The involvement of lipids in light-induced charge separation in the reaction center of photosystem II

Extraction of PS II particles with 50 mM cholate and 1 M NaCl releases several proteins (33-, 23-, 17- and 13 kDa) and lipids from the thylakoid membrane which are essential for O₂ evolution, dichlorophenolindophenol (DCIP) reduction and for stable charge separation between P680⁺ and Q_A ^-. This work correlates the results on the loss of steady-state rates for O₂ evolution and PS II mediated DCIP photo-reduction with flash absorption changes directly monitoring the reaction center charge separation at 830 nm due to P680⁺, the chlorophyll a donor. Reconstitution of the extracted lipids to the depleted membrane restores the ability to photo-oxidize P680 reversibly and to reduce DCIP, while stimulating O₂ evolution minimally. Addition of the extracted proteins of masses 33-, 23- and 17- kDa produces no further stimulation of DCIP reduction in the presence of an exogenous donor like DPC, but does enhance this rate in the absence of exogenous donors while also stimulating O₂ evolution. The proteins alone in the absence of lipids have little influence on charge separation in the reaction center. Thus lipids are essential for stable charge separation within the reaction center, involving formation of P680⁺ and Q_A ^-.Abbreviations A₈₃₀ Absorption change at 830 nm - Chl Chlorophyll - D₁ primary electron donor to P680 - DCIP 2,6-dichlorophenolindophenol - DPC 1,5-diphenylcarbazide - MOPS 3-(N-morpholino)propanesulfonic acid - P680 reaction center chlorophyll a molecule of photosystem II - PPBQ Phenyl-p-benzoquinone - PS II Photosystem II - Q_A, Q_B first and second quinone acceptors in PS II - V-DCIP rate of DCIP reduction - V-O₂ rate of oxygen evolution - Y water-oxidizing enzyme system - CHAPS 3-Cyclohexylamino-propanesulfonic acid     

Characterization of the photosystem II centers inactive in plastoquinone reduction by fluorescence induction

In order to characterize the photosystem II (PS II) centers which are inactive in plastoquinone reduction, the initial variable fluorescence rise from the non-variable fluorescence level F_o to an intermediate plateau level F_i has been studied. We find that the initial fluorescence rise is a monophasic exponential function of time. Its rate constant is similar to the initial rate of the fastest phase (-phase) of the fluorescence induction curve from DCMU-poisoned chloroplasts. In addition, the initial fluorescence rise and the -phase have the following common properties: their rate constants vary linearly with excitation light intensity and their fluorescence yields are lowered by removal of Mg⁺⁺ from the suspension medium. We suggest that the inactive PS II centers, which give rise to the fluorescence rise from F_o to F_i, belong to the -type PS II centers. However, since these inactive centers do not display sigmoidicity in fluorescence, they thus do not allow energy transfer between PS II units like PS II.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - DMQ 2,5-dimethyl-p-benzoquinone - F_o initial non-variable fluorescence yield - F_m maximum fluorescence yield - F_i intermediate fluorescence yield - PS II photosystem II - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II     

Measurement of stoichiometry of photosystem II to photosystem I reaction centers

A wide range of values for the photosystem II to photosystem I stoichiometry have been reported. It is likely that some of this variation is due to measurement artifacts, which are discussed. Careful measurements of photosystem II reactions by absorption change at 325 nm, and flash yields of oxygen evolution, of protons from oxidation of water and of reduction of dichloroindophenol give equivalent results. Stoichiometries other than 1:1 are routinely found, and they vary with growth conditions as well as plant type. Two atrazine binding sites are found for every photosystem II reaction center that is active in oxygen evolution.     

Site-directed mutagenesis of the CP 47 protein of photosystem II: 167W in the lumenally exposed loop C is required for photosystem II assembly and stability

The intrinsic chlorophyll-protein CP 47 is a component of photosystem II which functions in both light-harvesting and oxygen evolution. Using site-directed mutagenesis we have produced the mutant W167S which lies in loop C of CP 47. This strain exhibited a 75% loss in oxygen evolution activity and grew extremely slowly in the absence of glucose. Examination of normalized oxygen evolution traces indicated that the mutant was susceptible to photoinactivation. Analysis of the variable fluorescence yield indicated that the mutant accumulated very few functional PS II reaction centers. This was confirmed by immunoblotting experiments. Interestingly, when W167S was grown in the presence of 20 M DCMU, the mutant continued to exhibit these defects. These results indicate that tryptophan 167 in loop C of CP 47 is important for the assembly and stability of the PS II reaction center.     

A current assessment of photosystem II structure

This review covers the recent progress in the elucidation of the structure of photosystem II (PSII). Because much of the structural information for this membrane protein complex has been revealed by electron microscopy (EM), the review will also consider the specific technical and interpretation problems that arise with EM where they are of particular relevance to the structural data. Most recent reviews of photosystem II structure have concentrated on molecular studies of the PSII genes and on the likely roles of the subunits that they encode or they were mainly concerned with the biophysical data and fast absorption spectroscopy largely relating to electron transfer in various purified PSII preparations. In this review, we will focus on the approaches to the three-dimensional architecture of the complex and the lipid bilayer in which it is located (the thylakoid membrane) with special emphasis placed upon electron microscopical studies of PSII-containing thylakoid membranes. There are a few reports of 3D crystals of PSII and of associated X-ray diffraction measurements and although little structural information has so far been obtained from such studies (because of the lack of 3D crystals of sufficient quality), the prospects for such studies are also assessed.Abbreviations ATP adenosine triphosphate - Chl chlorophyll - CP chlorophyll-binding protein - EM electron microscopy - LHC light harvesting complex - NADP nicotinamide adenine dinucleotide phosphate - OEC oxygen evolution enhancing complex - PS photosystem - Tris tris-hydroxymethyl aminomethane     

Stoichiometric determination of pheophytin in photosystem II of oxygenic photosynthesis

Pheophytin and chlorophyll extracted from oxygen-evolving photosystem II particles, chloroplast thylakoids and cyanobacterial cells were separated by column chromatography with DEAE-Toyopearl, and quantitatively determined by spectrophotometry. The molecular ratio of chlorophyll a+b to pheophytin a was about 100 in spinach photosystem II particles and about 140 in spinach thylakoids. Using flash spectrophotometry of P680 and measurement of flash-induced oxygen yield, the molecular ratio of the chlorophyll to the photochemical reaction center II was determined to be about 200 in the photosystem II particles. These findings suggest that the stoichiometry in photosystem II particles is one reaction center II and two pheophytin a molecules per about 200 chlorophyll molecules. The same stoichiometry for pheophytin to the reaction center II was obtained in the cyanobacteria, Anacystis nidulans and Synechocystis PCC 6714. A quantitative determination of pheophytin a and the electron donor P700 in stroma thylakoids from pokeweed suggests that photosystem I does not contain pheophytin.Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.     

The physiological significance of photosystem II heterogeneity in chloroplasts

Photosystem II in green plant chloroplasts displays heterogeneity both in the composition of its light-harvesting antenna and in the ability to reduce the plastoquinone pool. These two features are discussed in terms of chloroplast development and in view of a proposed photosystem II repair cycle.     

Effect of ozone on photosystem II in Spinacia oleracea chloroplasts

Spinach plants were treated with 0.35 ppm ozone and the photochemical activity of photosystem II determined after separation from the isolated chloroplasts using digitonin. Ozone impaired the activity of the system and also reduced the amount of β-carotene in the chloroplasts.     

A thermoluminescence study of the effects of nitrite on photosystem II in spinach thylakoids.

The site of action of nitrite on PS II was investigated by measuring the TL profile of nitrite-treated spinach thylakoid membranes. Three bands were observed in control, which were identified as the Q band (7 degrees C), the B band (24 degrees C) and the C band (57 degrees C). In the presence of 20 mmol/L nitrite, the intensity of the Q band decreased, the B band upshifted to 46 degrees C but the C band disappeared. The suppression of the Q band and the upshift of the B band suggested that nitrite caused inhibition at the water oxidizing complex. The effects of nitrite also remained the same in the presence of chloride. In case of ion-sufficient thylakoid membranes, nitrite decreased the Q band peak intensity and caused an upshift in the B band peak temperature. Nitrite showed similar effects in the presence of DCMU. This suggested that the site of action of nitrite is not at the acceptor side but at the donor side of PS II. The inhibition shown by nitrite has been found to be specific for nitrite anion. No other anions such as formate, fluoride or nitrate, were effective.     

Functional architecture of higher plant photosystem II supercomplexes

Photosystem II (PSII) is a large multiprotein complex, which catalyses water splitting and plastoquinone reduction necessary to transform sunlight into chemical energy. Detailed functional and structural studies of the complex from higher plants have been hampered by the impossibility to purify it to homogeneity. In this work, homogeneous preparations ranging from a newly identified particle composed by a monomeric core and antenna proteins to the largest C₂S₂M₂ supercomplex were isolated. Characterization by biochemical methods and single particle electron microscopy allowed to relate for the first time the supramolecular organization to the protein content. A projection map of C₂S₂M₂ at 12 Å resolution was obtained, which allowed determining the location and the orientation of the antenna proteins. Comparison of the supercomplexes obtained from WT and Lhcb‐deficient plants reveals the importance of the individual subunits for the supramolecular organization. The functional implications of these findings are discussed and allow redefining previous suggestions on PSII energy transfer, assembly, photoinhibition, state transition and non‐photochemical quenching.     

Kinetics and pathways of charge recombination in photosystem II

The mechanism of charge recombination of the S(2)Q(A)(-) state in photosystem II was investigated by modifying the free energy gap between the quinone acceptor Q(A) and the primary pheophytin acceptor Ph. This was done either by changing the midpoint potential of Ph (using mutants of the cyanobacterium Synechocystis with a modified hydrogen bond to this cofactor), or that of Q(A) (using different inhibitors of the Q(B) pocket). The results show that the recombination rate is dependent on the free energy gap between Ph and Q(A), which confirms that the indirect recombination pathway involving formation of Ph(-) has a significant contribution. In the mutant with the largest free energy gap, direct electron transfer from Q(A)(-) to P(+) predominates. The temperature dependence of the recombination rate was investigated, showing a lower activation enthalpy in this mutant compared with the WT. The data allow the determination of the rate of the direct route and of its relative weight in the various strains. The set of currently accepted values for the midpoint potentials of the Q(A)/Q(A)(-), Ph/Ph(-), and P(+)/P* couples is not consistent with the relatively rapid rate of the indirect recombination pathway found here, nor with the 3% yield of delayed fluorescence as previously estimated by de Grooth and van Gorkom (1981, Biochim. Biophys. Acta 635, 445-456). It is argued that a likely explanation is that the midpoint potentials of the two latter couples are more positive than believed due to electrostatic interactions. If such is the case, the estimation of the midpoint potential of the P(+)/P and S(2)/S(1) couples must also be revised upward, with values of 1260 and 1020 mV, respectively.     

Copper and photosystem II: A controversial relationship

Copper is an essential micronutrient for higher plants and algae and has a direct impact on photosynthesis. It is a constituent of the primary electron donor in photosystem I, the Cu-protein plastocyanin. Many authors have also described Cu as a constituent of photosystem II (PSII). However, high Cu concentrations inhibit the photosynthetic electron transport, especially in PSII. In addition, both Cu deficiency and Cu toxicity interfere with pigment and lipid biosynthesis and, consequently, with chloroplast ultrastructure thus negatively influencing the photosynthetic efficiency.
In this review, the different functions proposed for the metal in PSII are reviewed. With reference to the effect of toxic Cu on PSII, the polemic results concerning its mechanism of action and Cu-binding sites are discussed. Other effects of Cu toxicity and Cu deprivation on the thylakoid membrane are also briefly described.     

Water oxidation chemistry of photosystem II

Photosystem II (PSII) uses light energy to split water into protons, electrons and O2. In this reaction, nature has solved the difficult chemical problem of efficient four-electron oxidation of water to yield O2 without significant amounts of reactive intermediate species such as superoxide, hydrogen peroxide and hydroxyl radicals. In order to use nature's solution for the design of artificial catalysts that split water, it is important to understand the mechanism of the reaction. The recently published X-ray crystal structures of cyanobacterial PSII complexes provide information on the structure of the Mn and Ca ions, the redox-active tyrosine called YZ and the surrounding amino acids that comprise the O2-evolving complex (OEC). The emerging structure of the OEC provides constraints on the different hypothesized mechanisms for O2 evolution. The water oxidation mechanism of PSII is discussed in the light of biophysical and computational studies, inorganic chemistry and X-ray crystallographic information.     

EPR evidence for an acceptor functioning in photosystem II when the pheophytin acceptor is reduced

Photosystem II particles have been poised at redox potentials where the pheophytin acceptor is reduced. Illumination of these particles at 200K results in the formation of radical signal in the g?2.00 region. This is attributed to the photoreduction of another acceptor. This acceptor may function between the primary donor, P₆₈₀, and pheophytin in forward electron transfer.     

Photoconsumption of oxygen in photosystem II preparations under impairment of the water-oxidizing complex

Oxygen consumption in photosystem II (PSII) preparations in the light was 2 mol O₂/h per mg Chl at weakly acidic and at neutral pH values. It increased fourfold to fivefold at pH 8.5-9.0. The addition of either artificial electron donors for PSII such as MnCl₂ or diphenylcarbazide, or diuron as an inhibitor of electron transfer from Q_A, the primary bound quinone acceptor, to Q_B, the secondary bound quinone acceptor of PSII, resulted in a decrease in oxygen consumption rate at basic pH to value close to ones measured at pH 6.5. Such additions did not affect oxygen consumption at lower pH values. The induction of variable chlorophyll fluorescence yield in the light differed greatly at pH 6.5 and 8.5. While at pH 6.5 the fluorescence yield, after an initial fast rise almost to F_max, only slightly decreased, at pH 8.5 after such a rise it dropped promptly to a low value. The additions of the artificial electron donors at pH 8.5 resulted in the induction kinetics close to that observed at pH 6.5. These data indicate impairment of electron donation to P680⁺ that could be caused by damage to the water oxidation system at basic pH values. In experiments with PSII preparations treated with Tris to destroy the water-oxidizing complex, photoconsumption of oxygen in the entire pH region was close to the values in untreated preparations at basic pH. In untreated preparations the rate of light-induced oxygen consumption decreased in the presence of catalase, which decomposes H₂O₂, as well as in the presence of electron acceptor potassium ferricyanide. From these data it is suggested that the light-induced oxygen consumption in PSII is caused by two processes, by an interaction of O₂ with organic radicals, which were formed due to oxidation of components of the donor side of this photosystem (proteins, lipids, pigments) by cation-radical P680⁺, as well as by oxygen reduction by still unidentified components of PSII.     

AtCYP38 ensures early biogenesis, correct assembly and sustenance of photosystem II

AtCYP38 is a thylakoid lumen protein comprising the immunophilin domain and the phosphatase inhibitor module. Here we show the association of AtCYP38 with the photosystem II (PSII) monomer complex and address its functional role using AtCYP38-deficient mutants. The dynamic greening process of etiolated leaves failed in the absence of AtCYP38, due to specific problems in the biogenesis of PSII complexes. Also the development of leaves under short-day conditions was severely disturbed. Detailed biophysical and biochemical analysis of mature AtCYP38-deficient plants from favorable growth conditions (long photoperiod) revealed: (i) intrinsic malfunction of PSII, which (ii) occurred on the donor side of PSII and (iii) was dependent on growing light intensity. AtCYP38 mutant plants also showed decreased accumulation of PSII, which was shown not to originate from impaired D1 synthesis or assembly of PSII monomers, dimers and supercomplexes as such but rather from the incorrect fine-tuning of the oxygen-evolving side of PSII. This, in turn, rendered PSII centers extremely susceptible to photoinhibition. AtCYP38 deficiency also drastically decreased the in vivo phosphorylation of PSII core proteins, probably related to the absence of the AtCYP38 phosphatase inhibitor domain. It is proposed that during PSII assembly AtCYP38 protein guides the proper folding of D1 (and CP43) into PSII, thereby enabling the correct assembly of the water-splitting Mn₄–Ca cluster even with high turnover of PSII.     

EPR studies on the photosystem II donor side in salt-washed and reconstituted inside-out thylakoids

EPR measurements on inside-out thylakoids revealed that salt-washing, known to inhibit oxygen evolution and release a 23 and a 16 kDa protein, induced a Signal II_f and decreased the EPR signal from state S₂. Readdition of the released 23 kDa protein restored the oxygen evolution and decreased the Signal II_f, but did not relieve the decrease in the state S₂ signal. It is suggested that salt-washing inhibits the electron transfer from the oxygen-evolving site to Z, the physiological donor to P680. In inhibited photosystem II units lacking Signal II_f, Z⁺ is rapidly reduced, possibly by a modified S-cycle unable to evolve oxygen.     

Strong-light photoinhibition treatment accelerates the changes of protein secondary structures in triton-treated photosystem I and photosystem II complexes

Changes in the protein secondary structure and electron transport activity of the Triton X-100-treated photosystem I (PSI) and photosystem II (PSII) complexes after strong illumination treatment were studied using Fourier transform-infrared (FT-IR) spectroscopy and an oxygen electrode. Short periods of photoinhibitory treatment led to obvious decreases in the rates of PSI-mediated electron transport activity and PSII-mediated oxygen evolution in the native or Triton-treated PSI and PSII complexes. In the native PSI and PSII complexes, the protein secondary structures had little changes after the photoinhibitory treatment. However, in both Triton-treated PSI and PSII complexes, short photoinhibition times caused significant loss of -helical content and increase of -sheet structure, similar to the conformational changes in samples of Triton-treated PSI and PSII complexes after long periods of dark incubation. Our results demonstrate that strong-light treatment to the Triton-treated PSI and PSII complexes accelerates destruction of the transmembrane structure of proteins in the two photosynthetic membranes.     

Polypeptides of photosystem II and their role in oxygen evolution

The linear, four-step oxidation of water to molecular oxygen by photosystem II requires cooperation between redox reactions driven by light and a set of redox reactions involving the S-states within the oxygen-evolving complex. The oxygenevolving complex is a highly ordered structure in which a number of polypeptides interact with one another to provide the appropriate environment for productive binding of cofactors such as manganese, chloride and calcium, as well as for productive electron transfer within the photoact. A number of recent advances in the knowledge of the polypeptide structure of photosystem II has revealed a correlation between primary photochemical events and a core complex of five hydrophobic polypeptides which provide binding sites for chlorophyll a, pheophytin a, the reaction center chlorophyll (P680), and its immediate donor, denoted Z. Although the core complex of photosystem II is photochemically active, it does not possess the capacity to evolve oxygen. A second set of polypeptides, which are water-soluble, have been discovered to be associated with photosystem II; these polypeptides are now proposed to be the structural elements of a special domain which promotes the activities of the loosely-bound cofactors (manganese, chloride, calcium) that participate in oxygen evolution activity. Two of these proteins (whose molecular weights are 23 and 17 kDa) can be released from photosystem II without concurrent loss of functional manganese; studies on these proteins and on the membranes from which they have been removed indicate that the 23 and 17 kDa species from part of the structure which promotes retention of chloride and calcium within the oxygen-evolving complex. A third water-soluble polypeptide of molecular weight 33 kDa is held to the photosystem II core complex by a series of forces which in some circumstances may include ligation to manganese. The 33 kDa protein has been studied in some detail and appears to promote the formation of the environment which is required for optimal participation by manganese in the oxygen evolving reaction. This minireview describes the polypeptides of photosystem II, places an emphasis on the current state of knowledge concerning these species, and discusses current areas of uncertainty concerning these important polypeptides.Abbreviations A 23187 ionophore that exchanges divalent cations with H⁺ - Chl chlorophyll - cyt cytochrome - DCPIP dichlorophenolindophenol - DPC diphenylcarbazide - EGTA ethyleneglycoltetraacetic acid - P680 the chlorophyll a reaction center of photosystem II - pheo pheophytin - PQ plastoquinone - PS photosystem - Q_A and Q_B primary and secondary plastoquinone electron acceptors of photosystem II - Sn (n=0, 1, 2, 3, 4) charge accumulating state of the oxygen evolving system - Signals II_vf, II_f and II_s epr-detectable free radicals associated with the oxidizing side of photosystem II - Z primary electron donor to the photosystem II reaction center The survey of literature for this review ended in September, 1984.     

The appearance of quenching centres in photosystem II

The variable fluorescence quenching found in the presence of DCMU with isolated chloroplasts which have been exposed previously to a prolonged low light intensity (Sinclair and Spence 1988), is accompanied by a loss of the sigmoidal appearance of the fluorescence induction transient. About 80% of the fluorescence decrease is due to the PS II units and 50% of the centres are inactivated by light exposure. Light incubation slows the PS II partial reaction while the PS I partial reaction is unaffected. We propose that in the light, normal PS II centres change into quenching centres which degrade excitation energy to thermal energy. This change can be reversed by 30 min of darkness. A higher flash intensity is needed to saturate the steady state O₂ flash yield from light-incubated chloroplasts indicating a light-induced decrease of the average photosynthetic unit size as would happen if PS II units were preferentially inactivated. These light-induced changes may relate to an adaptation in leaves to increasing light intensity.Abbreviations Chl Chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCPIP 2,6-Dichlorophenol-Indophenol - EDTA ethylaminediaminetetraacetic acid - F_v Level of variable fluorescence emission - F_o Initial level of fluorescence - Hepes buffer N-[2-Hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]