Lipids in oxygen-evolving photosystem II complexes of cyanobacteria and higher plants

Lipids in dimeric photosystem II complexes prepared from two species of cyanobacteria, Thermosynechococcus vulcanus and Synechocystis sp. PCC6803, and two higher plants, spinach and rice, were analyzed to determine how many lipid molecules and what class of lipids are present in the photosystem II complexes. It was estimated that 27, 20, 8, and 7 lipid molecules per monomer are bound to the dimeric photosystem II complexes of T. vulcanus, Synechocystis, spinach, and rice, respectively. In each of the organisms, the lipid composition of the photosystem II complexes was quite different from that of the thylakoid membranes used for preparation of the complexes. The content of phosphatidylglycerol in the photosystem II complexes of each organism was much higher than that in the thylakoid membranes. Phospholipase A2 treatment of the photosystem II complexes of Synechocystis that degraded phosphatidylglycerol resulted in impairment of QB-mediated but not QA-mediated electron transport. These findings suggest that phosphatidylglycerol plays important roles in the electron transport at the QB-binding site in photosystem II complexes.     

Study on the effects of chloride depletion on photosystem II using different chloride depletion methods

Chloride is an indispensable factor for the functioning of oxygen evolving complex (OEC) and has protective and activating effects on photosystem II. In this study we have investigated mainly by EPR, the properties of chloride-sufficient, chloride-deficient and chloride-depleted thylakoid membranes and photosystem II enriched membranes from spinach. The results on the effects of different chloride depletion methods on the structural and functional aspects of photosystem II showed that chloride-depletion by treating PS II membranes with high pH is a relatively harsh way causing a significant and irreparable damage to the PS II donor side. Damage to the acceptor side of PS II was recovered almost fully in chloride-deficient as well as chloride-depleted PS II membranes.     

Multiple types of association of photosystem II and its light-harvesting antenna in partially solubilized photosystem II membranes

Photosystem II is a multisubunit pigment-protein complex embedded in the thylakoid membranes of chloroplasts. It utilizes light for photochemical energy conversion, and is heavily involved in the regulation of the energy flow. We investigated the structural organization of photosystem II and its associated light-harvesting antenna by electron microscopy, multivariate statistical analysis, and classification procedures on partially solubilized photosystem II membranes from spinach. Observation by electron microscopy shortly after a mild disruption of freshly prepared membranes with the detergent n-dodecyl-alpha,D-maltoside revealed the presence of several large supramolecular complexes. In addition to the previously reported supercomplexes [Boekema, E. J., van Roon, H., and Dekker, J. P. (1998) FEBS Lett. 424, 95-99], we observed complexes with the major trimeric chlorophyll a/b protein (LHCII) in a third, L-type of binding position (C2S2M0-2L1-2), and two different types of megacomplexes, both identified as dimeric associations of supercomplexes with LHCII in two types of binding sites (C4S4M2-4). We conclude that the association of photosystem II and its associated light-harvesting antenna is intrinsically heterogeneous, and that the minor CP26 and CP24 proteins play a crucial role in the supramolecular organization of the complete photosystem. We suggest that different types of organization form the structural basis for photosystem II to specifically react to changing light and stress conditions, by providing different routes of excitation energy transfer.     

N,N-diethylhydroxylamine: a new electron donor to photosystem II

Diethylhydroxylamine, when added to beet spinach thylakoid membranes in the reaction mixture enhanced both photosystem II mediated dichlorophenolindophenol photoreduction and whole chain electron transport supported by methyl viologen. Diethylhydroxylamine supports dichlorophenolindophenol photoreduction when oxygen evolving complex is inactivated by hydroxylamine washings. All the electron transport assays were found to be highly sensitive to diuron, indicating that diethylhydroxylamine donates electrons to the photosystem II before the herbicide binding site. The stimulation of the photochemical activity by diethylhydroxylamine is not solely due to its action as an uncoupler. It was also observed that the action of diethylhydroxylamine was not altered by preincubations of thylakoids in light in the presence of diethylhydroxylamine. Also, thylakoid membranes did not lose their benzoquinone Hill activity by the pre-incubations with diethylhydroxylamine either in light or in dark. Thus, unlike the photosystem II electron donor, hydroxylamine, diethylhydroxylamine was found to donate electrons without the inactivations of oxygen evolving complex. It is suggested that diethylhydroxylamine is a useful electron donor to the photosystem II.     

The interaction of thylakoid lipids with everted photosystem II membranes

Spinach ( Spinacia oleracea L. cv. Viking II) thylakoid membranes enriched in photosystem II were disrupted by incubation at 35°C for 5 min followed by sonication. This treatment caused the loss of manganese and the release of extrinsic photosystem II proteins of 16, 23 and 33 kdaltons, resulting in retention of only 12–18% of the oxygen-evolving activity. Prior addition of exogenous digalactosyldiacylglycerol to the membranes afforded protection against the release of manganese and extrinsic proteins and enabled the retention of up to 47% of the activity. Monogalactosyldiacylglycerol was less effective in both respects and enabled retention of only 30% of the activity. Addition of charged thylakoid lipids had a severely damaging effect on photosystem II.     

Heptameric association of light-harvesting complex II trimers in partially solubilized photosystem II membranes.

We report a structural characterization by electron microscopy and image analysis of a supramolecular complex consisting of seven trimeric light-harvesting complex II proteins. The complex was readily observed in partially-solubilized Tris-washed photosystem II membranes from spinach but was also found to occur, with a low frequency, in oxygen-evolving photosystem II membranes. The structure reveals six peripheral trimers with the same rotational orientation and a central trimer with the opposite orientation. We conclude that the heptamer represents a naturally occurring aggregation state of part of the light-harvesting complex II trimers in the thylakoid membranes.     

Mobilization of photosystem II induced by intense red light in the Cyanobacterium Synechococcus sp PCC7942

We use confocal fluorescence microscopy and fluorescence recovery after photobleaching to show that a specific light signal controls the diffusion of a protein complex in thylakoid membranes of the cyanobacterium Synechococcus sp PCC7942 in vivo. In low light, photosystem II appears completely immobile in the membrane. However, exposure to intense red light triggers rapid diffusion of up to approximately 50% of photosystem II reaction centers. Particularly intense or prolonged red light exposure also leads to the redistribution of photosystem II to specific zones within the thylakoid membranes. The mobilization does not result from photodamage but is triggered by a specific red light signal. We show that mobilization of photosystem II is required for the rapid initiation of recovery from photoinhibition. Thus, intense red light triggers a switch from a static to a dynamic configuration of thylakoid membrane protein complexes, and this facilitates the rapid turnover and repair of the complexes. The localized concentrations of photosystem II seen after red light treatment may correspond to specific zones where the repair cycle is active.     

Restoration of light induced photosystem II inhibition without de novo protein synthesis

Illumination of isolated spinach thylakoid membranes under anaerobic conditions gave rise to severe inhibition of photosystem II electron transport but did not result in D1-protein degradation. When these photoinhibited thylakoids were incubated in total darkness the photosystem II activity could be fully restored in vitro in a process that required 1-2 h for completion.     

Statcking and separatio of photosystem I and photosystem II in plant thylak oid membranes: A physico-chemical view

The main objective of this article is to highlight important experimental findings and discuss current theories on photosystem segregation and stacking in thylakoid membranes. The facts are put in a framework of recent theoretical developments in the field of membrane biochemistry and biophysics. Some important experiments not considered by the currently accepted theories are discussed. Modifications of these theories in order to incorporate new results are proposed. The currently accepted theories on formation of membrane domains (probably responsible for segregation of photosystem 1 and II) and on interlamellar interactions (stacking) are discussed. Finally, a scheme is put forward summarising the forces which are responsible for organisation of functional structure of thylakoid membranes.     

Interaction of a phenolic inhibitor with photosystem II particles

Photosystem II particles have been prepared from spinach and Chlamydomonas reinhardii CW 15 thylakoids. Photosynthetic electron transport in these particles is inhibited by phenolic compounds like dinoseb, but not by atrazine and diuron. The labeling patterns obtained by photoaffinity labels derived from either atrazine (azido-atrazine) or the phenolic herbicide dinoseb (azido-dinoseb) were compared in photosystem II particles and thylakoids. Whereas azido-atrazine in thylakoids of spinach as well as of Chlamydomonas labels a 32-kilodalton peptide, this label does not react in photosystem II particle preparations. Azido-dinoseb, however, labels both the thylakoid membranes and the particles, predominantly polypeptides in the 40-53 kilodalton molecular weight region. Since the latter polypeptides are probably part of the reaction center of photosystem II, it is suggested that phenolic compounds have their inhibition site within the reaction center complex. This indicates that the atrazine-binding 32-kilodalton peptide is either absent or functionally inactive in photosystem II particles, whereas the phenol inhibitor-binding peptides are not.     

Isolation and characterization of photosystem II subcomplexes from cyanobacteria lacking photosystem I.

A photosystem II (PSII) core complex lacking the internal antenna CP43 protein was isolated from the photosystem II of Synechocystis PCC6803, which lacks photosystem I (PSI). CP47-RC and reaction centre (RCII) complexes were also obtained in a single procedure by direct solubilization of whole thylakoid membranes. The CP47-RC subcore complex was characterized by SDS/PAGE, immunoblotting, MALDI MS, visible and fluorescence spectroscopy, and absorption detected magnetic resonance. The purity and functionality of RCII was also assayed. These preparations may be useful for mutational analysis of PSII RC and CP47-RC in studying primary reactions of oxygenic photosynthesis.     

Characterization of photosystem II mutants of Chlamydomonas reinhardii lacking the psbA gene

Summary We have examined 78 chloroplast mutants of Chlamydomonas reinhardii lacking photosystem II activity. Most of them are unable to synthesize the 32 Kdalton protein. Analysis of 22 of these mutants reveals that they have deleted both copies of the psbA gene (which codes for the 32 Kdalton protein) in their chloroplast genome. Although these mutants are able to synthesize and to integrate the other photosystem II polypeptides in the thylakoid membranes, they are unable to assemble a stable functional photosystem II complex. The 32 Kprotein appears therefore to play an important role not only in photosystem II function, but also in stabilizing this complex.     

A red-shifted antenna protein associated with photosystem II in Physcomitrella patens

Antenna systems of plants and green algae are made up of pigment-protein complexes belonging to the light-harvesting complex (LHC) multigene family. LHCs increase the light-harvesting cross-section of photosystems I and II and catalyze photoprotective reactions that prevent light-induced damage in an oxygenic environment. The genome of the moss Physcomitrella patens contains two genes encoding LHCb9, a new antenna protein that bears an overall sequence similarity to photosystem II antenna proteins but carries a specific motif typical of photosystem I antenna proteins. This consists of the presence of an asparagine residue as a ligand for Chl 603 (A5) chromophore rather than a histidine, the common ligand in all other LHCbs. Asparagine as a Chl 603 (A5) ligand generates red-shifted spectral forms associated with photosystem I rather than with photosystem II, suggesting that in P. patens, the energy landscape of photosystem II might be different with respect to that of most green algae and plants. In this work, we show that the in vitro refolded LHCb9-pigment complexes carry a red-shifted fluorescence emission peak, different from all other known photosystem II antenna proteins. By using a specific antibody, we localized LHCb9 within PSII supercomplexes in the thylakoid membranes. This is the first report of red-shifted spectral forms in a PSII antenna system, suggesting that this biophysical feature might have a special role either in optimization of light use efficiency or in photoprotection in the specific environmental conditions experienced by this moss.     

Light-dependent degradation of the D1 protein in photosystem II is accelerated after inhibition of the water splitting reaction

Strong illumination of oxygen-evolving organisms inhibits the electron transport through photosystem II (photoinhibition). In addition the illumination leads to a rapid turnover of the D1 protein in the reaction center of photosystem II. In this study the light-dependent degradation of the D1 reaction center protein and the light-dependent inhibition of electron-transport reactions have been studied in thylakoid membranes in which the oxygen evolution has been reversibly inhibited by Cl- depletion. The results show that Cl(-)-depleted thylakoid membranes are very vulnerable to damage induced by illumination. Both the D1 protein and the inhibition of the oxygen evolution are 15-20 times more sensitive to illumination than in control thylakoid membranes. The presence, during the illumination, of the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) prevented both the light-dependent degradation of the D1 protein and the inhibition of the electron transport. The protection exerted by DCMU is seen only in Cl(-)-depleted thylakoid membranes. These observations lead to the proposal that continuous illumination of Cl(-)-depleted thylakoid membranes generates anomalously long-lived, highly oxidizing radicals on the oxidizing side of photosystem II, which are responsible for the light-induced protein damage and inhibition. The presence of DCMU during the illumination prevents the formation of these radicals, which explains the protective effects of the herbicide. It is also observed that in Cl(-)-depleted thylakoid membranes, oxygen evolution (measured after the readdition of Cl-) is inhibited before electron transfer from diphenylcarbazide to dichlorophenolindophenol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Damage to photosystem II by lipid peroxidation products

BackgroundPhotosystem II proteins of higher plant chloroplasts are prone to oxidative stress, and most prominently the reaction center-binding D1 protein is damaged under abiotic stress. The reactive oxygen species produced under these stress conditions have been suggested to be responsible for the protein injury.Scope of reviewRecently, it has been shown that the primary and secondary products of non-enzymatic and enzymatic lipid peroxidation have a capability to modify photosystem II proteins. Here, we give an overview showing how lipid peroxidation products formed under light stress and heat stress in the thylakoid membranes cause oxidative modification of proteins in higher plant photosystem II.Major conclusionsDamage to photosystem II proteins by lipid peroxidation products represents a new mechanism underlying photoinhibition and heat inactivation.General significanceComplete characterization of photosystem II protein damage is of crucial importance because avoidance of the damage makes plants to survive under various abiotic stresses. Further physiological significance of photosystem II protein oxidation by lipid peroxidation product should have a potential relevance to plant acclimation because the oxidized proteins might serve as signaling molecules.     

Effect of high-light on photosynthetic apparatus with different content of anionic lipids and organization of light-harvesting complex of photosystem II

The effect of the high-light at 4 °C on isolated thylakoid membranes with different organization of light-harvesting complex of photosystem II (LHCII) and lipid composition was investigated. Data revealed that the decreased amount of anionic lipids, which is correlated with the increase in the oligomeric form of LHCII stabilized the photosystem II complex against low temperature photoinhibition.     

Polyphenolic allelochemicals from the aquatic angiosperm Myriophyllum spicatum inhibit photosystem II

Myriophyllum spicatum (Haloragaceae) is a highly competitive freshwater macrophyte that produces and releases algicidal and cyanobactericidal polyphenols. Among them, beta-1,2,3-tri-O-galloyl-4,6-(S)-hexahydroxydiphenoyl-D-glucose (tellimagrandin II) is the major active substance and is an effective inhibitor of microalgal exoenzymes. However, this mode of action does not fully explain the strong allelopathic activity observed in bioassays. Lipophilic extracts of M. spicatum inhibit photosynthetic oxygen evolution of intact cyanobacteria and other photoautotrophs. Fractionation of the extract provided evidence for tellimagrandin II as the active compound. Separate measurements of photosystem I and II activity with spinach (Spinacia oleracea) thylakoid membranes indicated that the site of inhibition is located at photosystem II (PSII). In thermoluminescence measurements with thylakoid membranes and PSII-enriched membrane fragments M. spicatum extracts shifted the maximum temperature of the B-band (S(2)Q(B)(-) recombination) to higher temperatures. Purified tellimagrandin II in concentrations as low as 3 microM caused a comparable shift of the B-band. This demonstrates that the target site of this inhibitor is different from the Q(B)-binding site, a common target of commercial herbicides like 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Measurements with electron paramagnetic resonance spectroscopy suggest a higher redox midpoint potential for the non-heme iron, located between the primary and the secondary quinone electron acceptors, Q(A) and Q(B). Thus, tellimagrandin II has at least two modes of action, inhibition of exoenzymes and inhibition of PSII. Multiple target sites are a common characteristic of many potent allelochemicals.     

Quality control of photosystem II: reactive oxygen species are responsible for the damage to photosystem II under moderate heat stress

Moderate heat stress (40 degrees C for 30 min) on spinach thylakoid membranes induced cleavage of the reaction center-binding D1 protein of photosystem II, aggregation of the D1 protein with the neighboring polypeptides D2 and CP43, and release of three extrinsic proteins, PsbO, -P, and -Q. These heat-induced events were suppressed under anaerobic conditions or by the addition of sodium ascorbate, a general scavenger of reactive oxygen species. In accordance with this, singlet oxygen and hydroxyl radicals were detected in spinach photosystem II membranes incubated at 40 degrees C for 30 min with electron paramagnetic resonance spin-trapping spectroscopy. The moderate heat stress also induced significant lipid peroxidation under aerobic conditions. We suggest that the reactive oxygen species are generated by heat-induced inactivation of a water-oxidizing manganese complex and through lipid peroxidation. Although occurring in the dark, the damages caused by the moderate heat stress to photosystem II are quite similar to those induced by excessive illumination where reactive oxygen species are involved.     

Identification of the pheophytin-QA-Fe domain of the reducing side of the photosystem II as the Cu(II)-inhibitory binding site.

Oxygen evolution by photosystem II membranes was inhibited by Cu(II) when 2,6-dichlorobenzoquinone or ferricyanide, but not silicomolybdate, was used as electron acceptor. This indicated that Cu(II) affected the reducing side of the photosystem II. The inhibition curves of Cu(II), o-phenanthroline and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), were compared; the inhibitory patterns of Cu(II) and o-phenanthroline were very similar and different in turn from that of DCMU. Cu(II) did not eliminate or modify the electron paramagnetic resonance signal at g = 8.1 ascribed to the non-heme iron of the photosystem II reaction center, indicating that the inhibition by Cu(II) was not the result of the replacement of the iron by Cu(II). Controlled trypsin digestion of thylakoid membranes inhibited oxygen evolution using 2,6-dichlorobenzoquinone, but had no effect when using ferricyanide or silicomolybdate. Using ferricyanide, oxygen evolution of trypsin-treated thylakoids was insensitive to DCMU but became even more sensitive to Cu(II) and o-phenanthroline than nontreated thylakoids; however, trypsinized thylakoids were insensitive to inhibitors in the presence of silicomolybdate. We conclude that Cu(II) impaired the photosystem II electron transfer before the QB niche, most probably at the pheophytin-QA-Fe domain.     

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