analysis of different treatments that influence the relative content and the midpoint potential of HP Cyt b559 in PS II membrane fragments from higher plants. Two basically different types of irreversible modification effects are distinguished: the HP form of Cyt b559 is either predominantly affected when the heme group is oxidized (“O-type” effects) or when it is reduced (“R-type” effects). Transformation of HP Cyt b559 to lower potential redox forms (IP and LP forms) by the “O-type” mechanism is induced by high pH and detergent treatments. In this case the effects consist of a gradual decrease in the relative content of HP Cyt b559 while its midpoint potential remains unaffected. Transformation of HP Cyt b559 via an “R-type” mechanism is caused by a number of exogenous compounds denoted L: herbicides, ADRY reagents and tetraphenylboron. These compounds are postulated to bind to the PS II complex at a quinone binding site designated as QC which interacts with Cyt b559 and is clearly not the QB site. Binding of compounds L to the QC site when HP Cyt b559 is oxidized gives rise to a gradual decrease in the E m of HP Cyt b559 with increasing concentration of L (up to 10 K ox(L) values) while the relative content of HP Cyt b559 is unaffected. Higher concentrations of compounds L required for their binding to QC site when HP Cyt b559 is reduced (described by K red(L)) induce a conversion of HP Cyt b559 to lower potential redox forms (“R-type” transformation). Two reaction pathways for transitions of Cyt b559 between the different protein conformations that are responsible for the HP and IP/LP redox forms are proposed and new insights into the functional regulation of Cyt b559 via the QC site are discussed.
Keywords: Photosystem II; Cyt b559; Redox potential; Herbicide;

Examination of chlorophyll fluorescence decay kinetics in sulfur deprived algae Chlamydomonas reinhardtii by A.A. Volgusheva; V.E. Zagidullin; T.K. Antal; B.N. Korvatovsky; T.E. Krendeleva; V.Z. Paschenko; A.B. Rubin (559-564).
Chlorophyll fluorescence decay kinetics was measured in sulfur deprived cells of green alga Chlamydomonas reinhardtii with a home made picosecond fluorescence laser spectrometer. The measurements were carried out on samples either shortly adapted to the dark (‘Fo conditions’) or treated to reduce Qa (‘Fm conditions’). Bi-exponential fitting of decay kinetics was applied to distinguish two components one of them related to energy trapping (fast component) and the other to charge stabilization and recombination in PS 2 reaction centers (slow component). It was found that the slow component yield increased by 2.0 and 1.2 times when measured under ‘Fo’ and ‘Fm conditions’, respectively, in sulfur deprived cells as compared to control ones. An additional rapid rise of the slow component yield was observed when incubation was carried out in a sealed bioreactor and cell culture turned to anaerobic conditions. The obtained results strongly indicate the existence of the redox control of PS 2 activity during multiphase adaptation of C. reinhardtii to sulfur deficiency stress. Probable mechanisms responsible for the observed increased recombinant fluorescence yield in starved cells are discussed.
Keywords: Chlorophyll fluorescence; Laser spectrometry; Photosystem 2; Sulfur deprivation; Chlamydomonas reinhardtii;

The analysis of the time-resolved delayed fluorescence (DF) measurements represents an important tool to study quantitatively light-induced electron transfer as well as associated processes, e.g. proton movements, at the donor side of photosystem II (PSII). This method can provide, inter alia, insights in the functionally important inner-protein proton movements, which are hardly detectable by conventional spectroscopic approaches. The underlying rationale and experimental details of the method are described. The delayed emission of chlorophyll fluorescence of highly active PSII membrane particles was measured in the time domain from 10 μs to 60 ms after each flash of a train of nanosecond laser pulses. Focusing on the oxygen-formation step induced by the third flash, we find that the recently reported formation of an S4-intermediate prior to the onset of O–O bond formation [M. Haumann, P. Liebisch, C. Müller, M. Barra, M. Grabolle, H. Dau, Science 310, 1019–1021, 2006] is a multiphasic process, as anticipated for proton movements from the manganese complex of PSII to the aqueous bulk phase. The S4-formation involves three or more likely sequential steps; a tri-exponential fit yields time constants of 14, 65, and 200 μs (at 20 °C, pH 6.4). We determine that S4-formation is characterized by a sizable difference in Gibbs free energy of more than 90 meV (20 °C, pH 6.4). In the second part of the study, the temperature dependence (− 2.7 to 27.5 °C) of the rate constant of dioxygen formation (600/s at 20 °C) was investigated by analysis of DF transients. If the activation energy is assumed to be temperature-independent, a value of 230 meV is determined. There are weak indications for a biphasicity in the Arrhenius plot, but clear-cut evidence for a temperature-dependent switch between two activation energies, which would point to the existence of two distinct rate-limiting steps, is not obtained.
Keywords: Chlorophyll fluorescence; Delayed fluorescence; Oxygen evolution; Oxygenic photosynthesis; Photosystem II; Water oxidation;

Oxygen-evolving extrinsic proteins (PsbO,P,Q,R): Bioinformatic and functional analysis by Javier De Las Rivas; Pedro Heredia; Angel Roman (575-582).
The water-splitting and oxygen-evolving (OE) reaction is carried out by a large multisubunit protein complex, Photosystem II (PSII), that has two distinct regions: a membrane intrinsic-region that includes most of the PSII subunits and a lumenal extrinsic-region that is in close association to the manganese catalytic center. The recently determined PSII 3D structures from cyanobacteria provide a considerable amount of new knowledge about the OE architecture (K.N. Ferreira, T.M. Iverson, K. Maghlaoui, J. Barber, S. Iwata, Architecture of the photosynthetic oxygen-evolving center, Science 303 (2004) 1831–1838; B. Loll, J. Kern, W. Saenger, A. Zouni, J. Biesiadka, Towards complete cofactor arrangement in the 3.0 A resolution structure of photosystem II, Nature 438 (2005) 1040–1044). Most of the intrinsic core PSII polypeptides have been well conserved through evolution from ancient cyanobacteria to modern plants, keeping the essence of PSII light driven reactions from prokaryotes to eukaryotes; but what is striking is the large number of changes that have occurred in the oxygen-evolving extrinsic proteins (OEEp) associated to PSII lumenal side. For unknown reasons plant PSII has required the “invention” of three OEEps: PsbP (23 kDa), PsbQ (16 kDa) and PsbR (10 kDa); associated to the ubiquitous OEEp PsbO (33 kDa). This set of proteins seems to be required in plants for the full activity and stability of the OE center in vivo, but their specific function is not clear. In this paper, bioinformatics and functional data show that the OEEps present in plants and green algae are very distinct from their prokaryotic counterparts. Moreover, clear differences are found for PsbQ from higher plants and green algae; and a relationship has been found between PsbR and the Mn cluster.
Keywords: Oxygen-evolving complex; Photosystem II complex; PsbO; PsbP; PsbQ; PsbR; Protein structure; Protein evolution; Bioinformatics;

Manganese binding to the 23 kDa extrinsic protein of Photosystem II by Natallia Bondarava; Sun Un; Anja Krieger-Liszkay (583-588).
The recombinant form of the extrinsic 23 kDa protein (psbP) of Photosystem II (PSII) was studied with respect to its capability to bind Mn. The stoichiometry was determined to be one manganese bound per protein. A very high binding constant, K A  = 10− 17 M− 1, was determined by dialysis of the Mn containing protein against increasing EDTA concentration. High Field EPR spectroscopy was used to distinguish between specific symmetrically ligated Mn(II) from those non-specifically Mn(II) attached to the protein surface. Upon Mn binding PsbP exhibited fluorescence emission with maxima at 415 and 435 nm when tryptophan residues were excited. The yield of this blue fluorescence was variable from sample to sample. It was likely that different conformational states of the protein were responsible for this variability. The importance of Mn binding to PsbP in the context of photoactivation of PSII is discussed.
Keywords: Photosynthesis; Photosystem II; 23 kDa protein; Manganese;

Both chlorophylls a and d are essential for the photochemistry in photosystem II of the cyanobacteria, Acaryochloris marina by Eberhard Schlodder; Marianne Çetin; Hann-Jörg Eckert; Franz-Josef Schmitt; James Barber; Alison Telfer (589-595).
We have measured the flash-induced absorbance difference spectrum attributed to the formation of the secondary radical pair, P+Q, between 270 nm and 1000 nm at 77 K in photosystem II of the chlorophyll d containing cyanobacterium, Acaryochloris marina. Despite the high level of chlorophyll d present, the flash-induced absorption difference spectrum of an approximately 2 ms decay component shows a number of features which are typical of the difference spectrum seen in oxygenic photosynthetic organisms containing no chlorophyll d. The spectral shape in the near-UV indicates that a plastoquinone is the secondary acceptor molecule (QA). The strong C-550 change at 543 nm confirms previous reports that pheophytin a is the primary electron acceptor. The bleach at 435 nm and increase in absorption at 820 nm indicates that the positive charge is stabilized on a chlorophyll a molecule. In addition a strong electrochromic band shift, centred at 723 nm, has been observed. It is assigned to a shift of the Qy band of the neighbouring accessory chlorophyll d, ChlD1. It seems highly likely that it accepts excitation energy from the chlorophyll d containing antenna. We therefore propose that primary charge separation is initiated from this chlorophyll d molecule and functions as the primary electron donor. Despite its lower excited state energy (0.1 V less), as compared to chlorophyll a, this chlorophyll d molecule is capable of driving the plastoquinone oxidoreductase activity of photosystem II. However, chlorophyll a is used to stabilize the positive charge and ultimately to drive water oxidation.
Keywords: Photosystem II; Chlorophyll d; Water oxidation; Charge separation; Acaryochloris marina;

Redox potential of chlorophyll d in vitro by Masami Kobayashi; Shunsuke Ohashi; Koji Iwamoto; Yoshihiro Shiraiwa; Yuki Kato; Tadashi Watanabe (596-602).
Chlorophyll (Chl) d is a major chlorophyll in a novel oxygenic prokaryote Acaryochloris marina. Here we first report the redox potential of Chl d in vitro. The oxidation potential of Chl d was + 0.88 V vs. SHE in acetonitrile; the value was higher than that of Chl a (+ 0.81 V) and lower than that of Chl b (+ 0.94 V). The oxidation potential order, Chl b  > Chl d  > Chl a, can be explained by inductive effect of substituent groups on the conjugated π-electron system on the macrocycle. Corresponding pheophytins showed the same order; Phe b (+ 1.25 V) > Phe d (+ 1.21 V) > Phe a (+ 1.14 V), but the values were significantly higher than those of Chls, which are rationalized in terms of an electron density decrease in the π-system by the replacement of magnesium with more electronegative hydrogen. Consequently, oxidation potential of Chl a was found to be the lowest among Chls and Phes. The results will help us to broaden our views on photosystems in A. marina.
Keywords: Acaryochloris marina; Chlorophyll a; Chlorophyll d; Photosynthesis; Redox potential;

The chlorophyll d containing cyanobacterium, Acaryochloris marina has provided a model system for the study of chlorophyll replacement in the function of oxygenic photosynthesis. Chlorophyll d replaces most functions of chlorophyll a in Acaryochloris marina. It not only functions as the major light-harvesting pigment, but also acts as an electron transfer cofactor in the primary charge separation reaction in the two photosystems. The Mg-chlorophyll d-peptide coordinating interaction between the amino acid residues and chlorophylls using the latest semi-empirical PM5 method were examined. It is suggested that chlorophyll d possesses similar coordination ligand properties to chlorophyll a, but chlorophyll b possesses different ligand properties. Compared with other studies involving theoretical correlation and our prior experiments, this study suggests that the chlorophyll a-bound proteins will bind chlorophyll d without difficulty when chlorophyll d is available.
Keywords: Chlorophyll d; Synthetic Peptide; Heats of Formation; Molecular modelling;

Photochemically induced dynamic nuclear polarization in the reaction center of the green sulphur bacterium Chlorobium tepidum observed by 13C MAS NMR by Esha Roy; Alia; Peter Gast; Hans van Gorkom; Huub J.M. de Groot; Gunnar Jeschke; Jörg Matysik (610-615).
Photochemically induced dynamic nuclear polarization has been observed in reaction centres of the green sulphur bacterium Chlorobium tepidum by 13C magic-angle spinning solid-state NMR under continuous illumination with white light. An almost complete set of chemical shifts of the aromatic ring carbons of a BChl a molecule has been obtained. All light-induced 13C NMR signals appear to be emissive, which is similar to the pattern observed in the reaction centers of plant photosystem I and purple bacterial reaction centres of Rhodobacter sphaeroides wild type. The donor in RCs of green sulfur bacteria clearly differs from the substantially asymmetric special pair of purple bacteria and appears to be similar to the more symmetric donor of photosystem I.
Keywords: Green sulphur bacteria; Photosystem I; Solid-state NMR; Photo-CIDNP; Reaction centres;

The thylakoid carbonic anhydrase associated with photosystem II is the component of inorganic carbon accumulating system in cells of halo- and alkaliphilic cyanobacterium Rhabdoderma lineare by Marina V. Dudoladova; Elena V. Kupriyanova; Alexandra G. Markelova; Maria P. Sinetova; Suleyman I. Allakhverdiev; Natalia A. Pronina (616-623).
The organization of carbonic anhydrase (CA) system in halo- and alkaliphilic cyanobacterium Rhabdoderma lineare was studied by Western blot analysis and immunocytochemical electron microscopy. The presence of putative extracellular α-CA of 60 kDa in the glycocalyx, forming a tight sheath around the cell, and of two intracellular β-CA is reported. We show for the first time that the β-CA of 60 kDa is expressed constitutively and associated with polypeptides of photosystem II (β-CA-PS II). Another soluble β-CA of 25 kDa was induced in low-bicarbonate medium. Induction of synthesis of the latter β-CA was accompanied by an increase in the intracellular pool of inorganic carbon, which suggests an important role of this enzyme in the functioning of a CO2-concentrating mechanism.
Keywords: Rhabdoderma lineare; Alkaliphilic cyanobacteria; Soda lakes; Carbonic anhydrase immunolocalization; Photosystem II; Intracellular pool of Ci;

A protective effect of bicarbonate (BC) against extraction of the extrinsic proteins, predominantly the Mn-stabilizing protein (PsbO protein), during treatment of Photosystem II (PS II) membrane fragment from pea with 2 M urea, and at low pH (using incubation in 0.2 M glycine–HCl buffer, pH 3.5 or 0.5 M citrate buffer, pH 4.0–4.5) was detected. It was shown that the extraction of the proteins with Mw 24 kDa (PsbP protein) and 18 kDa (PsbQ protein) by the use of highly concentrated solutions of NaCl does not depend on the presence of BC in the medium. An optimal concentration of BC at which it produces the maximum protecting effect was shown to be between 1 mM and 10 mM. The addition of formate did not influence the protein extraction but it reduced the stabilizing effect of BC. Independence of the stabilizing effect on the presence of the functionally active Mn within the water-oxidizing complex indicates that the protecting effect of BC is not related to its interaction with Mn ions. The fact that there is a preferable sensitivity of the PsbO protein to the absence of BC in the medium during all the treatments makes it possible to suggest that either BC interacts directly with the PsbO protein or it binds to some other sites within PS II and this binding facilitates the preservation of the native structure of this protein.
Keywords: Photosystem II; Extrinsic proteins; Manganese-stabilizing protein (PsbO protein); 24 kDa protein (PsbP protein); 18 kDa protein (PsbQ protein); Bicarbonate;

The effects of Cl, Mn2+, Ca2+, and pH on extrinsic and intrinsic photosystem II carbonic anhydrase activity were compared. Under the conditions of our in vitro experiments, extrinsic CA activity, located on the OEC33 protein, was optimum at about 30 mM Cl, and strongly inhibited above this concentration. This enzyme is activated by Mn2+ and stimulated somewhat by Ca2+. The OEC33 showed dehydration activity that is optimum at pH 6 or below. In contrast, intrinsic CA activity found in the PSII complex after removal of extrinsic proteins was stimulated by Cl up to 0.4 M. Ca2+ appears to be the required cofactor, which implies that the location of the intrinsic CA activity is in the immediate vicinity of the CaMn4 complex. Up to now, intrinsic CA has shown only hydration activity that is nearly pH independent.
Keywords: Bicarbonate; Carbonic anhydrase; Chloride; OEC33; Manganese Stabilizing protein; Photosystem II;

A quantitative assessment of the carbonic anhydrase activity in photosystem II by I.L. McConnell; M.R. Badger; T. Wydrzynski; W. Hillier (639-647).
Using a carbonic anhydrase assay based on membrane inlet mass spectrometry (MIMS), we have extended our earlier investigations of Photosystem II (PSII)-associated carbonic anhydrase activity in spinach PSII preparations (W. Hillier, I. McConnell, M. R. Badger, A. Boussac, V.V. Klimov G. C. Dismukes, T. Wydrzynski Biochemistry 2006, 45:2094). The relationship between the carbonic anhydrase activity and O2 evolution has been evaluated in terms of the effects of metal ion addition, preparation type, light, and response to specific inhibitors. The results indicate that the PSII-associated carbonic anhydrase activity is variable and appears not to be associated specifically with the oxygen evolving activity nor the 33 kDa extrinsic manganese stabilising protein.
Keywords: Bicarbonate; Carbonic anhydrase; Manganese stabilising protein; Oxygen evolving complex; Photosystem II;

Molecules with one porphyrin and multiple quinone groups are described. The molecules are based on dendritic frameworks, with the quinone groups attached at the “internal” positions and the porphyrin attached at the focal point, leading to a characteristic layer architecture. When irradiated with visible light in the presence of 4-tert-butylthiophenol, the quinones were converted to quinols. Such a behavior mimics the function of quinone pools in photosynthesis.
Keywords: Quinone pool; Photoinduced electron transfer; Dendrimer; Porphyrin;

Bio-photosensor: Cyanobacterial photosystem I coupled with transistor via molecular wire by Nao Terasaki; Noritaka Yamamoto; Kaoru Tamada; Mineyuki Hattori; Takashi Hiraga; Akihiko Tohri; Ikutaro Sato; Masako Iwai; Michinao Iwai; Shunpei Taguchi; Isao Enami; Yasunori Inoue; Yoshinori Yamanoi; Tetsu Yonezawa; Katsuya Mizuno; Masaki Murata; Hiroshi Nishihara; Satoshi Yoneyama; Makoto Minakata; Tsutomu Ohmori; Makoto Sakai; Masaaki Fujii (653-659).
We report on the first successful output of electrons directly from photosystem I (PSI) of thermophilic cyanobacteria to the gate of a field-effect transistor (FET) by bypassing electron flow via a newly designed molecular wire, i.e., artificial vitamin K1, and a gold nanoparticle; in short, this newly manufactured photosensor employs a bio-functional unit as the core of the device. Photo-electrons generated by the irradiation of molecular complexes composed of reconstituted PSI on the gate were found to control the FET. This PSI-bio-photosensor can be used to interpret gradation in images. This PSI-FET system is moreover sufficiently stable for use exceeding a period of 1 year.
Keywords: Cyanobacterial photosystem I; Gold nanoparticle; Molecular wire; Field-effect transistor; Bio-photosensor;

Artificial model of photosynthetic oxygen evolving complex: Catalytic O2 production from water by di-μ-oxo manganese dimers supported by clay compounds by Masayuki Yagi; Komei Narita; Syou Maruyama; Koji Sone; Takayuki Kuwabara; Ken-ichi Shimizu (660-665).
Adsorption of [(OH2)(terpy)Mn(μ-O)2Mn(terpy)(OH2)]3+ (terpy = 2,2′:6′,2ʺ-terpyridine) (1) onto montmorillonite K10 (MK10) yielded catalytic dioxygen (O2) evolution from water using a CeIV oxidant. The Mn K-edge X-ray absorption near edge structure (XANES) of the 1/MK10 hybrid suggested that the oxidation state of the di-μ-oxo Mn2 core could be MnIII–MnIV. However the pre-edge peak in the XANES spectrum of 1 adsorbed on MK10 is different from the neat 1 powder. The kinetic analysis of O2 evolution showed that the catalysis requires cooperation of two equivalents of 1 adsorbed on MK10. The reaction of the [(bpy)2Mn(μ-O)2Mn(bpy)2]3+ (bpy = 2,2′-bipyridine) (2)/MK10 hybrid with a CeIV oxidant evolved O2. However, the turnover number value was less than unity for 2/MK10, showing that 2 adsorbed on MK10 does not work as a catalyst. The terminal water ligands could be an important for the catalysis by adsorbed 1. The mechanism of O2 production by photosynthetic oxygen evolving complex is discussed based on catalytic O2 evolution by 1 adsorbed on MK10.
Keywords: Oxygen evolving complex; Photosystem II; Manganese complex; Oxygen evolution; Water oxidation; Photosynthesis;

The synthesis and electrochemistry of half-sandwich type of Co(III) complexes [(C5Me5)Co(bidentate)(CH3CN)](BF4)2 {bidentate = dppe (1,2-bis(diphenylphosphino)ethane), dppp (1,3-bis(diphenylphosphino)propane), bpy (2,2′-bipyridine), en (ethylenediamine)) are reported. Cyclic voltammograms of [(C5Me5)Co(bidentate)(CH3CN)](BF4)2 in CH3CN s showed two redox couples assignable to Co(II)/Co(III) and Co(I)/Co(II). The Co(I) complex having C5Me5 and dppe was also prepared. Two redox couples of this Co(I) complex, (C5Me5)Co(dppe), in CH3CN coincided with those of [(C5Me5)Co(dppe)(CH3CN)](BF4)2 in spite of the structural change around the metal center.
Keywords: Cobalt; Pentamethylcyclopentadienyl; Bidentate ligand; Cyclic voltammograms;

The role of Hox hydrogenase in the H2 metabolism of Thiocapsa roseopersicina by Gábor Rákhely; Tatyana V. Laurinavichene; Anatoly A. Tsygankov; Kornél L. Kovács (671-676).
The purple sulfur phototrophic bacterium Thiocapsa roseopersicina BBS synthesizes at least three NiFe hydrogenases (Hox, Hup, Hyn). We characterized the physiological H2 consumption/evolution reactions in mutants having deletions of the structural genes of two hydrogenases in various combinations. This made possible the separation of the functionally distinct roles of the three hydrogenases. Data showed that Hox hydrogenase (unlike the Hup and Hyn hydrogenases) catalyzed the dark fermentative H2 evolution and the light-dependent H2 production in the presence of thiosulfate. Both Hox+ and Hup+ mutants demonstrated light-dependent H2 uptake stimulated by CO2 but only the Hup+ mutant was able to mediate O2-dependent H2 consumption in the dark. The ability of the Hox+ mutant to evolve or consume hydrogen was found to depend on a number of interplaying factors including both growth and reaction conditions (availability of glucose, sulfur compounds, CO2, H2, light). The study of the redox properties of Hox hydrogenase supported the reversibility of its action. Based on the results a scheme is suggested to describe the role of Hox hydrogenase in light-dependent and dark hydrogen metabolism in T. roseopersicina BBS.
Keywords: H2 metabolism; Hydrogenase Hox; Hup; Hyn; Thiocapsa roseopersicina;

Insights into the function of PsbR protein in Arabidopsis thaliana by Yagut Allahverdiyeva; Fikret Mamedov; Marjaana Suorsa; Stenbjörn Styring; Imre Vass; Eva-Mari Aro (677-685).
The functional state of the Photosystem (PS) II complex in Arabidopsis psbR T-DNA insertion mutant was studied. The ΔPsbR thylakoids showed about 34% less oxygen evolution than WT, which correlates with the amounts of PSII estimated from YD ox radical EPR signal. The increased time constant of the slow phase of flash fluorescence (FF)-relaxation and upshift in the peak position of the main TL-bands, both in the presence and in the absence of DCMU, confirmed that the S2QA and S2QB charge recombinations were stabilized in ΔPsbR thylakoids. Furthermore, the higher amount of dark oxidized Cyt-b559 and the increased proportion of fluorescence, which did not decay during the 100s time span of the measurement thus indicating higher amount of YD +QA recombination, pointed to the donor side modifications in ΔPsbR. EPR measurements revealed that S1-to-S2-transition and S2-state multiline signal were not affected by mutation. The fast phase of the FF-relaxation in the absence of DCMU was significantly slowed down with concomitant decrease in the relative amplitude of this phase, indicating a modification in QA to QB electron transfer in ΔPsbR thylakoids. It is concluded that the lack of the PsbR protein modifies both the donor and the acceptor side of the PSII complex.
Keywords: Arabidopsis; Photosynthesis; Photosystem II; PsbR; Thylakoid membrane;

Cytochrome (cyt) b559 has been proposed to play an important role in the cyclic electron flow processes that protect photosystem II (PSII) from light-induced damage during photoinhibitory conditions. However, the exact role(s) of cyt b559 in the cyclic electron transfer pathway(s) in PSII remains unclear. To study the exact role(s) of cyt b559, we have constructed a series of site-directed mutants, each carrying a single amino acid substitution of one of the heme axial-ligands, in the cyanobacterium Synechocystis sp. PCC6803. In these mutants, His-22 of the α or the β subunit of cyt b559 was replaced with either Met, Glu, Tyr, Lys, Arg, Cys or Gln. On the basis of oxygen-evolution and chlorophyll a fluorescence measurements, we found that, among all mutants that were constructed, only the H22Kα mutant grew photoautotrophically, and accumulated stable PSII reaction centers (∼ 81% compared to wild-type cells). In addition, we isolated one pseudorevertant of the H22Yβ mutant that regained the ability to grow photoautotrophically and to assemble stable PSII reaction centers (∼ 79% compared to wild-type cells). On the basis of 77 K fluorescence emission measurements, we found that energy transfer from the phycobilisomes to PSII reaction centers was uncoupled in those cyt b559 mutants that assembled little or no stable PSII. Furthermore, on the basis of immunoblot analyses, we found that in thylakoid membranes of cyt b559 mutants that assembled little or no PSII, the amounts of the D1, D2, cyt b559α and β polypeptides were very low or undetectable but their CP47 and PsaC polypeptides were accumulated to the wild-type level. We also found that the amounts of cyt b559β polypeptide were significantly increased (larger than two folds) in thylakoid membranes of cyt b559 H22YβPS+ mutant cells. We suspected that the increase in the amounts of cyt b559 H22YβPS+ mutant polypeptides in thylakoid membranes might facilitate the assembly of functional PSII in cyt b559 H22YβPS+ mutant cells. Moreover, we found that isolated His-tagged PSII particles from H22Kα mutant cells gave rise to redox-induced optical absorption difference spectra of cyt b559. Therefore, our results concluded that significant fractions of H22Kα mutant PSII particles retained the heme of cyt b559. Finally, this work is the first report of cyt b559 mutants having substitutions of an axial heme-ligands that retain the ability to grow photoautotrophically and to assemble stable PSII reaction centers. These two cyt b559 mutants (H22Kα and H22YβPS+) and their PSII reaction centers will be very suitable for further biophysical and biochemical studies of the functional role(s) of cyt b559 in PSII.
Keywords: Photosystem II; Cytochrome b559; Photoinhibition; Chlorophyll a fluorescence; Site-directed mutagenesis; Synechocystis;

Changes in photosynthetic electron transfer and state transitions in an herbicide-resistant D1 mutant from soybean cell cultures by Mercedes Roncel; Inmaculada Yruela; Diana Kirilovsky; Fernando Guerrero; Miguel Alfonso; Rafael Picorel; José M. Ortega (694-702).
Anomalies in photosynthetic activity of the soybean cell line STR7, carrying a single mutation (S268P) in the chloroplastic gene psbA that codes for the D1 protein of the photosystem II, have been examined using different spectroscopic techniques. Thermoluminescence emission experiments have shown important differences between STR7 mutant and wild type cells. The afterglow band induced by both white light flashes and far-red continuous illumination was downshifted by about 4 °C and the Q band was upshifted by 5 °C. High temperature thermoluminescence measurements suggested a higher level of lipid peroxidation in mutant thylakoid membranes. In addition, the reduction rate of P700+ was significantly accelerated in STR7 suggesting that the mutation led to an activation of the photosystem I cyclic electron flow. Modulated fluorescence measurements performed at room temperature as well as fluorescence emission spectra at 77 K revealed that the STR7 mutant is defective in state transitions. Here, we discuss the hypothesis that activation of the cyclic electron flow in STR7 cells may be a mechanism to compensate the reduced activity of photosystem II caused by the mutation. We also propose that the impaired state transitions in the STR7 cells may be due to alterations in thylakoid membrane properties induced by a low content of unsaturated lipids.
Keywords: Cyclic and linear electron flow; Photosystem I; Photosystem II; Thermoluminescence; State transitions; D1 mutant;

Evidences for interaction of PsbS with photosynthetic complexes in maize thylakoids by Enrico Teardo; Patrizia Polverino de Laureto; Elisabetta Bergantino; Francesca Dalla Vecchia; Fernanda Rigoni; Ildikò Szabò; Giorgio Mario Giacometti (703-711).
The PsbS subunit of Photosystem II (PSII) has received much attention in the past few years, given its crucial role in photoprotection of higher plants. The exact location of this small subunit in thylakoids is also debated. In this work possible interaction partners of PsbS have been identified by immunoaffinity and immunoprecipitation, performed with mildly solubilized whole thylakoid membrane. The interacting proteins, as identified by mass spectrometry analysis of the immunoaffinity eluate, include CP29, some LHCII components, but also components of Photosystem I, of the cytochrome b6f complex as well as of ATP synthase. These proteins can be co-immunoprecipitated by using highly specific anti-PsbS antibodies and, vice-versa, PsbS is co-immunoprecipitated by antisera against components of the interacting complexes. We also find that PsbS co-migrates with bands containing PSII, ATP synthase and cytochrome b6f as well as with LHCII-containing bands on non-denaturing Deriphat PAGE. These results suggest multiple location of PsbS in the thylakoid membrane and point to an unexpected lateral mobility of this PSII subunit. As revealed by immunogold labelling with antibody against PsbS, the protein is associated either with granal membranes or prevalently with stroma lamellae in low or high-intensity light-treated intact leaves, respectively. This finding is consistent with the capability of PsbS to interact with complexes located in stroma lamellae, even though the exact physiological condition(s) under which these interactions may take place remain to be clarified.
Keywords: PsbS; Photosynthetic complex; Maize thylakoid;

Chemical rescue of a site-modified ligand to a [4Fe–4S] cluster in PsaC, a bacterial-like dicluster ferredoxin bound to Photosystem I by Mikhail L. Antonkine; Estelle M. Maes; Roman S. Czernuszewicz; Christoph Breitenstein; Eckhard Bill; Christopher J. Falzone; Ramakrishnan Balasubramanian; Carolyn Lubner; Donald A. Bryant; John H. Golbeck (712-724).
Chemical rescue of site-modified amino acids using externally supplied organic molecules represents a powerful method to investigate structure–function relationships in proteins. Here we provide definitive evidence that aryl and alkyl thiolates, reagents typically used for in vitro iron–sulfur cluster reconstitutions, serve as rescue ligands to a site-specifically modified [4Fe–4S]1+,2+ cluster in PsaC, a bacterial dicluster ferredoxin-like subunit of Photosystem I. PsaC binds two low-potential [4Fe–4S]1+,2+ clusters termed FA and FB. In the C13G/C33S variant of PsaC, glycine has replaced cysteine at position 13 creating a protein that is missing one of the ligating amino acids to iron–sulfur cluster FB. Using a variety of analytical techniques, including non-heme iron and acid-labile sulfur assays, and EPR, resonance Raman, and Mössbauer spectroscopies, we showed that the C13G/C33S variant of PsaC binds two [4Fe–4S]1+,2+ clusters, despite the absence of one of the biological ligands. 19F NMR spectroscopy indicated that the external thiolate replaces cysteine 13 as a substitute ligand to the FB cluster. The finding that site-modified [4Fe–4S]1+,2+ clusters can be chemically rescued with external thiolates opens new opportunities for modulating their properties in proteins. In particular, it provides a mechanism to attach an additional electron transfer cofactor to the protein via a bound, external ligand.
Keywords: Photosystem I; PsaC; S  ≥ 3/2; [4Fe–4S] cluster; Iron–sulfur protein; Chemical rescue;

Organisation of Photosystem I and Photosystem II in red alga Cyanidium caldarium: Encounter of cyanobacterial and higher plant concepts by Zdenko Gardian; Ladislav Bumba; Adam Schrofel; Miroslava Herbstova; Jana Nebesarova; Frantisek Vacha (725-731).
Structure and organisation of Photosystem I and Photosystem II isolated from red alga Cyanidium caldarium was determined by electron microscopy and single particle image analysis. The overall structure of Photosystem II was found to be similar to that known from cyanobacteria. The location of additional 20 kDa (PsbQ′) extrinsic protein that forms part of the oxygen evolving complex was suggested to be in the vicinity of cytochrome c-550 (PsbV) and the 12 kDa (PsbU) protein. Photosystem I was determined as a monomeric unit consisting of PsaA/B core complex with varying amounts of antenna subunits attached. The number of these subunits was seen to be dependent on the light conditions used during cell cultivation. The role of PsaH and PsaG proteins of Photosystem I in trimerisation and antennae complexes binding is discussed.
Keywords: Photosynthesis; Red algae; Cyanidium caldarium; Photosystem I; Photosystem II; Electron microscopy;

Steady-state and transient polarized absorption spectroscopy of photosytem I complexes from the cyanobacteria Arthrospira platensis and Thermosynechococcus elongatus by Eberhard Schlodder; Vladimir V. Shubin; Eithar El-Mohsnawy; Matthias Roegner; Navassard V. Karapetyan (732-741).
Core antenna and reaction centre of photosytem I (PS I) complexes from the cyanobacteria Arthrospira platensis and Thermosynechococcus elongatus have been characterized by steady-state polarized absorption spectroscopy, including linear dichroism (LD) and circular dichroism (CD). CD spectra and the second derivatives of measured 77 K CD spectra reveal the spectral components found in the polarized absorption spectra indicating the excitonic origin of the spectral forms of chlorophyll in the PS I complexes. The CD bands at 669–670(+), 673(+), 680(−), 683–685(−), 696–697(−), and 711(−) nm are a common feature of used PSI complexes. The 77 K CD spectra of the trimeric PS I complexes exhibit also low amplitude components around 736 nm for A. platensis and 720 nm for T. elongatus attributed to red-most chlorophylls. The LD measurements indicate that the transition dipole moments of the red-most states are oriented parallel to the membrane plane. The formation of P700+A1 or 3P700 was monitored by time-resolved difference absorbance and LD spectroscopy to elucidate the spectral properties of the PS I reaction centre. The difference spectra give strong evidence for the delocalization of the excited singlet states in the reaction centre. Therefore, P700 cannot be considered as a dimer but should be regarded as a multimer of the six nearly equally coupled reaction centre chlorophylls in accordance with structure-based calculations. On the basis of the results presented in this work and earlier work in the literature it is concluded that the triplet state is localized most likely on PA, whereas the cation is localized most likely on PB.
Keywords: Photosystem I; P700; Linear and circular dichroism; Excitonic coupling; Long-wavelength antenna chlorophyll; Arthrospira platensis; Thermosynechococcus elongatus;

Phycobilisomes (PBS) are the major accessory light-harvesting complexes in cyanobacteria and their mobility affects the light energy distribution between the two photosystems. We investigated the effect of PBS mobility on state transitions, photosynthetic and respiratory electron transport, and various fluorescence parameters in Synechocystis sp. strain PCC 6803, using glycinebetaine to immobilize and couple PBS to photosystem II (PSII) or photosystem I (PSI) by applying under far-red or green light, respectively. The immobilization of PBS at PSII inhibited the increase in cyclic electron flow, photochemical and non-photochemical quenching, and decrease in respiration that occurred during the movement of PBS from PSII to PSI. In contrast, the immobilization of PBS at PSI inhibited the increase in respiration and photochemical quenching and decrease in cyclic electron flow and non-photochemical quenching that occurred when PBS moved from PSI to PSII. Linear electron transport did not change during PBS movement but increased or decreased significantly during longer illumination with far-red or green light, respectively. This implies that PBS movement is completed in a short time but it takes longer for the overall photosynthetic reactions to be tuned to a new state.
Keywords: Chlorophyll fluorescence parameters; Phycobilisome mobility; State transitions; Synechocystis sp. strain PCC 6803; The electron transport;

Heat- and light-induced reorganizations in the phycobilisome antenna of Synechocystis sp. PCC 6803. Thermo-optic effect by Katerina Stoitchkova; Ottó Zsiros; Tamás Jávorfi; Tibor Páli; Atanaska Andreeva; Zoltán Gombos; Győző Garab (750-756).
By using absorption and fluorescence spectroscopy, we compared the effects of heat and light treatments on the phycobilisome (PBS) antenna of Synechocystis sp. PCC 6803 cells. Fluorescence emission spectra obtained upon exciting predominantly PBS, recorded at 25 °C and 77 K, revealed characteristic changes upon heat treatment of the cells. A 5-min incubation at 50 °C, which completely inactivated the activity of photosystem II, led to a small but statistically significant decrease in the F 680/F 655 fluorescence intensity ratio. In contrast, heat treatment at 60 °C resulted in a much larger decrease in the same ratio and was accompanied by a blue-shift of the main PBS emission band at around 655 nm (F 655), indicating an energetic decoupling of PBS from chlorophylls and reorganizations in its internal structure. (Upon exciting PBS, F 680 originates from photosystem II and from the terminal emitter of PBS.). Very similar changes were obtained upon exposing the cells to high light (600–7500 μmol photons m−2 s−1) for different time periods (10 min to 3 h). In cells with heat-inactivated photosystem II, the variations caused by light treatment could clearly be assigned to a similar energetic decoupling of the PBS from the membrane and internal reorganizations as induced at around 60 °C. These data can be explained within the frameworks of thermo-optic mechanism [Cseh et al. 2000, Biochemistry 39, 15250]: in high light the heat packages originating from dissipation might lead to elementary structural changes in the close vicinity of dissipation in heat-sensitive structural elements, e.g. around the site where PBS is anchored to the membrane. This, in turn, brings about a diminishment in the energy supply from PBS to the photosystems and reorganization in the molecular architecture of PBS.
Keywords: Energetic decoupling of phycobilisome from photosystems; Fluorescence spectroscopy; Structural changes in phycobilisomes; Synechocystis sp. PCC 6803; Thermo-optic effect;

Phycobilin/chlorophyll excitation equilibration upon carotenoid-induced non-photochemical fluorescence quenching in phycobilisomes of the cyanobacterium Synechocystis sp. PCC 6803 by Marina G. Rakhimberdieva; Dmitrii V. Vavilin; Wim F.J. Vermaas; Irina V. Elanskaya; Navassard V. Karapetyan (757-765).
To determine the mechanism of carotenoid-sensitized non-photochemical quenching in cyanobacteria, the kinetics of blue-light-induced quenching and fluorescence spectra were studied in the wild type and mutants of Synechocystis sp. PCC 6803 grown with or without iron. The blue-light-induced quenching was observed in the wild type as well as in mutants lacking PS II or IsiA confirming that neither IsiA nor PS II is required for carotenoid-triggered fluorescence quenching. Both fluorescence at 660 nm (originating from phycobilisomes) and at 681 nm (which, upon 440 nm excitation originates mostly from chlorophyll) was quenched. However, no blue-light-induced changes in the fluorescence yield were observed in the apcE mutant that lacks phycobilisome attachment. The results are interpreted to indicate that interaction of the Slr1963-associated carotenoid with – presumably – allophycocyanin in the phycobilisome core is responsible for non-photochemical energy quenching, and that excitations on chlorophyll in the thylakoid equilibrate sufficiently with excitations on allophycocyanin in wild type to contribute to quenching of chlorophyll fluorescence.
Keywords: Allophycocyanin; Carotenoid; Fluorescence quenching; IsiA protein; Photosystem II; Phycobilisome;

At room temperature, the chlorophyll (Chl) a fluorescence induction (FI) kinetics of plants, algae and cyanobacteria go through two maxima, P at ∼ 0.2–1 and M at ∼ 100–500 s, with a minimum S at ∼ 2–10 s in between. Thus, the whole FI kinetic pattern comprises a fast OPS transient (with O denoting origin) and a slower SMT transient (with T denoting terminal state). Here, we examined the phenomenology and the etiology of the SMT transient of the phycobilisome (PBS)-containing cyanobacterium Synechococcus sp PCC 7942 by modifying PBS → Photosystem (PS) II excitation transfer indirectly, either by blocking or by maximizing the PBS → PS I excitation transfer. Blocking the PBS → PS I excitation transfer route with N-ethyl-maleimide [NEM; A. N. Glazer, Y. Gindt, C. F. Chan, and K.Sauer, Photosynth. Research 40 (1994) 167–173] increases both the PBS excitation share of PS II and Chl a fluorescence. Maximizing it, on the other hand, by suspending cyanobactrial cells in hyper-osmotic media [G. C. Papageorgiou, A. Alygizaki-Zorba, Biochim. Biophys. Acta 1335 (1997) 1–4] diminishes both the PBS excitation share of PS II and Chl a fluorescence. Here, we show for the first time that, in either case, the slow SMT transient of FI disappears and is replaced by continuous P → T fluorescence decay, reminiscent of the typical P → T fluorescence decay of higher plants and algae. A similar P → T decay was also displayed by DCMU-treated Synechococcus cells at 2 °C. To interpret this phenomenology, we assume that after dark adaptation cyanobacteria exist in a low fluorescence state (state 2) and transit to a high fluorescence state (state 1) when, upon light acclimation, PS I is forced to run faster than PS II. In these organisms, a state 2 → 1 fluorescence increase plus electron transport-dependent dequenching processes dominate the SM rise and maximal fluorescence output is at M which lies above the P maximum of the fast FI transient. In contrast, dark-adapted plants and algae exist in state 1 and upon illumination they display an extended P → T decay that sometimes is interrupted by a shallow SMT transient, with M below P. This decay is dominated by a state 1 → 2 fluorescence lowering, as well as by electron transport-dependent quenching processes. When the regulation of the PBS → PS I electronic excitation transfer is eliminated (as for example in hyper-osmotic suspensions, after NEM treatment and at low temperature), the FI pattern of Synechococcus becomes plant-like.
Keywords: Chlorophyll fluorescence; Cyanobacteria; Fast and slow fluorescence induction; State transitions;

Dependence of reaction center-type energy-dependent quenching on photosystem II antenna size by Ismayil S. Zulfugarov; Ok-Kyung Ham; Sujata R. Mishra; Ji-Young Kim; Krishna Nath; Hee-Young Koo; Ho-Seung Kim; Yong-Hwan Moon; Gynheung An; Choon-Hwan Lee (773-780).
The effects of photosystem II antenna size on reaction center-type energy-dependent quenching (qE) were examined in rice plants grown under two different light intensities using both wild type and qE-less (OsPsbS knockout) mutant plants. Reaction center-type qE was detected by measuring non-photochemical quenching at 50 μmol photons m− 2 s− 1 white light intensity. We observed that in low light-grown rice plants, reaction center-type qE was higher than in high light-grown plants, and the amount of reaction center-type qE did not depend on zeaxanthin accumulation. This was confirmed in Arabidopsis npq1–2 mutant plants that lack zeaxanthin due to a mutation in the violaxanthin de-epoxidase enzyme. Although the electron transport rate measured at a light intensity of 50 μmol photons m− 2 s− 1 was the same in high light- and low light-grown wild type and mutant plants lacking PsbS protein, the generation of energy-dependent quenching was completely impaired only in mutant plants. Analyses of the pigment content, Lhcb proteins and D1 protein of PSII showed that the antenna size was larger in low light-grown plants, and this correlated with the amount of reaction center-type qE. Our results mark the first time that the reaction center-type qE has been shown to depend on photosystem II antenna size and, although it depends on the existence of PsbS protein, the extent of reaction center-type qE does not correlate with the transcript levels of PsbS protein. The presence of reaction center-type energy-dependent quenching, in addition to antenna-type quenching, in higher plants for dissipation of excess light energy demonstrates the complexity and flexibility of the photosynthetic apparatus of higher plants to respond to different environmental conditions.
Keywords: NPQ; qE; Photosystem II; Antenna size; PsbS; Zeaxanthin;

This study deals with effects of membrane excitation on photosynthesis and cell protection against excessive light, manifested in non-photochemical quenching (NPQ). In Chara corallina cells, NPQ and pericellular pH displayed coordinated spatial patterns along the length of the cell. The NPQ values were lower in H+-extruding cell regions (external pH ∼ 6.5) than in high pH regions (pH ∼ 9.5). Generation of an action potential by applying a pulse of electric current caused NPQ to increase within 30–60 s. This effect, manifested as a long-lived drop of maximum chlorophyll fluorescence (F m′), occurred at lower photosynthetic flux densities (PFD) in the alkaline as compared to acidic cell regions. The light response curve of NPQ shifted, after generation of an action potential, towards lower PFD. The release of NPQ by nigericin and the rapid reversal of action potential-triggered NPQ in darkness indicate its relation to thylakoid ΔpH. Generation of an action potential shortly after darkening converted the chloroplasts into a latent state with the F m identical to that of unexcited cells. This state transformed to the quenched state after turning on weak light that was insufficient for NPQ prior to membrane excitation of the cells. The ionophore, A23187, shifted NPQ plots similarly to the action potential effect, consistent with a likely role of a rise in the cytosolic Ca2+ level in the action potential-induced quenching. The results suggest that a rapid electric signal, across the plasma membrane, might exert long-lived effects on photosynthesis and chlorophyll fluorescence through ion flux-mediated pathways.
Keywords: Chlorophyll fluorescence; Action potential; Energy-dependent quenching; Thylakoid membrane; Ionophores; Chara corallina;

Chlamydomonas raudensis UWO 241 and SAG 49.72 represent the psychrophilic and mesophilic strains of this green algal species. This novel discovery was exploited to assess the role of psychrophily in photoacclimation to growth temperature and growth irradiance. At their optimal growth temperatures of 8 °C and 28 °C respectively, UWO 241 and SAG 49.72 maintained comparable photostasis, that is energy balance, as measured by PSII excitation pressure. Although UWO 241 exhibited higher excitation pressure, measured as 1-qL, at all growth light intensities, the relative changes in 1-qL were similar to that of SAG 49.72 in response to growth light. In response to suboptimal temperatures and increased growth irradiance, SAG 49.72 favoured energy partitioning of excess excitation energy through inducible, down regulatory processes (Φ NPQ) associated with the xanthophyll cycle and antenna quenching, while UWO 241 favoured xanthophyll cycle-independent energy partitioning through constitutive processes involved in energy dissipation (Φ NO). In contrast to SAG 49.72, an elevation in growth temperature induced an increase in PSI/PSII stoichiometry in UWO 241. Furthermore, SAG 49.72 showed typical threonine-phosphorylation of LHCII, whereas UWO 241 exhibited phosphorylation of polypeptides of comparable molecular mass to PSI reaction centres but the absence of LHCII phosphorylation. Thus, although both strains maintain an energy balance irrespective of their differences in optimal growth temperatures, the mechanisms used to maintain photostasis were distinct. We conclude that psychrophily in C. raudensis is complex and appears to involve differential energy partitioning, photosystem stoichiometry and polypeptide phosphorylation.
Keywords: Chlamydomonas raudensis; Chlorophyll fluorescence; Cold acclimation; Energy partitioning; Photostasis; Psychrophily;

Association of chlorophyll a/b-binding Pcb proteins with photosystems I and II in Prochlorothrix hollandica by Vladimir A. Boichenko; Alexander V. Pinevich; Igor N. Stadnichuk (801-806).
Action spectra for photosystem II (PSII)-driven oxygen evolution and of photosystem I (PSI)-mediated H2 photoproduction and photoinhibition of respiration were used to determine the participation of chlorophyll (Chl) a/b-binding Pcb proteins in the functions of pigment apparatus of Prochlorothrix hollandica. Comparison of the in situ action spectra with absorption spectra of PSII and PSI complexes isolated from the cyanobacterium Synechocystis 6803 revealed a shoulder at 650 nm that indicated presence of Chl b in the both photosystems of P. hollandica. Fitting of two action spectra to absorption spectrum of the cells showed a chlorophyll ratio of 4:1 in favor of PSI. Effective antenna sizes estimated from photochemical cross-sections of the relevant photoreactions were found to be 192 ± 28 and 139 ± 15 chlorophyll molecules for the competent PSI and PSII reaction centers, respectively. The value for PSI is in a quite good agreement with previous electron microscopy data for isolated Pcb–PSI supercomplexes from P. hollandica that show a trimeric PSI core surrounded by a ring of 18 Pcb subunits. The antenna size of PSII implies that the PSII core dimers are associated with ∼ 14 Pcb light-harvesting proteins, and form the largest known Pcb–PSII supercomplexes.
Keywords: Photosystems I and II; Pcb protein; Action spectra; Antenna size;

The induction of CP43′ by iron-stress in Synechococcus sp. PCC 7942 is associated with carotenoid accumulation and enhanced fatty acid unsaturation by Alexander G. Ivanov; Marianna Krol; Eva Selstam; Prafullachandra Vishnu Sane; Dmitry Sveshnikov; Youn-Il Park; Gunnar Öquist; Norman P.A. Huner (807-813).
Comparative lipid analysis demonstrated reduced amount of PG (50%) and lower ratio of MGDG/DGDG in iron-stressed Synechococcus sp. PCC 7942 cells compared to cells grown under iron sufficient conditions. In parallel, the monoenoic (C:1) fatty acids in MGDG, DGDG and PG increased from 46.8%, 43.7% and 45.6%, respectively in control cells to 51.6%, 48.8% and 48.7%, respectively in iron-stressed cells. This suggests increased membrane dynamics, which may facilitate the diffusion of PQ and keep the PQ pool in relatively more oxidized state in iron-stressed compared to control cells. This was confirmed by chlorophyll fluorescence and thermoluminescence measurements. Analysis of carotenoid composition demonstrated that the induction of isiA (CP43′) protein in response to iron stress is accompanied by significant increase of the relative abundance of all carotenoids. The quantity of carotenoids calculated on a Chl basis increased differentially with nostoxanthin, cryptoxanthin, zeaxanthin and β-carotene showing 2.6-, 3.1-, 1.9- and 1.9-fold increases, respectively, while the relative amount of caloxanthin was increased only by 30%. HPLC analyses of the pigment composition of Chl–protein complexes separated by non-denaturating SDS-PAGE demonstrated even higher relative carotenoids content, especially of cryptoxanthin, in trimer and monomer PSI Chl–protein complexes co-migrating with CP43′ from iron-stressed cells than in PSI complexes from control cells where CP43′ is not present. This implies a carotenoid-binding role for the CP43′ protein which supports our previous suggestion for effective energy quenching and photoprotective role of CP43′ protein in cyanobacteria under iron stress.
Keywords: Cyanobacteria; Carotenoid; Chl–protein complex CP43′; Iron stress; Lipid; Thermoluminescence;

The mrgA protein of the cyanobacterium Synechocystis sp. PCC6803 is a member of the DPS Fe storage protein family. The physiological role of this protein was studied using a disruption mutant in the mrgA gene (slr1894) and by measuring intracellular Fe quotas, 77K chlorophyll fluorescence and growth rates. It was found that the deletion of the mrgA gene did not impair the Fe storage capacity, as the intracellular Fe quotas of the ΔmrgA cells were comparable to those of the wild type. Furthermore, the cellular response to decreasing external Fe concentrations, as detected by the emergence of the CP43′ 77K fluorescence band, was similar in wild type and mutant cultures. On the other hand, a considerable slow down in the growth rate of ΔmrgA cultures was observed upon transfer from Fe replete to Fe depleted medium, indicating impeded utilization of the plentiful intracellular Fe. Based on these results, we suggest that mrgA plays an important role in the transport of intracellular Fe from storage (within bacterioferritins) to biosynthesis of metal cofactors throughout the cell's growth.
Keywords: Cyanobacteria; DPS family protein; Fe; Synechocystis sp.;

The role of the FtsH and Deg proteases in the repair of UV-B radiation-damaged Photosystem II in the cyanobacterium Synechocystis PCC 6803 by Otilia Cheregi; Cosmin Sicora; Peter B. Kós; Myles Barker; Peter J. Nixon; Imre Vass (820-828).
The photosystem two (PSII) complex found in oxygenic photosynthetic organisms is susceptible to damage by UV-B irradiation and undergoes repair in vivo to maintain activity. Until now there has been little information on the identity of the enzymes involved in repair. In the present study we have investigated the involvement of the FtsH and Deg protease families in the degradation of UV-B-damaged PSII reaction center subunits, D1 and D2, in the cyanobacterium Synechocystis 6803. PSII activity in a ΔFtsH (slr0228) strain, with an inactivated slr0228 gene, showed increased sensitivity to UV-B radiation and impaired recovery of activity in visible light after UV-B exposure. In contrast, in ΔDeg-G cells, in which all the three deg genes were inactivated, the damage and recovery kinetics were the same as in the WT. Immunoblotting showed that the loss of both the D1 and D2 proteins was retarded in ΔFtsH (slr0228) during UV-B exposure, and the extent of their restoration during the recovery period was decreased relative to the WT. However, in the ΔDeg-G cells the damage and recovery kinetics of D1 and D2 were the same as in the WT. These data demonstrate a key role of FtsH (slr0228), but not the Deg proteases, for the repair of PS II during and following UV-B radiation at the step of degrading both of the UV-B damaged D1 and D2 reaction center subunits.
Keywords: Photosystem II; D1-protein; D2-protein; FtsH protease; UV-B damage;

Cleavage after residue Ala352 in the C-terminal extension is an early step in the maturation of the D1 subunit of Photosystem II in Synechocystis PCC 6803 by Josef Komenda; Stanislava Kuviková; Bernhard Granvogl; Lutz A. Eichacker; Bruce A. Diner; Peter J. Nixon (829-837).
We have investigated the pathway by which the 16 amino-acid C-terminal extension of the D1 subunit of photosystem two is removed in the cyanobacterium Synechocystis sp. PCC 6803 to leave Ala344 as the C-terminal residue. Previous work has suggested a two-step process involving formation of a processing intermediate of D1, termed iD1, of uncertain origin. Here we show by mass spectrometry that a synthetic peptide mimicking the C- terminus of the D1 precursor is cleaved by cellular extracts or purified CtpA processing protease after residue Ala352, making this a likely site for formation of iD1. Characteristics of D1 site-directed mutants with either the Leu353 residue replaced by Pro or with a truncation after Ala352 are in agreement with this assignment. Interestingly, analysis of various CtpA and CtpB null mutants further indicate that the CtpA protease plays a crucial role in forming iD1 but that, surprisingly, low levels of C-terminal processing occur in vivo in the absence of CtpA and CtpB, possibly catalysed by other related proteases. A possible role for two-step maturation of D1 in the assembly of PSII is discussed.
Keywords: CtpA protease; D1 maturation; Photosystem II; psbA gene; Synechocystis PCC 6803;

Quality control of Photosystem II: Cleavage and aggregation of heat-damaged D1 protein in spinach thylakoids by Keisuke Komayama; Mahbuba Khatoon; Daichi Takenaka; Junko Horie; Amu Yamashita; Miho Yoshioka; Yohsuke Nakayama; Mari Yoshida; Satoshi Ohira; Noriko Morita; Maya Velitchkova; Isao Enami; Yasusi Yamamoto (838-846).
Moderate heat stress (40 °C, 30 min) on spinach thylakoids induced cleavage of the D1 protein, producing an N-terminal 23-kDa fragment, a C-terminal 9-kDa fragment, and aggregation of the D1 protein. A homologue of Arabidopsis FtsH2 protease, which is responsible for degradation of the damaged D1 protein, was abundant in the stroma thylakoids. Two processes occurred in the thylakoids in response to heat stress: dephosphorylation of the D1 protein in the stroma thylakoids, and aggregation of the phosphorylated D1 protein in the grana. Heat stress also induced the release of the extrinsic PsbO, P and Q proteins from Photosystem II, which affected D1 degradation and aggregation significantly. The cleavage and aggregation of the D1 protein appear to be two alternative processes influenced by protein phosphorylation/dephosphorylation, distribution of FtsH, and intactness of the thylakoids.
Keywords: Photosystem II; D1 protein; FtsH proteases; Phosphatases; Protein aggregation; Spinach;

Importance of trimer–trimer interactions for the native state of the plant light-harvesting complex II by Petar H. Lambrev; Zsuzsanna Várkonyi; Sashka Krumova; László Kovács; Yuliya Miloslavina; Alfred R. Holzwarth; Győző Garab (847-853).
Aggregates and solubilized trimers of LHCII were characterized by circular dichroism (CD), linear dichroism and time-resolved fluorescence spectroscopy and compared with thylakoid membranes in order to evaluate the native state of LHCII in vivo. It was found that the CD spectra of lamellar aggregates closely resemble those of unstacked thylakoid membranes whereas the spectra of trimers solubilized in n-dodecyl-β,d-maltoside, n-octyl-β,d-glucopyranoside, or Triton X-100 were drastically different in the Soret region. Thylakoid membranes or LHCII aggregates solubilized with detergent exhibited CD spectra similar to the isolated trimers. Solubilization of LHCII was accompanied by profound changes in the linear dichroism and increase in fluorescence lifetime. These data support the notion that lamellar aggregates of LHCII retain the native organization of LHCII in the thylakoid membranes. The results indicate that the supramolecular organization of LHCII, most likely due to specific trimer–trimer contacts, has significant impact on the pigment interactions in the complexes.
Keywords: Thylakoid membrane; Light-harvesting complex; Circular dichroism; Linear dichroism; Time-resolved fluorescence;

In our study, EPR spin-trapping technique was employed to study dark production of two reactive oxygen species, hydroxyl radicals (OH) and singlet oxygen (1O2), in spinach photosystem II (PSII) membrane particles exposed to elevated temperature (47 °C). Production of OH, evaluated as EMPO-OH adduct EPR signal, was suppressed by the enzymatic removal of hydrogen peroxide and by the addition of iron chelator desferal, whereas externally added hydrogen peroxide enhanced OH production. These observations reveal that OH is presumably produced by metal-mediated reduction of hydrogen peroxide in a Fenton-type reaction. Increase in pH above physiological values significantly stimulated the formation of OH, whereas the presence of chloride and calcium ions had the opposite effect. Based on our results it is proposed that the formation of OH is linked to the thermal disassembly of water-splitting manganese complex on PSII donor side. Singlet oxygen production, followed as the formation of nitroxyl radical TEMPO, was not affected by OH scavengers. This finding indicates that the production of these two species was independent and that the production of 1O2 is not closely linked to PSII donor side.
Keywords: EMPO; EPR spin-trapping; Heat; Hydroxyl radical; Singlet oxygen; Photosystem II;

Treatment with the herbicide acifluorfen-sodium (AF-Na), an inhibitor of protoporphyrinogen oxidase, caused an accumulation of protoporphyrin IX (Proto IX) , light-induced necrotic spots on the cucumber cotyledon within 12–24 h, and photobleaching after 48–72 h of light exposure. Proto IX-sensitized and singlet oxygen (1O2)-mediated oxidative stress caused by AF-Na treatment impaired photosystem I (PSI), photosystem II (PSII) and whole chain electron transport reactions. As compared to controls, the F v/F m (variable to maximal chlorophyll a fluorescence) ratio of treated samples was reduced. The PSII electron donor NH2OH failed to restore the F v/F m ratio suggesting that the reduction of F v/F m reflects the loss of reaction center functions. This explanation is further supported by the practically near-similar loss of PSI and PSII activities. As revealed from the light saturation curve (rate of oxygen evolution as a function of light intensity), the reduction of PSII activity was both due to the reduction in the quantum yield at limiting light intensities and impairment of light-saturated electron transport. In treated cotyledons both the Q (due to recombination of QA with S2) and B (due to recombination of QB with S2/S3) band of thermoluminescence decreased by 50% suggesting a loss of active PSII reaction centers. In both the control and treated samples, the thermoluminescence yield of B band exhibited a periodicity of 4 suggesting normal functioning of the S states in centers that were still active. The low temperature (77 K) fluorescence emission spectra revealed that the F 695 band (that originates in CP-47) increased probably due to reduced energy transfer from the CP47 to the reaction center. These demonstrated an overall damage to the PSI and PSII reaction centers by 1O2 produced in response to photosensitization reaction of protoporphyrin IX in AF-Na-treated cucumber seedlings.
Keywords: Acifluorfen; Chlorophyll biosynthesis; Chlorophyll fluorescence; Cucumis sativus; Diphenyl ether herbicide; Oxidative stress; Photodynamic reaction; Photosystem I; Photosystem II; Protoporphyrin IX; Singlet oxygen; Thermoluminescence;

Structural–functional state of thylakoid membranes of wheat genotypes under water stress by Irada M. Huseynova; Saftar Y. Suleymanov; Jalal A. Aliyev (869-875).
Plants were grown in field conditions in the wide area under normal water supply and severe water deficit. Two wheat (Triticum aestivum L.) genotypes contrasting by architectonics and differing in drought-resistance were used: Giymatli-2/17, short stature, with broad and drooping leaves, drought-sensitive, and Azamatli-95, short stature, with vertically oriented small leaves, drought-tolerant). It was found out that Giymatli-2/17 was characterized by relatively low content of Chl a-protein of PS I (CP I) and β-subunit of ATP-synthase complex, the high content of proteins in the 33–30.5 kDa region and LHC polypeptides (28–24.5 kDa), the intensive fluorescence at 740 nm and more high photochemical activity of PS II under normal irrigation compared with Azamatli-95. However, the content of CP I (M r 115 kDa) and apoprotein of P700 with M r 63 kDa insignificantly increases in the drought-resistant genotype Azamatli-95 under extreme water supply condition while their content decreases in drought-sensitive cv Giymatli-2/17. Intensity of synthesis α- and β-subunits of CF1 (55 and 53.5 kDa) also decreases in Giymatli-2/17. The levels of the core antenna polypeptides of FS II with M r 46 and 44.5 kDa (CP47 and CP43) remains stable both in normal, and stressful conditions. At the same time the significant reduction is observed in the content of polypeptides in the 33–30.5 kDa region in the more sensitive genotype Giymatli-2/17. There is an increase in the LHC II polypeptides level in tolerant genotype Azamatli-95 in contrast to Giymatli-2/17 (where the content of these subunits is observed decreasing). The intensity of short wavelength peaks at 687 and 695 nm sharply increases in the fluorescence spectra (77 K) of chloroplasts from sensitive genotype Giymatli-2/17 under water deficiency and there is a stimulation of the ratio of fluorescence band intensity F687/F740. After exposure to drought, cv Giymatli-2/17 shows a larger reduction in the actual PS II photochemical efficiency of chloroplasts than cv Azamatli-95.
Keywords: Drought; Wheat genotype; Thylakoid membrane; Polypeptide; Fluorescence; Adaptation;

The water-oxidizing complex of Photosystem II is an important target of ultraviolet-B (280–320 nm) radiation, but the mechanistic background of the UV-B induced damage is not well understood. Here we studied the UV-B sensitivity of Photosystem II in different oxidation states, called S-states of the water-oxidizing complex. Photosystem II centers of isolated spinach thylakoids were synchronized to different distributions of the S0, S1, S2 and S3 states by using packages of visible light flashes and were exposed to UV-B flashes from an excimer laser (λ  = 308 nm). The loss of oxygen evolving activity showed that the extent of UV-B damage is S-state-dependent. Analysis of the data obtained from different synchronizing flash protocols indicated that the UV-sensitivity of Photosystem II is significantly higher in the S3 and S2 states than in the S1 and S0 states. The data are discussed in terms of a model where UV-B-induced inhibition of water oxidation is caused either by direct absorption within the catalytic manganese cluster or by damaging intermediates of the water oxidation process.
Keywords: UV-damage; Photosystem II; Oxygen evolution; S-states;