Photosynthesis Research (v.98, #1-3)

A brief introduction of Kimiyuki Satoh by Isao Enami; Jian-Ren Shen (7-11).
In this Special Issue of Photosynthesis Research (Structure, Function, and Dynamics of Photosystem II) in honor of Kimiyuki Satoh and Thomas J. Wydrzynski, we present here a brief introduction to the scientific career and achievements of Kimiyuki Satoh, a great scientist with numerous important contributions in photosynthesis research, especially in the field of photosystem II.
Keywords: Kimiyuki Satoh; Hirosi Huzisige; Warren Butler; Photosystem II; Reaction center; Pigment–protein complexes; Urea–SDS-PAGE

In appreciation of his contribution to the Photosystsem II research and commemoration of the book Photosystem II: The Light-Driven Water-Plastoquinone Oxido-Reductase, co-edited with Kimiyuki Satoh, I present here some of my recollections of Thomas John Wydrzynski and by several others with whom he has associated over the years at Urbana (Illinois), Berkeley (California), Standard Oil Company-Indiana (Illinois), Berlin (Germany), Gothenburg (Sweden), and Canberra (Australia). We not only recognize him for his unique career path in Photosystem II research, but also for his qualities as a collaborative scientist working on the only system on Earth that has the ability to oxidize water to molecular oxygen using the energy of sunlight.
Keywords: Contributions of Thomas Wydrzynski; First application of NMR to photosynthesis; Artificial photosynthesis; Mechanism of water oxidation; Bicarbonate in Photosystem II

This article provides a glimpse into the dawning of research on chlorophyll–protein complexes and a brief recollection of the path that led us to the identification of the photosystem II reaction center, i.e., the polypeptides that carry the site of primary charge separation in oxygenic photosynthesis. A preliminary version of the personal review on the latter topic has already appeared in this journal (Satoh Photosynth Res 76:233–240, 2003).
Keywords: Primary charge separation; Chlorophyll; Chlorophyll–protein complex; D1 protein; Photosystem II; P-680; Reaction center

As this special issue shows, we know quite a lot about the workings of Photosystem II and the oxidation of water to molecular O2. However, there are still many questions and details that remain to be answered. In this article, I very briefly outline some aspects of Photosystem II electron transport that are crucial for the efficient oxidation of water and require further studies. To fully understand Photosystem II reactions is not only a satisfying intellectual pursuit, but is also an important goal as we develop new solar technologies for the splitting of water into pure O2 and H2 for use as a potential fuel source. “As Students of the Past, We Send Greetings to the Students of the Future.”*
Keywords: Photosynthesis; Photosystem II; O2 evolution; Water splitting; Future studies

Photosystem II: The machinery of photosynthetic water splitting by Gernot Renger; Thomas Renger (53-80).
This review summarizes our current state of knowledge on the structural organization and functional pattern of photosynthetic water splitting in the multimeric Photosystem II (PS II) complex, which acts as a light-driven water: plastoquinone-oxidoreductase. The overall process comprises three types of reaction sequences: (1) photon absorption and excited singlet state trapping by charge separation leading to the ion radical pair $$ { ext{P}}680^{ + ullet } { ext{Q}}_{ ext{A}}^{ - ullet } left( { overset{wedge}{=}{ ext{P}}_{ ext{D1}}^{ + ullet } { ext{Q}}_{ ext{A}}^{ - ullet } } ight) $$ formation, (2) oxidative water splitting into four protons and molecular dioxygen at the water oxidizing complex (WOC) with $$ { ext{P}}680^{ + ullet } $$ as driving force and tyrosine YZ as intermediary redox carrier, and (3) reduction of plastoquinone to plastoquinol at the special QB binding site with $$ { ext{Q}}_{ ext{A}}^{ - ullet } $$ acting as reductant. Based on recent progress in structure analysis and using new theoretical approaches the mechanism of reaction sequence (1) is discussed with special emphasis on the excited energy transfer pathways and the sequence of charge transfer steps: $$ ^{1} left( { ext{RC-PC}} ight)^{ *} { ext{Q}}_{ ext{A}} o { ext{P}}_{{{ ext{D}}2}} { ext{P}}_{{{ ext{D}}1}} { ext{Chl}}_{{{ ext{D}}1}}^{ + ullet } { ext{Pheo}}_{{{ ext{D}}1}}^{ - ullet } { ext{Q}}_{ ext{A}} o { ext{P}}_{{{ ext{D}}2}} { ext{P}}_{{{ ext{D}}1}}^{ + ullet } { ext{Chl}}_{{{ ext{D}}1}} { ext{Pheo}}_{{{ ext{D}}1}}^{ - ullet } { ext{Q}}_{ ext{A}} o { ext{P}}_{{{ ext{D}}2}} { ext{P}}_{{{ ext{D}}1}}^{ + ullet } { ext{Chl}}_{ ext{D1}} { ext{Pheo}}_{ ext{D1}} { ext{Q}}_{ ext{A}}^{ - ullet } , $$ where 1(RC-PC)* denotes the excited singlet state 1P680* of the reaction centre pigment complex. The structure of the catalytic Mn4O X Ca cluster of the WOC and the four step reaction sequence leading to oxidative water splitting are described and problems arising for the electronic configuration, in particular for the nature of redox state S3, are discussed. The unravelling of the mode of O–O bond formation is of key relevance for understanding the mechanism of the process. This problem is not yet solved. A multistate model is proposed for S3 and the functional role of proton shifts and hydrogen bond network(s) is emphasized. Analogously, the structure of the QB site for PQ reduction to PQH2 and the energetic and kinetics of the two step redox reaction sequence are described. Furthermore, the relevance of the protein dynamics and the role of water molecules for its flexibility are briefly outlined. We end this review by presenting future perspectives on the water oxidation process.
Keywords: Photosystem II; Charge separation; Oxidative water splitting; Reductive plastoquinol formation; Mechanisms

Primary light-energy conversion in tetrameric chlorophyll structure of photosystem II and bacterial reaction centers: I. A review by Ravil A. Khatypov; Anton Yu. Khmelnitskiy; Maria M. Leonova; Lyudmila G. Vasilieva; Vladimir A. Shuvalov (81-93).
The purpose of the review is to show that the tetrameric (bacterio)chlorophyll ((B)Chl) structures in reaction centers of photosystem II (PSII) of green plants and in bacterial reaction centers (BRCs) are similar and play a key role in the primary charge separation. The Stark effect measurements on PSII reaction centers have revealed an increased dipole moment for the transition at ~730 nm (Frese et al., Biochemistry 42:9205–9213, 2003). It was found (Heber and Shuvalov, Photosynth Res 84:84–91, 2005) that two fluorescent bands at 685 and 720 nm are observed in different organisms. These two forms are registered in the action spectrum of QA photoreduction. Similar results were obtained in core complexes of PSII at low temperature (Hughes et al., Biochim Biophys Acta 1757: 841–851, 2006). In all cases the far-red absorption and emission can be interpreted as indication of the state with charge transfer character in which the chlorophyll monomer plays a role of an electron donor. The role of bacteriochlorophyll monomers (BA and BB) in BRCs can be revealed by different mutations of axial ligand for Mg central atoms. RCs with substitution of histidine L153 by tyrosine or leucine and of histidine M182 by leucine (double mutant) are not stable in isolated state. They were studied in antennaless membrane by different kinds of spectroscopy including one with femtosecond time resolution. It was found that the single mutation (L153HY) was accompanied by disappearance of BA molecule absorption near 802 nm and by 14-fold decrease of photochemical activity measured with ms time resolution. The lifetime of P870* increased up to ~200 ps in agreement with very low rate of the electron transfer to A-branch. In the double mutant L153HY + M182HL, the BA appears to be lost and BB is replaced by bacteriopheophytin ΦB with the absence of any absorption near 800 nm. Femtosecond measurements have revealed the electron transfer to B-branch with a time constant of ~2 ps. These results are discussed in terms of obligatory role of BA and ΦB molecules located near P for efficient electron transfer from P*.
Keywords: Bacterial and photosystem II reaction centers; Charge separation; Charge transfer state; Primary electron donor and acceptor; Femtosecond spectroscopy

Primary light-energy conversion in tetrameric chlorophyll structure of photosystem II and bacterial reaction centers: II. Femto- and picosecond charge separation in PSII D1/D2/Cyt b559 complex by I. V. Shelaev; F. E. Gostev; V. A. Nadtochenko; A. Ya. Shkuropatov; A. A. Zabelin; M. D. Mamedov; A. Yu. Semenov; O. M. Sarkisov; V. A. Shuvalov (95-103).
In Part I of the article, a review of recent data on electron-transfer reactions in photosystem II (PSII) and bacterial reaction center (RC) has been presented. In Part II, transient absorption difference spectroscopy with 20-fs resolution was applied to study the primary charge separation in PSII RC (DI/DII/Cyt b 559 complex) excited at 700 nm at 278 K. It was shown that the initial electron-transfer reaction occurs within 0.9 ps with the formation of the charge-separated state P680+ChlD1 , which relaxed within 14 ps as indicated by reversible bleaching of 670-nm band that was tentatively assigned to the ChlD1 absorption. The subsequent electron transfer from ChlD1 within 14 ps was accompanied by a development of the radical anion band of PheoD1 at 445 nm, attributable to the formation of the secondary radical pair P680+PheoD1 . The key point of this model is that the most blue Q y transition of ChlD1 in RC is allowing an effective stabilization of separated charges. Although an alternative mechanism of charge separation with ChlD1* as a primary electron donor and PheoD1 as a primary acceptor can not be ruled out, it is less consistent with the kinetics and spectra of absorbance changes induced in the PSII RC preparation by femtosecond excitation at 700 nm.
Keywords: Chlorophyll; Pheophytin; Photosystem II; Primary charge separation; Reaction center

PS II model-based simulations of single turnover flash-induced transients of fluorescence yield monitored within the time domain of 100 ns–10 s on dark-adapted Chlorella pyrenoidosa cells by N. E. Belyaeva; F.-J. Schmitt; R. Steffen; V. Z. Paschenko; G. Yu. Riznichenko; Yu. K. Chemeris; G. Renger; A. B. Rubin (105-119).
The set up described in Steffen et al. (Biochemistry 40:173–180, 2001) was used to monitor in the time domain from 100 ns to 10 s single turnover flash-induced transients of the normalized fluorescence yield (SFITFY) on dark-adapted cells of the thermophilic algae Chlorella pyrenoidosa Chick. Perfect data fit was achieved within the framework of a previously proposed model for the PS II reaction pattern (Lebedeva et al., Biophysics 47:968–980, 2002; Belyaeva et al., Biophysics 51:860–872, 2006) after its modification by taking into account nonradiative decay processes including nonphotochemical quenching due to time-dependent populations of P680+• and 3Car. On the basis of data reported in the literature, a consistent set of rate constants was obtained for electron transfer at the donor and acceptor sides of PS II, pH in lumen and stroma, the initial redox state of plastoquinone pool and the rate of plastoquinone oxidation. The evaluation of the rate constant values of dissipative processes due to quenching by carotenoid triplets in antennae and P680+•QA −• recombination as well as the initial state populations after excitation with a single laser flash are close to that outlined in (Steffen et al., Biochemistry 44:3123–3133, 2005a). The simulations based on the model of the PS II reaction pattern provide information on the time courses of population probabilities of different PS II states. We analyzed the maximum ( $$ F_{ ext{m}}^{ ext{STF}} $$ ) and minimum (F 0) of the normalized FL yield dependence on the rate of the recombination processes (radiative and dissipative nonradiative) and of P680+• reduction. The developed PS II model provides a basis for theoretical comparative analyses of time-dependent fluorescence signals, observed at different photosynthetic samples under various conditions (e.g. presence of herbicides, other stress conditions, excitation with actinic pulses of different intensity, and duration).
Keywords: Fluorescence yield; Single turnover flash; Photosystem II; Model simulation; Electron transfer; Dissipative energy losses

Electrogenic reactions and dielectric properties of photosystem II by Alexey Semenov; Dmitry Cherepanov; Mahir Mamedov (121-130).
This review is focused on the mechanism of photovoltage generation involving the photosystem II turnover. This large integral membrane enzyme catalyzes the light-driven oxidation of water and reduction of plastoquinone. The data discussed in this work show that there are four main electrogenic steps in native complexes: (i) light-induced charge separation between special pair chlorophylls P680 and primary quinone acceptor QA; (ii) P 680 + reduction by the redox-active tyrosine YZ of polypeptide D1; (iii) oxidation of Mn cluster by Y Z ox followed by proton release, and (iv) protonation of double reduced secondary quinone acceptor QB. The electrogenicity related to (i) proton-coupled electron transfer between Q A and preoxidized non-heme iron (Fe3+) in native and (ii) electron transfer between protein–water boundary and Y Z ox in the presence of redox-dye(s) in Mn-depleted samples, respectively, were also considered. Evaluation of the dielectric properties using the electrometric data and the polarity profiles of reaction center from purple bacteria Blastochloris viridis and photosystem II are presented. The knowledge of the profile of dielectric permittivity along the photosynthetic reaction center is important for understanding of the mechanism of electron transfer between redox cofactors.
Keywords: Photosystem II; Proteoliposomes; Photovoltage; Iron–quinone complex; Oxygen-evolving complex; Dielectric properties

We studied the charge recombination characteristics of Photosystem II (PSII) redox components in whole cells of the chlorophyll (Chl) d-dominated cyanobacterium, Acaryochloris marina, by flash-induced chlorophyll fluorescence and thermoluminescence measurements. Flash-induced chlorophyll fluorescence decay was retarded in the μs and ms time ranges and accelerated in the s time range in Acaryochloris marina relative to that in the Chl a-containing cyanobacterium, Synechocystis PCC 6803. In the presence of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea, which blocks the QB site, the relaxation of fluorescence decay arising from S2QA recombination was somewhat faster in Acaryochloris marina than in Synechocystis PCC 6803. Thermoluminescence intensity of the so called B band, arising from the recombination of the S2QB charge separated state, was enhanced significantly (2.5 fold) on the basis of equal amounts of PSII in Acaryochloris marina as compared with Synechocystis 6803. Our data show that the energetics of charge recombination is modified in Acaryochloris marina leading to a ~15 meV decrease of the free energy gap between the QA and QB acceptors. In addition, the total free energy gap between the ground state and the excited state of the reaction center chlorophyll is at least ~25–30 meV smaller in Acaryochloris marina, suggesting that the primary donor species cannot consist entirely of Chl a in Acaryochloris marina, and there is a contribution from Chl d as well.
Keywords: Photosystem II; Charge separation; Electron transfer; Acaryochloris marina ; Chlorophyll d ; Thermoluminescence; Fluorescence

Unique photosystems in Acaryochloris marina by Shunsuke Ohashi; Hideaki Miyashita; Naoki Okada; Tatsuya Iemura; Tadashi Watanabe; Masami Kobayashi (141-149).
A short overview is given on the discovery of the chlorophyll d-dominated cyanobacterium Acaryochloris marina and the minor pigments that function as key components therein. In photosystem I, chlorophyll d′, chlorophyll a, and phylloquinone function as the primary electron donor, the primary electron acceptor and the secondary electron acceptor, respectively. In photosystem II, pheophytin a serves as the primary electron acceptor. The oxidation potential of chlorophyll d was higher than that of chlorophyll a in vitro, while the oxidation potential of P740 was almost the same as that of P700. These results help us to broaden our view on the questions about the unique photosystems in Acaryochloris marina.
Keywords: Acaryochloris marina ; Chlorophyll a ; Chlorophyll d ; Chlorophyll d′ ; Cyanobacteria; Pheophytin a

Electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) were performed to investigate the difference in microenvironments and functions between tyrosine Z (YZ) and tyrosine D (YD). Mn-depletion or Ca2+-depletion causes extension of the lifetime of tyrosine radical YZ , which can be trapped by rapid freezing after illumination at about 250 K. Above pH 6.5, YZ radical in Mn-depleted PS II shows similar EPR and ENDOR spectra similar to that of YD radical, which are ascribed to a typical neutral tyrosine radical. Below pH 6.5, YZ radical shows quite different EPR and ENDOR spectra. ENDOR spectra show the spin density distribution of the low-pH form of YZ that has been quite different from the high-pH form of YZ . The spin density distribution of the low-pH YZ can be explained by a cation radical or the neutral radical induced by strong electrostatic interaction. The pH dependence of the activation energy of the recombination rate between YZ and QA shows a gap of 4.4 kJ/mol at pH 6.0–6.5. In the Ca2+-depleted PS II, YZ signal was the mixture of the cation-like and normal neutral radicals, and the pH dependence of YZ spectrum in Ca2+-depleted PS II is considerably different from the neutral radical found in Mn-depleted PS II. Based on the recent structure data of cyanobacterial PS II, the pH dependence of YZ could be ascribed to the modification of the local structure and hydrogen-bonding network induced by the dissociation of ASP170 near YZ.
Keywords: Photosystem II; Manganese cluster; EPR; ENDOR; Tyrosine radicals

The redox potential of QA in photosystem II (PSII) is known to be lower by ∼100 mV in the presence of phenolic herbicides compared with the presence of DCMU-type herbicides. In this study, the structural basis underlying the herbicide effects on the QA redox potential was studied using Fourier transform infrared (FTIR) spectroscopy. Light-induced $$ { ext{Q}}^{ - }_{{ ext{A}}} $$ /QA FTIR difference spectra of Mn-depleted PSII membranes in the presence of DCMU, atrazine, terbutryn, and bromacil showed a strong CO stretching peak of $$ { ext{Q}}^{ - }_{{ ext{A}}} $$ at 1,479 cm−1, while binding of phenolic herbicides, bromoxynil and ioxynil, induced a small but clear downshift by ∼1 cm−1. The CO peak positions and the small frequency difference were reproduced in the $$ { ext{S}}_{{ ext{2}}} { ext{Q}}^{ - }_{{ ext{A}}} $$ /S1QA spectra of oxygen-evolving PSII membranes with DCMU and bromoxynil. The relationship of the CO frequency with herbicide species correlated well with that of the peak temperatures of thermoluminescence due to $$ { ext{S}}_{{ ext{2}}} { ext{Q}}^{ - }_{{ ext{A}}} $$ recombination. Density functional theory calculations of model hydrogen-bonded complexes of plastoquinone radical anion showed that the small shift of the CO frequency is consistent with a change in the hydrogen-bond structure most likely as a change in its strength. The $$ { ext{Q}}^{ - }_{{ ext{A}}} $$ /QA spectra in the presence of bromoxynil, and ioxynil, which bear a nitrile group in the phenolic ring, also showed CN stretching bands around 2,210 cm−1. Comparison with the CN frequencies of bromoxynil in solutions suggested that the phenolic herbicides take a phenotate anion form in the QB pocket. It was proposed that interaction of the phenolic C–O with D1-His215 changes the strength of the hydrogen bond between the CO of QA with D2-His214 via the iron-histidine bridge, causing the decrease in the QA redox potential.
Keywords: FTIR; Herbicide; Plastoquinone; Photosystem II; Thermoluminescence

Over the last 10 years, studies of enzyme systems have demonstrated that, in many cases, H-transfers occur by a quantum mechanical tunneling mechanism analogous to long-range electron transfer. H-transfer reactions can be described by an extension of Marcus theory and, by substituting hydrogen with deuterium (or even tritium), it is possible to explore this theory in new ways by employing kinetic isotope effects. Because hydrogen has a relatively short deBroglie wavelength, H-transfers are controlled by the width of the reaction barrier. By coupling protein dynamics to the reaction coordinate, enzymes have the potential ability to facilitate more efficient H-tunneling by modulating barrier properties. In this review, we describe recent advances in both experimental and theoretical studies of enzymatic H-transfer, in particular the role of protein dynamics or promoting motions. We then discuss possible consequences with regard to tyrosine oxidation/reduction kinetics in Photosystem II.
Keywords: Hydrogen tunneling; Protein dynamics; Kinetic isotope effects; Tyrosine; Proton-coupled electron transfer

Differential kinetic absorption spectra were measured during actinic illumination of photosystem II reaction centres and core complexes in the presence of electron acceptors silicomolybdate and ferricyanide. The spectra of samples with ferricyanide differ from those with both ferricyanide and silicomolybdate. Near-infrared spectra show temporary β-carotene and peripheral chlorophyll oxidation during room temperature actinic illumination. Peripheral chlorophyll is photooxidized even after decay of β-carotene oxidation activity and significant reduction of β-carotene content in both reaction centres and photosystem II core complexes. Besides, new carotenoid cation is observed after about 1 s of actinic illumination in the reaction centres when silicomolybdate is present. Similar result was observed in PSII core complexes. HPLC analyses of illuminated reaction centres reveal several novel carotenoids, whereas no new carotenoid species were observed in HPLC of illuminated core complexes. Our data support the proposal that pigments of inner antenna are a sink of cations originating in the photosystem II reaction centre.
Keywords: Photosystem II; Reaction centre; β-Carotene; Oxidation; Silicomolybdate; Ferricyanide

β-carotene (Car) and chlorophyll (Chl) function as secondary electron donors in photosystem II (PS II) under conditions, such as low temperature, when electron donation from the O2-evolving complex is inhibited. In prior studies of the formation and decay of Car•+ and Chl•+ species at low temperatures, cytochrome b 559 (Cyt b 559 ) was chemically oxidized prior to freezing the sample. In this study, the photochemical formation of Car•+ and Chl•+ is characterized at low temperature in O2-evolving Synechocystis PS II treated with ascorbate to reduce most of the Cyt b 559 . Not all of the Cyt b 559 is reduced by ascorbate; the remainder of the PS II reaction centers, containing oxidized low-potential Cyt b 559 , give rise to Car•+ and Chl•+ species after illumination at low temperature that are characterized by near-IR spectroscopy. These data are compared to the measurements on ferricyanide-treated O2-evolving Synechocystis PS II in which the Car•+ and Chl•+ species are generated in PS II centers containing mostly high- and intermediate-potential Cyt b 559 . Spectral differences observed in the ascorbate-reduced PS II samples include decreased intensity of the Chl•+ and Car•+ absorbance peaks, shifts in the Car•+ absorbance maxima, and lack of formation of a 750 nm species that is assigned to a Car neutral radical. These results suggest that different spectral forms of Car are oxidized in PS II samples containing different redox forms of Cyt b 559 , which implies that different secondary electron donors are favored depending on the redox form of Cyt b 559 in PS II.
Keywords: Carotenoid radical cation; Chlorophyll radical cation; Cytochrome b 559 ; Photosystem II

Quantum efficiency distributions of photo-induced side-pathway donor oxidation at cryogenic temperature in photosystem II by Joseph L. Hughes; A. William Rutherford; Miwa Sugiura; Elmars Krausz (199-206).
We monitored illuminated-minus-dark absorption difference spectra in the range of 450–1100 nm induced by continuous illumination at 8 K of photosystem II (PSII) core complexes from Thermosynechococcus elongatus. The photo-induced oxidation of the side-path donors Cytb559, β-carotene and chlorophyll Z, as well as the concomitant stable (t 1/2 > 1 s) reduction of the first plastoquinone electron acceptor, QA (monitored by the well-known ‘C550’ shift), were quantified as a function of the absorbed photons per PSII. The QA photo-induced reduction data can be described by three distinct quantum efficiency distributions: (i) a very high efficiency of ~0.5–1, (ii) a middle efficiency with a very large range of ~0.014–0.2, and (iii) a low efficiency of ~0.002. Each of the observed side-path donors exhibited similar quantum efficiency distributions, which supports a branched pathway model for side-path oxidation where β-carotene is the immediate electron donor to the photo-oxidized chlorophyll (P680+). The yields of the observed side-path donors account quantitatively for the wide middle efficiency range of photo-induced QA reduction, but not for the PSII fractions that exhibit the highest and lowest efficiencies. The high-efficiency component may be due to TyrZ oxidation. A donor that does not exhibit an identified absorption in the visible-near-IR region is mainly responsible for the lowest efficiency component.
Keywords: Photosystem II; Side-path donor; Secondary donor; Quantum efficiency

The discovery that the native PS II enzyme undergoes charge separation via an absorption extending to 730 nm has led us to re-examine the low-temperature absorption spectra of Nanba-Satoh PS II reaction centre preparations with particular focus on the long wavelength region. It is shown that these preparations do not exhibit absorption in the 700–730 nm region at 1.7 K. Absorption in the Nanba-Satoh type preparations analogous to the ‘red tail’ as observed in functional PS II core complexes is likely shifted to higher energy by >20 nm. Spectral changes associated with the stable reduction of pheoa in chemically treated reaction centre preparations are also revisited. Dithionite treatment of PS II preparations in the dark leads to changes of pigment–pigment and/or pigment–protein interactions, as evidenced by changes in absorption and CD spectra. Absorption and CD changes associated with stable PheoD1 photo-reduction in PS II core complexes and Nanba-Satoh preparations are compared. For Nanba-Satoh preparations, Qy bleaches are ~3× broader than in PS II core complexes and are blue-shifted by ~4 nm. These data are discussed in terms of current models of PS II, and suggest a need to consider protein-induced changes of some electronic properties of reaction centre pigments.
Keywords: Absorption; CD; PS II; Charge separation; Spectral assignment

Possible role of high-energy bosons (virtual photons) is discussed with respect to the formation of elementary particles and their interaction in nucleus, many-electron atom, and molecule including photoreaction centers. Using properties of the photons, the expressions for calculations of the mass of particles, of the energy of electrons and their distances from nucleus in atoms, of the dissociation energy and distances between atoms in molecules were found which give results in good agreement with experimental data. This approach allows doing calculations in rather complicated system like photoreaction centers in which chlorophyll molecules form electron transfer chain.
Keywords: Bacterial and photosystem II reaction centers; Charge separation; Charge transfer state; Primary electron donor and acceptor; Femtosecond spectroscopy; Photons; Elementary particles; Many-electron atoms

Cyanobacteria, algae, and plants produce dioxygen from water. Driven and clocked by light quanta, the catalytic Mn4Ca Tyrosine centre accumulates four oxidizing equivalents before it abstracts four electrons from water and liberates dioxygen and protons. Intermediates of this reaction cascade are short-lived (<100 μs) and difficult to detect. By application of high oxygen pressure to cyanobacterial PSII-core-complexes, we have previously suppressed the transition from the highest oxidation state of the centre to the lowest by stabilizing a (peroxy) intermediate. Here, we investigated the inhibitory interplay of acidification and augmented oxygen pressure. Starting from pH 6.5, acidification increasingly inhibited the reduction of the highest oxidized state and resulted in a lower oxygen partial pressure for half inhibition. Oxygen and proton interfere with different steps of the reaction cascade.
Keywords: Photosynthesis; Water oxidation; Proton release; Photosystem II; Intermediate; Oxygen

Decoupling of the processes of molecular oxygen synthesis and electron transport in Ca2+-depleted PSII membranes by Boris K. Semin; Lira N. Davletshina; Il’ya I. Ivanov; Andrei B. Rubin; Michael Seibert (235-249).
Extraction of Ca2+ from the O2-evolving complex (OEC) of photosystem II (PSII) membranes with 2 M NaCl in the light (PSII(–Ca/NaCl)) results in 90% inhibition of the O2-evolution reaction. However, electron transfer from the donor to acceptor side of PSII, measured as the reduction of the exogenous acceptor 2,6-dichlorophenolindophenol (DCIP) under continuous light, is inhibited by only 30%. Thus, calcium extraction from the OEC inhibits the synthesis of molecular O2 but not the oxidation of a substrate we term X, the source of electrons for DCIP reduction. The presence of electron transfer across PSII(–Ca/NaCl) membranes was demonstrated using fluorescence induction kinetics, a method that does not require an artificial acceptor. The calcium chelator, EGTA (5 mM), when added to PSII(–Ca/NaCl) membranes, does not affect the inhibition of O2 evolution by NaCl but does inhibit DCIP reduction up to 92% (the reason why electron transport in Ca2+-depleted materials has not been noticed before). Another chelator, sodium citrate (citrate/low pH method of calcium extraction), also inhibits both O2 evolution and DCIP reduction. The role of all buffer components (including bicarbonate and sucrose) as possible sources of electrons for PSII(–Ca/NaCl) membranes was investigated, but only the absence of chloride anions strongly inhibited the rate of DCIP reduction. Substitution of other anions for chloride indicates that Cl serves its well-known role as an OEC cofactor, but it is not substrate X. Multiple turnover flash experiments have shown a period of four oscillations of the fluorescence yield (both the maximum level, F max, and the fluorescence level measured 50 s after an actinic flash in the presence of DCMU) in native PSII membranes, reflecting the normal function of the OEC, but the absence of oscillations in PSII(–Ca/NaCl) samples. Thus, PSII(–Ca/NaCl) samples do not evolve O2 but do transfer electrons from the donor to acceptor sides and exhibit a disrupted S-state cycle. We explain these results as follows. In Ca2+-depleted PSII membranes, obtained without chelators, the oxidation of the OEC stops after the absorption of three quanta of light (from the S1 state), which should convert the native OEC to the S4 state. An one-electron oxidation of the water molecule bound to the Mn cluster then occurs (the second substrate water molecule is absent due to the absence of calcium), and the OEC returns to the S3 state. The appearance of a sub-cycle within the S-state cycle between S3-like and S4-like states supplies electrons (substrate X is postulated to be OH), explains the absence of O2 production, and results in the absence of a period of four oscillation of the normal functional parameters, such as the fluorescence yield or the EPR signal from S2. Chloride anions probably keep the redox potential of the Mn cluster low enough for its oxidation by Y Z .
Keywords: Calcium; Chloride; Manganese; Fluorescence induction kinetics; Oxygen-evolving complex; Photosystem II

Effects of methanol on the S i -state transitions in photosynthetic water-splitting by Birgit Nöring; Dmitriy Shevela; Gernot Renger; Johannes Messinger (251-260).
From a chemical point of view methanol is one of the closest analogues of water. Consistent with this idea EPR spectroscopy studies have shown that methanol binds at—or at least very close to—the Mn4O x Ca cluster of photosystem II (PSII). In contrast, Clark-type oxygen rate measurements demonstrate that the O2 evolving activity of PSII is surprisingly unaffected by methanol concentrations of up to 10%. Here we study for the first time in detail the effect of methanol on photosynthetic water-splitting by employing a Joliot-type bare platinum electrode. We demonstrate a linear dependence of the miss parameter for S i state advancement on the methanol concentrations in the range of 0–10% (v/v). This finding is consistent with the idea that methanol binds in PSII with similar affinity as water to one or both substrate binding sites at the Mn4O x Ca cluster. The possibility is discussed that the two substrate water molecules bind at different stages of the cycle, one during the S4 → S0 and the other during the S2 → S3 transition.
Keywords: Photosystem II; Manganese cluster; Water-splitting; Oxygen evolution; Methanol

This mini review presents a general introduction to photosystem II with an emphasis on the oxygen evolving complex. An attempt is made to summarise what is currently known about substrate interaction in the oxygen evolving complex of photosystem II in terms of the nature of the substrate, the timing and the location of its binding. As the nature of substrate water binding has a direct bearing on the mechanism of O–O bond formation in PSII, a discussion of O–O bond formation follows the summary of current opinion in substrate interaction.
Keywords: Water oxidation; Water oxidising complex; Substrate water; Photosystem II; Water exchange

Reconstitution of the water-oxidizing complex in manganese-depleted photosystem II preparations using synthetic Mn complexes: a fluorine-19 NMR study of the reconstitution process by Toshi Nagata; Sergei K. Zharmukhamedov; Andrei A. Khorobrykh; Vyacheslav V. Klimov; Suleyman I. Allakhverdiev (277-284).
Reconstitution of Mn-depleted photosystem II (PSII) particles was examined with synthetic trinuclear Mn complexes of newly developed tripod ligands. Rates of the electron transfer and oxygen evolution were up to 74–86 and 52–56% of those measured in native PSII. These values are higher than those for the PSII reconstituted by MnCl2. The role of the tripod ligands during the reconstitution process was examined by 19F NMR. Due to the high NMR sensitivity of the 19F nucleus and the low abundance of fluorine atoms in natural PSII, it was possible to selectively observe the fluorine atoms on the tripod ligand. It was shown that the tripod ligands were released from the Mn complex after the reconstitution. We propose that the primary step in the reconstitution process is the prebinding of the Mn complex to the hydrophobic part of the PSII particle.
Keywords: Manganese complex; Photosystem II; Reconstitution; Water-oxidizing complex; Fluorine-19 NMR

Isotopic labelling of photosystem II in Thermosynechococcus elongatus by Alain Boussac; Jean-Marc Verbavatz; Miwa Sugiura (285-292).
This report describes a protocol to incorporate isotopically labelled aromatic amino acids into the proteins of the thermophilic cyanobacterium Thermosynechoccus elongatus. By using the EPR signal of the two redox active tyrosines of Photosystem II, Tyr D and Tyr Z , as spectroscopic probes it is shown that labelled tyrosines can be incorporated with a high yield in this cyanobacterium. The production of a fully 13C- or 2H-labelled enzyme is also described.
Keywords: Isotopic labelling; Thermosynechococcus elongatus ; Photosystem II; Tyrosyl radical; EPR

Isolation and spectral characterization of Photosystem II reaction center from Synechocystis sp. PCC 6803 by Tatsuya Tomo; Seiji Akimoto; Tohru Tsuchiya; Michitaka Fukuya; Kazunori Tanaka; Mamoru Mimuro (293-302).
We isolated highly-purified photochemically active photosystem (PS) II reaction center (RC) complexes from the cyanobacterium Synechocystis sp. PCC 6803 using a histidine-tag introduced to the 47 kDa chlorophyll protein, and characterized their spectroscopic properties. Purification was carried out in a one-step procedure after isolation of PS II core complex. The RC complexes consist of five polypeptides, the same as in spinach. The pigment contents per two molecules of pheophytin a were 5.8 ± 0.3 chlorophyll (Chl) a and 1.8 ± 0.1 β-carotene; one cytochrome b 559 was found per 6.0 Chl a molecules. Overall absorption and fluorescence properties were very similar to those of spinach PS II RCs; our preparation retains the best properties so far isolated from cyanobacteria. However, a clear band-shift of pheophytin a and β-carotene was observed. Reasons for these differences, and RC composition, are discussed on the basis of the three-dimensional structure of complexes.
Keywords: Photosystem II; Reaction center; Cyanobacteria; Chlorophyll; Pheophytin; β-Carotene

Spectral properties of the CP43-deletion mutant of Synechocystis sp. PCC 6803 by Yuichiro Shimada; Tohru Tsuchiya; Seiji Akimoto; Tatsuya Tomo; Michitaka Fukuya; Kazunori Tanaka; Mamoru Mimuro (303-314).
Spectral properties, particularly fluorescence spectra and their time-dependent behavior, were investigated for a mutant of the cyanobacterium Synechocystis sp. PCC 6803 lacking the 43 kDa chlorophyll-protein (CP43, PsbC). Lack of CP43 was confirmed by a size shift of the corresponding gene and by Western blotting. The CP43-deletion mutant grown under heterotrophic conditions accumulated a small amount of photosystem (PS) II, but virtually no PS II fluorescence was observed. A 686-nm fluorescence band was clearly observed by phycocyanin excitation, coming from the terminal pigments of phycobilisomes. In contrast, no PS I fluorescence was detected by phycocyanin excitation when accumulation of PS II components was not proved by a fluorescence excitation spectrum, indicating that energy transfer to PS I chlorophyll a was mediated by PS II chlorophyll a. Direct connection of phycobilisomes with PS I was not suggested. Based on these fluorescence properties, the energy flow in the CP43-deletion mutant cells is discussed.
Keywords: Photosystem II; Cyanobacteria; CP43-deletion mutant; Fluorescence spectrum; Synechocystis sp. PCC 6803

The small hydrophobic polypeptide PsbT is associated with the photosystem II (PSII) reaction center (D1/D2 heterodimer). Here, we report the effect of the deletion of PsbT on the biogenesis of PSII complex during light-induced greening of y-1 mutants of the green alga Chlamydomonas reinhardtii. The y-1 is unable to synthesize chlorophylls in the dark but do so in the light. The dark-grown y-1 cells accumulated no major PSII proteins but a small amount of PsbT. Upon illumination, PsbT was immediately synthesized while chlorophylls, major PSII proteins, and O2-evolving activity increased after a 1-h lag. The y-1 cells without PsbT accumulated chlorophylls and PSI protein at a similar rate, whereas the accumulation of PSII complex was specifically retarded during greening. The absence of PsbT did not affect the synthesis of PSII proteins. These results indicate that PsbT is required for the efficient biogenesis of PSII complex.
Keywords: Photosynthesis; Chlorophyll–protein complex; Reaction center; Greening; Assembly; y-1 mutant; Pulse-labeling

Evidence for a stable association of Psb30 (Ycf12) with photosystem II core complex in the cyanobacterium Synechocystis sp. PCC 6803 by Natsuko Inoue-Kashino; Takeshi Takahashi; Akiko Ban; Miwa Sugiura; Yuichiro Takahashi; Kazuhiko Satoh; Yasuhiro Kashino (323-335).
Ycf12 (Psb30) is a small hydrophobic subunit of photosystem II (PS II) complexes found in the cyanobacterium, Thermosynechococcus elongatus. However, earlier intense proteomic analysis on the PS II complexes from the cyanobacterium, Synechocystis 6803, could not detect Psb30. In this work, we generated a mutant of Synechocystis 6803 in which a hexa-histidine tag was fused to the C-terminus of Synechocystis Psb30. The mutant accumulated fully functional PS II complexes. Purification of Psb30 by metal affinity chromatography from thylakoid extracts resulted in co-purification of an oxygen-evolving PS II complex with normal subunit composition. This result indicates that Psb30 is expressed and stably associated with the PS II complex in Synechocystis. The histidine-tagged Psb30 in the purified PS II complex was not detected by staining or anti-polyhistidine antibodies. We also generated a mutant in which ycf12 was disrupted. The mutant grew photosynthetically and showed no significant phenotype under moderate growth conditions. Purified PS II complexes from the disruptant showed an oxygen-evolving activity comparable to wild type under low irradiance. However, it showed a remarkably lower activity than wild type under high irradiance. Thus Psb30 is required for the efficient function of PS II complexes, particularly under high irradiance conditions.
Keywords: Low-molecular-weight polypeptide; Photosystem II; Psb30; PsbY; Synechocystis 6803; Ycf12

The PsbL protein is one of three low-molecular-weight subunits identified at the monomer–monomer interface of photosystem II (PSII) [Ferreira et al. (2004) Science 303:1831–1838; Loll et al. (2005) Nature 438:1040–1044]. We have employed site-directed mutagenesis to investigate the role of PsbL in Synechocystis sp. PCC 6803 cells. Truncation of the C-terminus by deleting the last four residues (Tyr-Phe-Phe-Asn) prevented association of PsbL with the CP43-less monomeric sub-complex and therefore blocked PSII assembly resulting in an obligate photoheterotrophic strain. Replacement of these residues with Ala created four photoautotrophic mutants. Compared to wild type, the F37A, F38A, and N39A strains had reduced levels of assembled PSII centers and F37A and F38A cells were readily photodamaged. In contrast, Y36A and Y36F mutants were similar to wild type. However, each of these strains had elevated levels of the CP43-less inactive monomeric complex. Mutations targeting a putative hydrogen bond between Arg-16 and sulfoquinovosyldiacylglycerol resulted in mutants that were also highly susceptible to photodamage. Similarly mutations targeting a conserved Tyr residue (Tyr-20) also destabilized PSII under high light and suggest that Tyr-20–lipid interactions or interactions of Tyr-20 with PsbT influence the ability of PSII to recover from photodamage.
Keywords: Photosystem II; Photodamage; PsbL; Site-directed mutagenesis; Synechocystis sp. PCC 6803

Structures and functions of the extrinsic proteins of photosystem II from different species by Isao Enami; Akinori Okumura; Ryo Nagao; Takehiro Suzuki; Masako Iwai; Jian-Ren Shen (349-363).
This minireview presents a summary of information available on the variety and binding properties of extrinsic proteins that form the oxygen-evolving complex of photosystem II (PSII) of cyanobacteria, red alga, diatom, green alga, euglena, and higher plants. In addition, the structure and function of extrinsic PsbO, PsbV, and PsbU proteins are summarized based on the crystal structure of thermophilic cyanobacterial PSII together with biochemical and genetic studies from various organisms.
Keywords: Oxygen-evolving complex; Photosystem II; Extrinsic proteins; PsbO; PsbP; PsbQ; PsbQ′; PsbV; PsbU; A novel extrinsic protein

The Manganese Stabilizing Protein (MSP) of Photosystem II (PSII) is a so-called extrinsic subunit, which reversibly associates with the other membrane-bound PSII subunits. The MSP is essential for maximum rates of O2 production under physiological conditions as stabilizes the catalytic [Mn4Ca] cluster, which is the site of water oxidation. The function of the MSP subunit in the PSII complex has been extensively studied in higher plants, and the structure of non-PSII associated MSP has been studied by low-resolution biophysical techniques. Recently, crystal structures of PSII from the thermophilic cyanobacterium Thermosynechococcus elongatus have resolved the MSP subunit in its PSII-associated state. However, neither any crystal structure is available yet for MSP from mesophilic organisms, higher plants or algae nor has the non-PSII associated form of MSP been crystallized. This article reviews the current understanding of the structure, dynamics, and function of MSP, with a particular focus on properties of the MSP from T. elongatus that may be attributable to the thermophilic ecology of this organism rather than being general features of MSP.
Keywords: Photosystem II; Manganese stabilizing protein; Oxygen evolution; Thermophile

Importance of a single disulfide bond for the PsbO protein of photosystem II: protein structure stability and soluble overexpression in Escherichia coli by Julia Nikitina; Tatiana Shutova; Bogdan Melnik; Sergey Chernyshov; Victor Marchenkov; Gennady Semisotnov; Vyacheslav Klimov; Göran Samuelsson (391-403).
PsbO protein is an important constituent of the water–oxidizing complex, located on the lumenal side of photosystem II. We report here the efficient expression of the spinach PsbO in E. coli where the solubility depends entirely on the formation of the disulfide bond. The PsbO protein purified from a pET32 system that includes thioredoxin fusion is properly folded and functionally active. Urea unfolding experiments imply that the reduction of the single disulfide bridge decreases stability of the protein. Analysis of inter-residue contact density through the PsbO molecule shows that Cys51 is located in a cluster with high contact density. Reduction of the Cys28–Cys51 bond is proposed to perturb the packing interactions in this cluster and destabilize the protein as a whole. Taken together, our results give evidence that PsbO exists in solution as a compact highly ordered structure, provided that the disulfide bridge is not reduced.
Keywords: PsbO; Overexpression; Thioredoxin; Disulfide bridge; Photosystem II

Towards understanding the functional difference between the two PsbO isoforms in Arabidopsis thaliana—insights from phenotypic analyses of psbo knockout mutants by Björn Lundin; Markus Nurmi; Marc Rojas-Stuetz; Eva-Mari Aro; Iwona Adamska; Cornelia Spetea (405-414).
The extrinsic PsbO subunit of the water-oxidizing photosystem II (PSII) complex is represented by two isoforms in Arabidopsis thaliana, namely PsbO1 and PsbO2. Recent analyses of psbo1 and psbo2 knockout mutants have brought insights into their roles in photosynthesis and light stress. Here we analyzed the two psbo mutants in terms of PsbOs expression pattern, organization of PSII complexes and GTPase activity. Both PsbOs are present in wild-type plants, and their expression is mutually controlled in the mutants. Almost all PSII complexes are in the monomeric form not only in the psbo1 but also in the psbo2 mutant grown under high-light conditions. This results either from an enhanced susceptibility of PSII to photoinactivation or from malfunction of the repair cycle. Notably, the psbo1 mutant displays such problems even under growth-light conditions. These results together with the finding that PsbO2 has a threefold higher GTPase activity than PsbO1 have significance for the turnover of the PSII D1 subunit in Arabidopsis.
Keywords: Arabidopsis thaliana ; Blue Native gel electrophoresis; D1 protein turnover; GTPase; High-light stress; Photosystem II organization; PsbO protein

The D1 and D2 proteins of dinoflagellates: unusually accumulated mutations which influence on PSII photoreaction by Satoko Iida; Atsushi Kobiyama; Takehiko Ogata; Akio Murakami (415-425).
Plastid encoded genes of the dinoflagellates are rapidly evolving and most divergent. The importance of unusually accumulated mutations on structure of PSII core protein and photosynthetic function was examined in the dinoflagellates, Symbiodinium sp. and Alexandrium tamarense. Full-length cDNA sequences of psbA (D1 protein) and psbD (D2 protein) were obtained and compared with the other oxygen-evolving photoautotrophs. Twenty-three amino acid positions (7%) for the D1 protein and 34 positions (10%) for the D2 were mutated in the dinoflagellates, although amino acid residues at these positions were conserved in cyanobacteria, the other algae, and plant. Many mutations were likely to distribute in the N-terminus and the D–E interhelical loop of the D1 protein and helix B of D2 protein, while the remaining regions were well conserved. The different structural properties in these mutated regions were supported by hydropathy profiles. The chlorophyll fluorescence kinetics of the dinoflagellates was compared with Synechocystis sp. PCC6803 in relation to the altered protein structure.
Keywords: Alexandrium ; Chlorophyll fluorescence; Hydropathy plot; psbA/D ; Symbiodinium ; Synechocystis sp. PCC6803

Structure, function, and evolution of the PsbP protein family in higher plants by Kentaro Ifuku; Seiko Ishihara; Ren Shimamoto; Kunio Ido; Fumihiko Sato (427-437).
The PsbP is a thylakoid lumenal subunit of photosystem II (PSII), which has developed specifically in higher plants and green algae. In higher plants, the molecular function of PsbP has been intensively investigated by release–reconstitution experiments in vitro. Recently, solution of a high-resolution structure of PsbP has enabled investigation of structure–function relationships, and efficient gene-silencing techniques have demonstrated the crucial role of PsbP in PSII activity in vivo. Furthermore, genomic and proteomic studies have shown that PsbP belongs to the divergent PsbP protein family, which consists of about 10 members in model plants such as Arabidopsis and rice. Characterization of the molecular function of PsbP homologs using Arabidopsis mutants suggests that each plays a distinct and important function in maintaining photosynthetic electron transfer. In this review, recent findings regarding the molecular functions of PsbP and other PsbP homologs in higher plants are summarized, and the molecular evolution of these proteins is discussed.
Keywords: Molecular evolution; Oxygen-evolving complex; Photosystem II; PsbP protein; PsbP-like protein; PsbP-domain protein

The effects of simultaneous RNAi suppression of PsbO and PsbP protein expression in photosystem II of Arabidopsis by Xiaoping Yi; Stefan R. Hargett; Laurie K. Frankel; Terry M. Bricker (439-448).
Interfering RNA was used to suppress simultaneously the expression of the four genes which encode the PsbO and PsbP proteins of Photosystem II in Arabidopsis (PsbO: At5g66570, At3g50820 and PsbP: At1g06680, At2g30790). A phenotypic series of transgenic plants was obtained that expressed variable amounts of the PsbO proteins and undetectable amounts of the PsbP proteins. Immunological studies indicated that the loss of PsbP expression was correlated with the loss of expression of the PsbQ, D2, and CP47 proteins, while the loss of PsbO expression was correlated with the loss of expression of the D1 and CP43 proteins. Q A reoxidation kinetics in the absence of DCMU indicated that the slowing of electron transfer from Q A to QB was correlated with the loss of the PsbP protein. Q A reoxidation kinetics in the presence of DCMU indicated that charge recombination between Q A and donor side components of the photosystem was retarded in all of the mutants. Decreasing amounts of the PsbO protein in the absence of the PsbP component also led to a progressive loss of variable fluorescence yield (FV/FM). During fluorescence induction, the loss of PsbP was correlated with a more rapid O to J transition and a loss of the J to I transition. These results indicate that the losses of the PsbO and PsbP proteins differentially affect separate protein components and different PS II functions and can do so, apparently, in the same plant.
Keywords: PsbO; PsbP; RNAi; Photosystem II; Arabidopsis

D1-arginine257 mutants (R257E, K, and Q) of Chlamydomonas reinhardtii have a lowered QB redox potential: analysis of thermoluminescence and fluorescence measurements by Stuart Rose; Jun Minagawa; Manfredo Seufferheld; Sean Padden; Bengt Svensson; Derrick R. J. Kolling; Antony R. Crofts; Govindjee (449-468).
Arginine257 (R257), in the de-helix that caps the QB site of the D1 protein, has been shown by mutational studies to play a key role in the sensitivity of Photosystem II (PS II) to bicarbonate-reversible binding of the formate anion. In this article, the role of this residue has been further investigated through D1 mutations (R257E, R257Q, and R257K) in Chlamydomonas reinhardtii. We have investigated the activity of the QB site by studying differences from wild type on the steady-state turnover of PS II, as assayed through chlorophyll (Chl) a fluorescence yield decay after flash excitation. The effects of p-benzoquinone (BQ, which oxidizes reduced QB, Q B ) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU, which blocks electron flow from Q A to QB) were measured. The equilibrium constants of the two-electron gate were obtained through thermoluminescence measurements. The thermoluminescence properties were changed in the mutants, especially when observed after pretreatment with 100 μM BQ. A theoretical analysis of the thermoluminescence data, based mainly on the recombination pathways model of Rappaport et al. (2005), led to the conclusion that the free-energy difference for the recombination of Q B with S2 was reduced by 20–40 mV in the three mutants (D1-R257K, D1-R257Q, and D1-R257E); this was interpreted to be due to a lowering of the redox potential of QB/Q B . Further, since the recombination of Q A with S2 was unaffected, we suggest that no significant change in redox potential of QA/Q A occurred in these three mutants. The maximum variable Chl a fluorescence yield is lowered in the mutants, in the order R257K > R257Q > R257E, compared to wild type. Our analysis of the binary oscillations in Chl a fluorescence following pretreatment of cells with BQ showed that turnover of the QB site was relatively unaffected in the three mutants. The mutant D1-R257E had the lowest growth rate and steady-state activity and showed the weakest binary oscillations. We conclude that the size and the charge of the amino acid at the position D1-257 play a role in PS II function by modulating the effective redox potential of the QB/Q B pair. We discuss an indirect mechanism mediated through electrostatic and/or surface charge effects and the possibility of more pleiotropic effects arising from decreased stability of the D1/D2 and D1/CP47 interfaces.
Keywords: D1-R257 mutants; Bicarbonate in Photosystem II; Thermoluminescence; Theory of thermoluminescence; Chlorophyll a fluorescence yield decay; Electron acceptor side of Photosystem II; Redox potentials of Q A /QA and Q B /QB ; Chlamydomonas reinhardtii ; Two-electron gate in Photosystem II; Benzoquinone

Lipids are important components of transmembrane protein complexes. In order to study the roles of lipids in photosystem II (PSII), we treated the PSII core dimer complex from a thermophilic cyanobacterium Thermosynechococcus vulcanus with phospholipase A2 (PLA2) and lipase, and examined their effects on PSII structure and function. PLA2-treatment decreased the content of phospholipid, phosphatidylglycerol (PG) by 59%, leading to a decrease of oxygen evolution by 40%. On the other hand, although treatment with lipase specifically decreased the content of monogalactosyldiacylglycerol (MGDG) by 52%, it decreased oxygen evolution only by 16%. This indicates that PG plays a more important role in PSII than MGDG. Both PLA2- and lipase-treatments induced neither the dissociation of PSII dimer, nor any loss of polypeptides. The degradation of PG resulted in a damage to the QB-binding site as demonstrated from photoreduction activity of 2,6-dichlorophenolindophenol and chlorophyll fluorescence yields in the absence or presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, and the dependencies of oxygen evolution on various electron acceptors before and after PLA2- or lipase-treatments. However, there were approximately three and five molecules of PG and MGDG per PSII reaction center left in the PSII dimeric complex after the PLA2- and lipase-treatments. These lipids are therefore bound to the interior of the protein matrix and resistant to the lipase treatments. The resistance of these lipids against PLA2- and lipase-treatments may be a specific feature of PSII from the thermophilic cyanobacterium, suggesting a possible correlation between binding of lipids and thermostability of PSII.
Keywords: Photosystem II; Lipids; Oxygen evolution; Phospholipid; Phospholipase-treatment; Cyanobacteria

The small CAB-like proteins of Synechocystis sp. PCC 6803 bind chlorophyll by Patrik Storm; Miguel A. Hernandez-Prieto; Laura L. Eggink; J. Kenneth Hoober; Christiane Funk (479-488).
The large family of light-harvesting-like proteins contains members with one to four membrane spanning helices with significant homology to the chlorophyll a/b-binding antenna proteins of plants. From structural as well as evolutionary perspective, it is likely that the members of this family bind chlorophylls and carotenoids. However, undisputable evidence is still lacking. The cyanobacterial small CAB-like proteins (SCPs) are one-helix proteins with compelling similarity to the first and third transmembrane helix of LHCII (LHCIIb) including the chlorophyll-binding motifs. They have been proposed to act as chlorophyll-carrier proteins. Here, we analyze the in vivo absorption spectra of single scp deletion mutants in Synechocystis sp. PCC 6803 and compare the in vitro pigment binding ability of the SCP pairs ScpC/D and ScpB/E with the one of LHCII and a synthetic peptide containing the chlorophyll-binding motif (Eggink LL, Hoober JK (2000) J Biol Chem 275:9087–9090). We demonstrate that deletion of scpB alters the pigmentation in the cyanobacterial cell. Furthermore, we are able to show that chlorophylls and carotenoids interact in vitro with the pairs of ScpC/D and ScpB/E, demonstrated by fluorescence resonance energy transfer and circular dichroism.
Keywords: Antenna; Chlorophyll-binding protein; Cyanobacteria; Early-light-induced proteins (ELIPs); High-light-induced proteins (HLIPs); Light-harvesting complex; Synechocystis sp. PCC 6803

Auxiliary proteins involved in the assembly and sustenance of photosystem II by Paula Mulo; Sari Sirpiö; Marjaana Suorsa; Eva-Mari Aro (489-501).
Chloroplast proteins that regulate the biogenesis, performance and acclimation of the photosynthetic protein complexes are currently under intense research. Dozens, possibly even hundreds, of such proteins in the stroma, thylakoid membrane and the lumen assist the biogenesis and constant repair of the water splitting photosystem (PS) II complex. During the repair cycle, assistance is required at several levels including the degradation of photodamaged D1 protein, de novo synthesis, membrane insertion, folding of the nascent protein chains and the reassembly of released protein subunits and different co-factors into PSII in order to guarantee the maintenance of the PSII function. Here we review the present knowledge of the auxiliary proteins, which have been reported to be involved in the biogenesis and maintenance of PSII.
Keywords: Chloroplast; Light reactions; Photoinhibition; Photosystem II biogenesis; Photosystem II maintenance; Photosystem II repair

Even prior to the publication of the crystal structures for photosystem II (PSII), it had already been suggested that water, O2 and H+ channels exist in PSII to achieve directed transport of these molecules, and to avoid undesirable side reactions. Computational efforts to uncover these channels and investigate their properties are still at early stages, and have so far only been based on the static PSII structure. The rationale behind the proposals for such channels and the computer modelling studies thus far are reviewed here. The need to take the dynamic protein into account is then highlighted with reference to the specific issues and techniques applicable to the simulation of each of the three channels. In particular, lessons are drawn from simulation studies on other protein systems containing similar channels.
Keywords: Photosystem II; Molecular dynamics; Channels; Substrate water access; H+ exit pathway; O2 exit pathway

Analysis of xenon binding to photosystem II by X-ray crystallography by J. W. Murray; K. Maghlaoui; J. Kargul; M. Sugiura; J. Barber (523-527).
In order to investigate oxygen binding and hydrophobic cavities in photosystem II (PSII), we have introduced xenon under pressure into crystals of PSII isolated from Thermosynechococcus elongatus and used X-ray anomalous diffraction analyses to identify the xenon sites in the complex. Under the conditions employed, 25 Xe-binding sites were identified in each monomer of the dimeric PSII complex. The majority of these were distributed within the membrane spanning portion of the complex with no obvious correlation with the previously proposed oxygen channels. One binding site was located close to the haem of cytochrome b559 in a position analogous to a Xe-binding site of myoglobin. The only Xe-binding site not associated with the intrinsic subunits of PSII was within the hydrophobic core of the PsbO protein.
Keywords: Photosystem II; Xenon-binding; X-ray crystallography; Oxygen channel; Cytochrome b559; PsbO protein

Salt stress inhibits photosystems II and I in cyanobacteria by Suleyman I. Allakhverdiev; Norio Murata (529-539).
Recent studies of responses of cyanobacterial cells to salt stress have revealed that the NaCl-induced decline in the photosynthetic activities of photosystems II and I involves rapid and slow changes. The rapid decreases in the activities of both photosystems, which occur within a few minutes, are reversible and are associated with osmotic effects, which induce the efflux of water from the cytosol through water channels and rapidly increase intracellular concentrations of salts. Slower decreases in activity, which occur within hours, are irreversible and are associated with ionic effects that are due to the influx of Na+ and Cl ions through K+(Na+) channels and, probably, Cl channels, with resultant dissociation of extrinsic proteins from photosystems. In combination with light stress, salt stress significantly stimulates photoinhibition by inhibiting repair of photodamaged photosystem II. Tolerance of photosystems to salt stress can be enhanced by genetically engineered increases in the unsaturation of fatty acids in membrane lipids and by intracellular synthesis of compatible solutes, such as glucosylglycerol and glycinebetaine. In this review, we summarize recent progress in research on the effects of salt stress on photosynthesis in cyanobacteria.
Keywords: Cyanobacteria; Membrane lipids; Photosystem II; Photosystem I; Salt stress; Tolerance

Heat stress: an overview of molecular responses in photosynthesis by Suleyman I. Allakhverdiev; Vladimir D. Kreslavski; Vyacheslav V. Klimov; Dmitry A. Los; Robert Carpentier; Prasanna Mohanty (541-550).
The primary targets of thermal damage in plants are the oxygen evolving complex along with the associated cofactors in photosystem II (PSII), carbon fixation by Rubisco and the ATP generating system. Recent investigations on the combined action of moderate light intensity and heat stress suggest that moderately high temperatures do not cause serious PSII damage but inhibit the repair of PSII. The latter largely involves de novo synthesis of proteins, particularly the D1 protein of the photosynthetic machinery that is damaged due to generation of reactive oxygen species (ROS), resulting in the reduction of carbon fixation and oxygen evolution, as well as disruption of the linear electron flow. The attack of ROS during moderate heat stress principally affects the repair system of PSII, but not directly the PSII reaction center (RC). Heat stress additionally induces cleavage and aggregation of RC proteins; the mechanisms of such processes are as yet unclear. On the other hand, membrane linked sensors seem to trigger the accumulation of compatible solutes like glycinebetaine in the neighborhood of PSII membranes. They also induce the expression of stress proteins that alleviate the ROS-mediated inhibition of repair of the stress damaged photosynthetic machinery and are required for the acclimation process. In this review we summarize the recent progress in the studies of molecular mechanisms involved during moderate heat stress on the photosynthetic machinery, especially in PSII.
Keywords: Acclimation; Heat stress; Photosynthesis; Photosystem II

Singlet oxygen production in photosystem II and related protection mechanism by Anja Krieger-Liszkay; Christian Fufezan; Achim Trebst (551-564).
High-light illumination of photosynthetic organisms stimulates the production of singlet oxygen by photosystem II (PSII) and causes photo-oxidative stress. In the PSII reaction centre, singlet oxygen is generated by the interaction of molecular oxygen with the excited triplet state of chlorophyll (Chl). The triplet Chl is formed via charge recombination of the light-induced charge pair. Changes in the midpoint potential of the primary electron donor P680 of the primary acceptor pheophytin or of the quinone acceptor QA, modulate the pathway of charge recombination in PSII and influence the yield of singlet oxygen formation. The involvement of singlet oxygen in the process of photoinhibition is discussed. Singlet oxygen is efficiently quenched by β-carotene, tocopherol or plastoquinone. If not quenched, it can trigger the up-regulation of genes, which are involved in the molecular defence response of photosynthetic organisms against photo-oxidative stress.
Keywords: Photoinhibition; Photosystem II; QA midpoint potential; Singlet oxygen

Photosystem II reaction centre quenching: mechanisms and physiological role by Alexander G. Ivanov; Prafullachandra V. Sane; Vaughan Hurry; Gunnar Öquist; Norman P. A. Huner (565-574).
Dissipation of excess absorbed light energy in eukaryotic photoautotrophs through zeaxanthin- and ΔpH-dependent photosystem II antenna quenching is considered the major mechanism for non-photochemical quenching and photoprotection. However, there is mounting evidence of a zeaxanthin-independent pathway for dissipation of excess light energy based within the PSII reaction centre that may also play a significant role in photoprotection. We summarize recent reports which indicate that this enigma can be explained, in part, by the fact that PSII reaction centres can be reversibly interconverted from photochemical energy transducers that convert light into ATP and NADPH to efficient, non-photochemical energy quenchers that protect the photosynthetic apparatus from photodamage. In our opinion, reaction centre quenching complements photoprotection through antenna quenching, and dynamic regulation of photosystem II reaction centre represents a general response to any environmental condition that predisposes the accumulation of reduced QA in the photosystem II reaction centres of prokaryotic and eukaryotic photoautotrophs. Since the evolution of reaction centres preceded the evolution of light harvesting systems, reaction centre quenching may represent the oldest photoprotective mechanism.
Keywords: Energy dissipation; Non-photochemical quenching; Photoinhibition; Photoprotection; Photosystem II; QA ; QB ; Reaction centre quenching

Grana are not essential for photosynthesis, yet they are ubiquitous in higher plants and in the recently evolved Charaphyta algae; hence grana role and its need is still an intriguing enigma. This article discusses how the grana provide integrated and multifaceted functional advantages, by facilitating mechanisms that fine-tune the dynamics of the photosynthetic apparatus, with particular implications for photosystem II (PSII). This dynamic flexibility of photosynthetic membranes is advantageous in plants responding to ever-changing environmental conditions, from darkness or limiting light to saturating light and sustained or intermittent high light. The thylakoid dynamics are brought about by structural and organizational changes at the level of the overall height and number of granal stacks per chloroplast, molecular dynamics within the membrane itself, the partition gap between appressed membranes within stacks, the aqueous lumen encased by the continuous thylakoid membrane network, and even the stroma bathing the thylakoids. The structural and organizational changes of grana stacks in turn are driven by physicochemical forces, including entropy, at work in the chloroplast. In response to light, attractive van der Waals interactions and screening of electrostatic repulsion between appressed grana thylakoids across the partition gap and most probably direct protein interactions across the granal lumen (PSII extrinsic proteins OEEp–OEEp, particularly PsbQ–PsbQ) contribute to the integrity of grana stacks. We propose that both the light-induced contraction of the partition gap and the granal lumen elicit maximisation of entropy in the chloroplast stroma, thereby enhancing carbon fixation and chloroplast protein synthesizing capacity. This spatiotemporal dynamic flexibility in the structure and function of active and inactive PSIIs within grana stacks in higher plant chloroplasts is vital for the optimization of photosynthesis under a wide range of environmental and developmental conditions.
Keywords: Entropy; Grana; Oxygen-evolving proteins; Photosystem II; Thylakoid membranes PsbQ–PsbQ interaction; Thylakoid lumen

Quality control of photosystem II: impact of light and heat stresses by Yasusi Yamamoto; Ryota Aminaka; Miho Yoshioka; Mahbuba Khatoon; Keisuke Komayama; Daichi Takenaka; Amu Yamashita; Nobuyoshi Nijo; Kayo Inagawa; Noriko Morita; Takayuki Sasaki; Yoko Yamamoto (589-608).
Photosystem II is vulnerable to various abiotic stresses such as strong visible light and heat. Under both stresses, the damage seems to be triggered by reactive oxygen species, and the most critical damage occurs in the reaction center-binding D1 protein. Recent progress has been made in identifying the protease involved in the degradation of the photo- or heat-damaged D1 protein, the ATP-dependent metalloprotease FtsH. Another important result has been the discovery that the damaged D1 protein aggregates with nearby polypeptides such as the D2 protein and the antenna chlorophyll-binding protein CP43. The degradation and aggregation of the D1 protein occur simultaneously, but the relationship between the two is not known. We suggest that phosphorylation and dephosphorylation of the D1 protein, as well as the binding of the extrinsic PsbO protein to Photosystem II, play regulatory roles in directing the damaged D1 protein to the two alternative pathways.
Keywords: Photosystem II; Light stress and heat stress; FtsH proteases; D1 protein; PsbO protein; Reactive oxygen species

D1-protein dynamics in photosystem II: the lingering enigma by Marvin Edelman; Autar K. Mattoo (609-620).
The D1/D2 heterodimer core is the heart of the photosystem II reaction center. A characteristic feature of this heterodimer is the differentially rapid, light-dependent degradation of the D1 protein. The D1 protein is possibly the most researched photosynthetic polypeptide, with aspects of structure–function, gene, messenger and protein regulation, electron transport, reactive oxygen species, photoinhibition, herbicide binding, stromal–granal translocations, reversible phosphorylation, and specific proteases, all under intensive investigation more than three decades after the protein’s debut in the literature. This review will touch on some treaded areas of D1 research that have, so far, defied clear resolution, as well as cutting edge research on mechanisms and consequences of D1 protein degradation.
Keywords: Photosynthesis; Protein turnover; Reaction center protein; Reactive oxygen species; Spirodela oligorrhiza ; Arabidopsis thaliana

The functionality of photosystem II (PS II) following high-light pre-treatment of leaf segments at a chilling temperature was monitored as F v /F m, the ratio of variable to maximum chlorophyll fluorescence in the dark-adapted state and a measure of the optimal photochemical efficiency in PS II. Recovery of PS II functionality in low light (LL) and at a favourable temperature was retarded by (1) water stress and (2) growth in LL, in both spinach and Alocasia macrorrhiza L. In spinach leaf segments, water stress per se affected neither F v /F m nor the ability of the adenosine triphosphate (ATP) synthase to be activated by far-red light for ATP synthesis, but it induced chloroplast shrinkage as observed in frozen and fractured samples by scanning electron microscopy. A common feature of water stress and growth of plants in LL is the enhanced anchoring of PS II complexes, either across the shrunken lumen in water-stress conditions or across the partition gap in larger grana due to growth in LL. We suggest that such enhanced anchoring restricts the mobility of PS II complexes in the thylakoid membrane system, and hence hinders the lateral migration of photoinactivated PS II reaction centres to the stroma-located ribosomes for repair.
Keywords: Photoinactivation; Photoinhibition; Photosystem II; Repair of photosystem II; Water stress

The effects brought about by growing Allochromatium (Alc.) minutissimum in the presence of different concentrations of the carotenoid (Car) biosynthetic inhibitor diphenylamine (DPA) have been investigated. A decrease of Car content (from ~70% to >5%) in the membranes was accompanied by an increase of the percentage of (immature) Cars with reduced numbers of conjugated C=C bonds (from neurosporene to phytoene). Based on the obtained results and the analysis of literature data, the conclusion is reached that accumulation of phytoene during inhibition did not occur. Surprisingly, DPA inhibited phytoene synthase instead of phytoene desaturase as generally assumed. The distribution of Cars in peripheral antenna (LH2) complexes and their effect on the stability of LH2 has been investigated using absorption spectroscopy and HPLC analysis. Heterogeneity of Car composition and contents in the LH2 pool is revealed. The Car contents in LH2 varied widely from control levels to complete absence. According to common view, the assembly of LH2 occurs only in the presence of Cars. Here, we show that the LH2 can be assembled without any Cars. The presence of Cars, however, is important for structural stability of LH2 complexes.
Keywords: Bacteriochlorophyll; Carotenoids; Photosynthesis; Pigment–protein complexes; Carotenoid biosynthesis inhibition

Comparison of bacterial reaction centers and photosystem II by László Kálmán; JoAnn C. Williams; James P. Allen (643-655).
In photosynthetic organisms, the utilization of solar energy to drive electron and proton transfer reactions across membranes is performed by pigment–protein complexes including bacterial reaction centers (BRCs) and photosystem II. The well-characterized BRC has served as a structural and functional model for the evolutionarily-related photosystem II for many years. Even though these complexes transfer electrons and protons across cell membranes in analogous manners, they utilize different secondary electron donors. Photosystem II has the unique ability to abstract electrons from water, while BRCs use molecules with much lower potentials as electron donors. This article compares the two complexes and reviews the factors that give rise to the functional differences. Also discussed are the modifications that have been performed on BRCs so that they perform reactions, such as amino acid and metal oxidation, which occur in photosystem II.
Keywords: Photosynthesis; Reaction center; Tyrosine oxidation; Manganese; Oxygen evolving complex; Rhodobacter sphaeroides ; Purple bacteria

Oxygen activation by cytochrome P450 monooxygenase by Djemel Hamdane; Haoming Zhang; Paul Hollenberg (657-666).
Unlike photosystem II (PSII) that catalyzes formation of the O–O bond, the cytochromes P450 (P450), members of a superfamily of hemoproteins, catalyze the scission of the O–O bond of dioxygen molecules and insert a single oxygen atom into unactivated hydrocarbons through a hydrogen abstraction-oxygen rebound mechanism. Hydroxylation of the unactivated hydrocarbons at physiological temperatures is vital for many cellar processes such as the biosynthesis of many endogenous compounds and the detoxification of xenobiotics in humans and plants. Even though it carries out the opposite of the water splitting reaction, P450 may share similarities to PSII in proton delivery networks, oxygen and water access channels, and consecutive electron transfer processes. In this article, we review recent advances in understanding the molecular mechanisms by which P450 activates dioxygen.
Keywords: P450; Oxygen intermediate; Oxygen splitting; Hydroxylation; P450 reductase

During the last decade the practice of laboratory-directed protein evolution has become firmly established as a versatile tool in biochemical research by enabling molecular evolution toward desirable phenotypes or detection of novel structure–function interactions. Applications of this technique in the field of photosynthesis research are still in their infancy, but recently first steps have been reported in the directed evolution of the CO2-fixing enzyme Rubisco and its helper protein Rubisco activase. Here we summarize directed protein evolution strategies and review the progressive advances that have been made to develop and apply suitable selection systems for screening mutant forms of these enzymes that improve the fitness of the host organism. The goal of increasing photosynthetic efficiency of plants by improving the kinetics of Rubisco has been a long-term goal scoring modest successes. We discuss how directed evolution methodologies may one day be able to circumvent the problems encountered during this venture.
Keywords: CO2-assimilation; Rubisco; Activase; Protein evolution; Sequence space; Mutagenesis

Artificial photoactive proteins by Reza Razeghifard (677-685).
Solar power is the most abundant source of renewable energy. In this respect, the goal of making photoactive proteins is to utilize this energy to generate an electron flow. Photosystems have provided the blueprint for making such systems, since they are capable of converting the energy of light into an electron flow using a series of redox cofactors. Protein tunes the redox potential of the cofactors and arranges them such that their distance and orientation are optimal for the creation of a stable charge separation. The aim of this review is to present an overview of the literature with regard to some elegant functional structures that protein designers have created by introducing cofactors and photoactivity into synthetic proteins.
Keywords: Protein design; Photosystem; Cofactor binding; Chlorophyll; Electron transfer; Reaction center

Biological photosynthesis utilizes membrane-bound pigment/protein complexes to convert light into chemical energy through a series of electron-transfer events. In the unique photosystem II (PSII) complex these electron-transfer events result in the oxidation of water to molecular oxygen. PSII is an extremely complex enzyme and in order to exploit its unique ability to convert sunlight into chemical energy it will be necessary to make a minimal model. Here we will briefly describe how PSII functions and identify those aspects that are essential in order to catalyze the oxidation of water into O2, and review previous attempts to design simple photo-catalytic proteins and summarize our current research exploiting the E. coli bacterioferritin protein as a scaffold into which multiple cofactors can be bound, to oxidize a manganese metal center upon illumination. Through the reverse engineering of PSII and light driven water splitting reactions it may be possible to provide a blueprint for catalysts that can produce clean green fuel for human energy needs.
Keywords: Artificial photosynthesis; Photosystem II; Manganese; Electron transfer; Protein engineering; Bacterioferritin; Zinc chlorin e6 ; Water splitting