Role of sulfoquinovosyl diacylglycerol for the maintenance of photosystem II in Chlamydomonas reinhardtii.

The physiological role of sulfoquinovosyl diacylglycerol (SQDG) in photosynthesis was investigated with a SQDG defective mutant (hf-2) of Chlamydomonas reinhardtii that did not have any detectable amount of SQDG. The mutant showed a lower rate of photosystem II (PSII) activity by approximately 40% and also a lower growth rate than those of the wild-type. Results of genetical analysis of hf-2 strongly suggest that the SQDG defect and the lowered PSII activity are due to a single gene mutation. The supplementation of SQDG to hf-2 cells restored the lowered PSII activity to the same level as that of wild-type cells, and also enabled the mutant to grow even in the presence of 135 nm 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Moreover, the incubation of isolated thylakoid membranes of hf-2 with SQDG raised the lowered PSII activity. Chemical modifications of SQDG impaired the recovery of PSII activity. The results suggest that SQDG is indispensable for PSII activity in Chlamydomonas by maintaining PSII complexes in their proper state.     

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.     

Structural studies on photosystem II of cyanobacteria

Photosynthesis is one of the most important chemical processes in the biosphere responsible for the maintenance of life on Earth. Light energy is converted into energy of chemical bonds in photoreaction centers, which, in particular, include photosystem II (PS II). PS II is a multisubunit pigment-protein complex located in the thylakoid membrane of cyanobacteria, algae and plants. PS II realizes the first stage of solar energy conversion that results in decomposition of water to molecular oxygen, protons, and bound electrons via a series of consecutive reactions. During recent years, considerable progress has been achieved in determination of the spatial structures of PS II from various cyanobacteria. In the present review, we outline the current state of crystallographic studies on PS II.     

Studies on the mechanism of photosystem II photoinhibition II. The involvement of toxic oxygen species

In a previous paper it was shown that photoinhibition of reaction centre II of spinach thylakoids was predominantly caused by the degradation of D1-protein. An initial inactivation step at the Q_B-site was distinguished from its breakdown. The present paper deals with the question as to whether this loss of Q_B-function is caused by oxygen radical attack. For this purpose the photoinhibition of thylakoids was induced at 20°C in the presence of either superoxide dismutase and catalase or the antioxidants glutathione and ascorbic acid. This resulted in comparable though not total protection of D1-protein, photochemistry and fluorescence from photoinhibition. The combined action of both the enzymatic and the non-enzymatic radical scavenging systems brought about an even more pronounced protective effect against photoinhibition than did either of the two systems singularly at saturating concentrations. The results signify a major contribution of activated oxygen species to the degradation process of D1-protein and the related phenomena of photoinhibition. Thylakoids treated with hydroxyl radicals generated through a Fenton reaction showed a loss of atrazine binding sites, electron transport capacity and variable fluorescence in a similar manner, though not to the same extent, as usually observed following photoinhibitory treatment.Abbreviations Asc ascorbate - Fecy ferricyanide - GSH reduced glutathione - PQ plastoquinone - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II - SOD superoxide dismutase     

Phycoerythrins of marine unicellular cyanobacteria. III. Sequence of a class II phycoerythrin

The genes for the alpha and beta subunits of a novel six bilin-bearing (class II) phycoerythrin were cloned from Synechococcus sp. WH8020 and sequenced. The cloned genes (mpeA and mpeB) were detected by homology with the genes for C-phycoerythrin from Pseudanabaena sp. PCC7409. The mpe locus occurs once in the genome and is arranged similarly to that of many other phycobiliproteins, with mpeA shortly 3' of mpeB. Sequence comparison suggests that this phycoerythrin (and perhaps all class II phycoerythrins) occupy a branch of the phycoerythrin family separate from five-chromophore per alpha beta (class I) phycoerythrins, C-phycoerythrin, and B-phycoerythrin. The position of the sixth chromophore of the class II phycoerythrin of WH8020 was determined by comparison of the amino acid sequence of the chromopeptides (Ong, L. J., and Glazer, A. N. (1991) J. Biol Chem. 266, 9515-9527) with the sequence deduced from the gene. This located the chromophore at residue 75 of the alpha subunit, very close to the alpha-83 chromophore in the primary structure and, presumably, in the three-dimensional structure.     

The orange carotenoid protein in photoprotection of photosystem II in cyanobacteria

Photoprotective mechanisms have evolved in photosynthetic organisms to cope with fluctuating light conditions. Under high irradiance, the production of dangerous oxygen species is stimulated and causes photo-oxidative stress. One of these photoprotective mechanisms, non photochemical quenching (qE), decreases the excess absorbed energy arriving at the reaction centers by increasing thermal dissipation at the level of the antenna. In this review we describe results leading to the discovery of this process in cyanobacteria (qE(cya)), which is mechanistically distinct from its counterpart in plants, and recent progress in the elucidation of this mechanism. The cyanobacterial photoactive soluble orange carotenoid protein is essential for the triggering of this photoprotective mechanism. Light induces structural changes in the carotenoid and the protein leading to the formation of a red active form. The activated red form interacts with the phycobilisome, the cyanobacterial light-harvesting antenna, and induces a decrease of the phycobilisome fluorescence emission and of the energy arriving to the reaction centers. The orange carotenoid protein is the first photoactive protein to be identified that contains a carotenoid as the chromophore. Moreover, its photocycle is completely different from those of other photoactive proteins. A second protein, called the Fluorescence Recovery Protein encoded by the slr1964 gene in Synechocystis PCC 6803, plays a key role in dislodging the red orange carotenoid protein from the phycobilisome and in the conversion of the free red orange carotenoid protein to the orange, inactive, form. This protein is essential to recover the full antenna capacity under low light conditions after exposure to high irradiance. This article is part of a Special Issue entitled: Photosystem II.     

Analysis of photosystem II using particle II preparation. I. Experimental evidence supporting the idea of the involvement of two light reactions in photosystem II of green plant photosynthesis

(1) To analyze the photoelectron flow related to photosystemII, particle II preparation, i.e., the chloroplast fragmenthaving only photosystem II activity, proved to be far betterthan the generally used chloroplast preparations having activitiesof both PS-I and PS-II. (2) By simultaneous measurements ofthe activities of O₂ evolution and DPIP- and ferricyanide photoreductionusing variously-treated particle II preparations, it was foundthat a noticeable activity of ferricyanide photoreduction wasstill observed, though the former two activities were completelylost in the course of treatments such as Tris-treatment, pre-illuminationand aging. (3) Besides this, differences were found betweenferricyanide- and DPIP-photoreduction in respect to susceptibilityto CCCP, availability of artificial electron donor, and theeffect of chloride addition. However, both photo-reductionswere equally inactivated by heat-treatment and addition of DCMU.(4) To explain the observed distinctions between DPIP and ferricyanidein their mode of action as electron acceptor for PS-II, a schemesuggesting the involvement of two light reactions in PS-II isproposed and the electron flow near PS-II is discussed. ¹ This work has been supported by Grants from the Ministry ofEducation (Nos. 8425- 70-'71; 4970l4-'69-'71), which are gratefullyacknowledged here. (Received January 12, 1972; )     

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.     

Phycoerythrins of marine unicellular cyanobacteria. II. Characterization of phycobiliproteins with unusually high phycourobilin content

A survey of marine unicellular cyanobacterial strains for phycobiliproteins with high phycourobilin (PUB) content led to a detailed investigation of Synechocystis sp. WH8501. The phycobiliproteins of this strain were purified and characterized with respect to their bilin composition and attachment sites. Amino-terminal sequences were determined for the alpha and beta subunits of the phycocyanin and the major and minor phycoerythrins. The amino acid sequences around the attachment sites of all bilin prosthetic groups of the phycocyanin and of the minor phycoerythrin were also determined. The phycocyanin from this strain carries a single PUB on the alpha subunit and two phycocyanobilins on the beta subunit. It is the only phycocyanin known to carry a PUB chromophore. The native protein, isolated in the (alpha beta)2 aggregation state, displays absorption maxima at 490 and 592 nm. Excitation at 470 nm, absorbed almost exclusively by PUB, leads to emission at 644 nm from phycocyanobilin. The major and minor phycoerythrins from strain WH8501 each carry five bilins per alpha beta unit, four PUBs and one phycoerythrobilin. Spectroscopic properties determine that the PUB groups function as energy donors to the sole phycoerythrobilin. Analysis of the bilin peptides unambiguously identifies the phycoerythrobilin at position beta-82 (residue numbering assigned by homology with B-phycoerythrin; Sidler, W., Kumpf, B., Suter, F., Klotz, A. V., Glazer, A. N., and Zuber, H. (1989) Biol. Chem. Hoppe-Seyler 370, 115-124) as the terminal energy acceptor in phycoerythrins.     

Production of reactive oxygen species in chloride- and calcium-depleted photosystem II and their involvement in photoinhibition

Mixed photosystem II (PSII) samples consisting of Cl(-)-depleted and active, or Ca(2+)-depleted and active PSII enriched membrane fragments, respectively, were investigated with respect to their susceptibility to light. In the presence of Cl(-)-depleted PSII, active centers were damaged more severely, most likely caused by a higher amount of reactive oxygen species formed in the nonfunctional centers. Cl(-) depletion led to an increased H(2)O(2) production, which seemed to be responsible for the stimulation of PSII activity loss. To distinguish between direct H(2)O(2) formation by partial water oxidation and indirect H(2)O(2) formation by oxygen reduction involving the prior formation of O(2)(-?), the production of reactive oxygen species was followed by spin trapping EPR spectroscopy. All samples investigated, i.e. PSII with a functional water splitting complex, Ca(2+)- and Cl(-)-depleted PSII, produced upon illumination O(2)(-?) and OH(?) radicals on the acceptor side, while Cl(-)-depleted PSII produced additionally OH(?) radicals originating from H(2)O(2) formed on the donor side of PSII.     

Sulfide inhibition of photosystem II in cyanobacteria (blue-green algae) and tobacco chloroplasts.

The present study shows that in the presence of 600 nm light, sulfide acts as a specific inhibitor of photosynthetic electron transport between water and Photosystem II in the cyanobacteria Aphanothece halophytica and Synechococcus 6311 as well as in tobacco chloroplasts. In the presence of 600 nm light sulfied affects the fast fluorescence transients as does a low concentration (10 mM) of hydroxylamine; the fluorescence yield decreases in the presence of either chemical and can be restored by the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. In chloroplasts, however, NH2OH, an electron donor at high concentrations (40 mM), relieves the sulfide effect. In the dark, sulfide affects the cyanobacterial fluorescence transients through decrease of oxygen tension. The fluorescence yield increases in a similar pattern to that observed under nitrogen flushing. Upon omission of sulfide in A. halophytica, the characteristic aerobic fluorescence transients return, consistent with the ease of alternation between oxygenic and sulfide-dependent anoxygenic photosynthesis in many cyanobacteria.     

Covalently closed circular DNAs in closely related unicellular cyanobacteria.

Plasmids of Synechococcus cedrorum and two Anacytsis nidulans strains were characterized physically, and a probable instance of spontaneous "curing" is described.     

Differing responses of the two forms of photosystem II carbonic anhydrase to chloride, cations, and pH

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

Differing responses of the two forms of photosystem II carbonic anhydrase to chloride, cations, and pH

The effects of Cl(-), Mn(2+), Ca(2+), and pH on extrinsic and intrinsic photosystem II carbonic anhydrase activity were compared. Under the conditions of our in vitro experiments, extrinsic CA activity, located on the OEC33 protein, was optimum at about 30 mM Cl(-), and strongly inhibited above this concentration. This enzyme is activated by Mn(2+) and stimulated somewhat by Ca(2+). The OEC33 showed dehydration activity that is optimum at pH 6 or below. In contrast, intrinsic CA activity found in the PSII complex after removal of extrinsic proteins was stimulated by Cl(-) up to 0.4 M. Ca(2+) appears to be the required cofactor, which implies that the location of the intrinsic CA activity is in the immediate vicinity of the CaMn(4) complex. Up to now, intrinsic CA has shown only hydration activity that is nearly pH independent.     

Oxygen and the light–dark cycle of nitrogenase activity in two unicellular cyanobacteria

Cyanobacteria capable of fixing dinitrogen exhibit various strategies to protect nitrogenase from inactivation by oxygen. The marine Crocosphaera watsonii WH8501 and the terrestrial Gloeothece sp. PCC6909 are unicellular diazotrophic cyanobacteria that are capable of aerobic nitrogen fixation. These cyanobacteria separate the incompatible processes of oxygenic photosynthesis and nitrogen fixation temporally, confining the latter to the dark. Although these cyanobacteria thrive in fully aerobic environments and can be cultivated diazotrophically under aerobic conditions, the effect of oxygen is not precisely known due to methodological limitations. Here we report the characteristics of nitrogenase activity with respect to well‐defined levels of oxygen to which the organisms are exposed, using an online and near real‐time acetylene reduction assay combined with sensitive laser‐based photoacoustic ethylene detection. The cultures were grown under an alternating 12–12 h light–dark cycle and acetylene reduction was recorded continuously. Acetylene reduction was assayed at 20%, 15%, 10%, 7.5%, 5% and 0% oxygen and at photon flux densities of 30 and 76 μmol m^?2 s^?1 provided at the same light–dark cycle as during cultivation. Nitrogenase activity was predominantly but not exclusively confined to the dark. At 0% oxygen nitrogenase activity in Gloeothece sp. was not detected during the dark and was shifted completely to the light period, while C. watsonii did not exhibit nitrogenase activity at all. Oxygen concentrations of 15% and higher did not support nitrogenase activity in either of the two cyanobacteria. The highest nitrogenase activities were at 5–7.5% oxygen. The highest nitrogenase activities in C. watsonii and Gloeothece sp. were observed at 29°C. At 31°C and above, nitrogenase activity was not detected in C. watsonii while the same was the case at 41°C and above in Gloeothece sp. The differences in the behaviour of nitrogenase activity in these cyanobacteria are discussed with respect to their presumed physiological strategies to protect nitrogenase from oxygen inactivation and to the environment in which they thrive.     

Organization of transmembrane helices in photosystem II: comparison of plants and cyanobacteria

Electron microscopy and X-ray crystallography are revealing the structure of photosystem II. Electron crystallography has yielded a 3D structure at sufficient resolution to identify subunit positioning and transmembrane organization of the reaction-centre core complex of spinach. Single-particle analyses are providing 3D structures of photosystem II-light-harvesting complex II supercomplexes that can be used to incorporate high-resolution structural data emerging from electron and X-ray crystallography. The positions of the chlorins and metal centres within photosystem II are now available. It can be concluded that photosystem II is a dimeric complex with the transmembrane helices of CP47/D2 proteins related to those of the CP43/D1 proteins by a twofold axis within each monomer. Further, both electron microscopy and X-ray analyses show that P(680) is not a 'special pair' and that cytochrome b559 is located on the D2 side of the reaction centres some distance from P(680). However, although comparison of the electron microscopy and X-ray models for spinach and Synechococcus elongatus show considerable similarities, there seem to be differences in the number and positioning of some small subunits.     

Hydrogen metabolism of three unicellular nitrogen-fixing cyanobacteria

Abstract The enzyme activities responsible for the evolution and consumption of hydrogen in three unicellular cyanobacteria were investigated. Gloeothece sp. 6909 and Cyanothece sp. 7822 performed an oxygen-tolerant nitrogen fixation, whereas the nitrogenase activity of Synechococcus sp. 7425 was much more sensitive to oxygen. While in Gloeothece the net hydrogen production during nitrogen fixation was relatively low due to recycling by an uptake hydrogenase, little hydrogen consumption was detected in Cyanothece and Synechococcu . On the other hand a reversible hydrogenase was demonstrated in the latter strains. However, only Cyanothece shows hydrogenase-catalysed hydrogen production in vivo under anaerobic conditions in the dark. It is suggested that hydrogen is a fermentation product, and that the physiological function of this reversible hydrogenase is the removal of excess reduction equivalents under such conditions.     

Isolation of the carotenoid-containing cell wall of three unicellular cyanobacteria.

A carotenoid-containing membrane fraction devoid of chlorophyll and phycobiliproteins was isolated from three unicellular cyanobacteria, Synechococcus sp., Synechococcus leopoliensis UTEX 625, and Anacystis nidulans R-2, by aqueous-phase separation, hydrophobic chromatography, and differential centrifugation. The presence of 2-keto-3-deoxyoctonate, muramic acid, and diaminopimelic acid suggests that the preparation is highly enriched in cell wall. Electron micrographs of thin sections of this material showed C-shaped membrane profiles similar to those seen in other gram-negative cell wall preparations. The inactivation of cyanophage AS-1 by this fraction confirmed its identity as cell wall. The cell wall contained approximately equal weights of total carbohydrate and protein. Absorption maxima at 434, 452, and 488 nm indicated the presence of carotenoids. These were in the outer membrane and were not due to contaminating cytoplasmic or thylakoid membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the preparations showed a broad band of approximately 50,000 molecular weight which contained 35% of the total outer membrane protein. This band was resolved into at least two components running at approximately 50,000 and 52,000 molecular weight. The smaller of these polypeptides was a glycoprotein. The polypeptide components were unaffected by protease or detergent treatment in either whole cells or isolated cell wall preparations, indicating that the polypeptide components were not exposed to the surface or easily removed from the hydrophobic environment.     

Variation in sulfide tolerance of photosystem II in phylogenetically diverse cyanobacteria from sulfidic habitats

Physiological and molecular phylogenetic approaches were used to investigate variation among 12 cyanobacterial strains in their tolerance of sulfide, an inhibitor of oxygenic photosynthesis. Cyanobacteria from sulfidic habitats were found to be phylogenetically diverse and exhibited an approximately 50-fold variation in photosystem II performance in the presence of sulfide. Whereas the degree of tolerance was positively correlated with sulfide levels in the environment, a strain's phenotype could not be predicted from the tolerance of its closest relatives. These observations suggest that sulfide tolerance is a dynamic trait primarily shaped by environmental variation. Despite differences in absolute tolerance, similarities among strains in the effects of sulfide on chlorophyll fluorescence induction indicated a common mode of toxicity. Based on similarities with treatments known to disrupt the oxygen-evolving complex, it was concluded that sulfide toxicity resulted from inhibition of the donor side of photosystem II.