A comparison of electron transport and photophosphorylation systems of Rhodopseudomonas capsulata and Rhodospirillum rubrum

The photophosphorylation systems of Rhodopseudomonas capsulata and Rhodospirillum rubrum chromatophores have been compared in respect to the effects of artificial electron carries [N-methyl-phenazonium methosulfate (PMS) and diaminodurene], reducing agents (ascorbate in particular), and various quinones in the absence and presence of the electron transport inhibitors antimycin A and dibromothymoquinone (DBMIB). In addition, the effects of both inhibitors on photosynthetic electron transport through cytochromes b and c has been followed. From the results obtained, it appears that in both organisms: a) ubiquinone functions as an electron carrier between the cytochromes, and b) both antimycin A and DBMIB inhibit cyclic electron flow in the segment ... cytochrome bubiquinone»cytochrome c ..., but at different sites. The systems apparently differ mainly in respect to the nature of the electron flow by-pass shunt that is evoked in the presence of PMS; thus, in R. rubrum, PMS catalyzes a shunt that by-passes both cytochrome b and ubiquinone, whereas in Rps. capsulata the PMS shunt seems to circumvent only ubiquinone.Abbreviations BChl bacteriochlorophyll - DAD diaminodurene=2,3,5,6-tetramethyl-p-phenylenediamine - DBMIB dibromothymoquinone=2,5-dibromo-6-isopropyl-3-methylbenzoquinone - HOQNO heptylhydroxyquinoline-N-oxide - PMS N-methylphenazonium methosulfate     

Light-induced electron transport pathways in membrane preparations from Rhodopseudomonas capsulata

The photosynthetic electron transport chain in Rhodopseudomonas capsulata cells was investigated by studying light-induced noncyclic electron transport from external donors to O₂. Two membrane preparations with opposite membrane polarity, heavy chromatophores and regular chromatophores, were used to characterize this electron transport. It was shown that with lipophylic electron donors such as dichloroindophenol, diaminobenzidine, and phenazine methosulfate the electron transport activities were similar in both types of chromatophores, whereas horse heart cytochrome c, K₄Fe(CN)₆, 3-sulfonic acid phenazine methosulfate, and ascorbate, which cannot penetrate the membrane, were more active in the heavy chromatophores than in the regular chromatophores. Partial depletion of cytochrome c₂ from the heavy chromatophores caused a decrease in the light-induced O₂ uptake from reduced dichloroindophenol or ascorbate. The activity could be restored with higher concentrations of dichloroindophenol or with purified cytochrome c₂ from Rps. capsulata. It is assumed that in the heavy chromatophores the artificial electron donors are oxidized on the cytochrome c₂ level which faces the outside medium. However, cytochrome c₂ is not exposed to the outside medium in the regular chromatophores. Therefore, only lipophylic donors would interact with cytochrome c₂ in this system, while hydrophylic donors would be oxidized by another component of the electron transport chain which is exposed to the external medium. Studies with inhibitors of photophosphorylation show that antimycin A enhances the light-dependent electron transport to O₂ whereas 1:10 phenanthroline inhibited the reaction, but dibromothymoquinone did not affect it. It is assumed that a nonheme iron protein is taking part in this electron transport but not a dibromothymoquinone-sensitive quinone. The terminal oxidase of the light-dependent pathway is different from the two oxidases of the respiratory chain. The ratio between electrons entering the system and molecules of O₂ consumed is 4, which means that the end product of O₂ reduction is H₂O.     

The effect of dibromothymoquinone on respiratory and photosynthetic electron transport in Rhodopseudomonas capsulata chromatophores

Dibromothymoquinone has been shown to inhibit light-induced cytochrome b reduction, and oxidation of succinate and NADH by chromatophores of Rhodopseudomonas capsulata. The half-inhibitory concentration of light-induced reactions and NADH oxidation is 2.5 M, but of succinate oxidation is 16 M. Hexane extraction inhibited oxidation of NADH and succinate equally. The results are interpreted to suggest that ubiquinone is concerned in all three processes described, but that the pools associated with NADH and succinate oxidation are not equally accessible to dibromothymoquinone.Abbreviations DBMIB Dibromothymoquinone - NADH Reduced nicotinamide adenine dinucleotide - Bchl Bacteriochlorophyll     

Nucleotide exchange in membrane vesicles from the photosynthetic bacterium Rhodopseudomonas capsulata

Membrane vesicles (heavy chromatophores) prepared from the photosynthetic bacteria Rhodopseudomonas capsulata catalyze photophosphorylation of exogenous ADP and also take up [³H]ADP from the external medium. The rate of uptake depends on the concentration of external ADP reaching half-maximal velocity at 2.7 mm. The rate increases also with the increase in the concentration of internal ADP. Vesicles, preloaded with [³H]ADP release the radioactive nucleotide when ADP is included in the external medium. Regular chromatophores, which are inside-out membrane vesicles also take up [³H]ADP from the external medium when preloaded with ADP. These results are interpreted to indicate the existence of nucleotide transport across the cytoplasmic membrane of these bacteria which is catalyzed by an ADP exchange carrier.     

Resolved difference spectra of redox centers involved in photosynthetic electron flow in Rhodopseudomonas capsulata and Rhodopseudomonas sphaeroides

1. In Rhodopseudomonas sphaeroides the Qx absorption band of the reaction center bacteriochlorophyll dimer which bleaches on photo-oxidation is both blue-shifted and has an increased extinction coefficient on solubilisation of the chromatophore membrane with lauryldimethylamine-N-oxide. These effects may be attributable in part to the particle flattening effect. 2. The difference spectrum of photo-oxidisable c type cytochrome in the chromatophore was found to have a slightly variable peak position in the alpha-band (lambda max at 551--551.25 nm); this position was always red-shifted in comparison to that of isolated cytochrome c2 (lambda max at 549.5 +/- 0.5 nm). The shift in wavelength maximum was not due to association with the reaction center protein. A possible heterogeneity in the c-type cytochromes of chromatophores is discussed. 3. Flash-induced difference spectra attributed to cytochrome b were resolved at several different redox potentials and in the presence and absence of antimycin. Under most conditions, one major component, cytochrome b50 appeared to be involved. However, in some circumstances, reduction of a component with the spectral characteristics of cytochrome b-90 was observed. 4. Difference spectra attributed to (BChl)2, (Formula: see text), c type cytochrome and cytochrome b50 were resolved in the Soret region for Rhodopseudomonas capsulata. 5. A computer-linked kinetic spectrophotometer for obtaining automatically the difference spectra of components functioning in photosynthetic electron transfer chains is described. The system incorporates a novel method for automatically adjusting and holding the photomultiplier supply voltage.     

Divalent cation transport systems of Rhodopseudomonas capsulata.

Separate divalent cation transport systems for energy-dependent uptake of Mg2+ and Mn2+ were found both with aerobically and heterotrophically grown and with photosynthetically grown cells of Rhodopseudomonas capsulata. The maximum rate of Mg2+ uptake differed between photosynthetic and aerobic cells, while the Km for the Mg2+ transport system was constant. Photosynthetic midlog-phase cells exhibited Km's for uptake of about 55 micrometer Mg2+ and 0.5 micrometer Mn2+. The Vmax's also differed between the two systems: 0.6 to 1.8 mumol/min per g (dry weight) of cells for Mg2+, but only 0.020 mumol/min per g for Mn2+, making the distinction between a "macro-requirement" system and a system functioning at trace nutrient levels. Calcium was not normally taken up by intact cells of R. capsulata. However, chromatophore membranes isolated from photosynthetic cells took up Ca2+ by an energy-dependent process.     

Potassium transport system of Rhodopseudomonas capsulata

Rhodopseudomonas capsulata required potassium (or rubidium or cesium as analogs of potassium) for growth. These cations were actively accumulated by the cells by a process following Michaelis-Menten saturation kinetics. The monovalent cation transport system had Km's of 0.2 mM K+, 0.5 mM Rb+, and 2.6 mM Cs+. The rates of uptake of substrates by the potassium transport system varied with the age of the culture, although the affinity constant for the substrates remained constant. The maximal velocity of uptake of K+ was lower in aerobically grown cells than in photosynthetically grown cells, although the Km's for K+ and for Rb+ were about the same.     

Phospholipid composition of photosynthetic membranes of Rhodopseudomonas capsulata

The phospholipids and the fatty acids present in membranes of cells of Rhodopseudomonas capsulata, grown photosynthetically in anaerobiosis, were analyzed by thin layer chromatography and gas chromatography-mass spectrometry. The three phospholipids detected, phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol, contained about 80% of a single monounsaturated C18 fatty acid, cis-vaccenic acid. These membranes offer therefore a naturally occurring model system endowed with an extremely simplified phospholipid complement. The data indicate moreover that the biosynthetic pathway of unsaturated fatty acids present in these facultative aerobic bacteria proceeds only via the 3-hydroxydecanoyl acyl carrier protein dehydratase (E.C. 4.2.1.60).     

Cyclic photophosphorylation by chromatophores of the facultative phototroph,Rhodopseudomonas capsulata

Summary Cyclic photophosphorylation catalyzed by chromatophores derived from the facultative phototroph, Rhodopseudomonas capsulata was investigated. In the absence of an external electron donor such as succinate, cyclic photophosphorylation is strongly inhibited by O₂. Maximal phosphorylation rates are obtained in the presence of molecular hydrogen. Cytochrome c and bovine serum albumin have no significant effects on the reaction. However, dichlorophenolindophenol and phenazonium methosulfate are inhibitory to cyclic photophosphorylation. Cyclic photophosphorylation is sensitive to antimycin A, but highly resistant to heptylhydroxy-quinoline-N-oxide. Neither phenazonium methosulfate, nor dichlorophenolindophenol or tetramethyl-p-phenylenediamine can effect antimycin-insensitive cyclic photophosphorylation. Oligomycin strongly inhibits the phosphorylation. Overreduction caused by the ascorbate-dichlorophenolindophenol couple results in strong inhibition of phosphorylation. Addition of fumarate decreases the inhibition caused by overreduction. However, the fumarate mediated phosphorylation is nearly completely inhibited by antimycin A. Atebrine is a strong inhibitor for cyclic photophosphorylation, whereas dinitrophenol is only a weak inhibitor.

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Zusammenfassung Die durch Chromatophoren aus dem fakultativ phototrophen Rhodopseudomonas capsulata katalysierte cyclische Photophosphorylierung wurde untersucht. In der Abwesenheit eines zusätzlichen Elektronendonators wie Succinat wird die cyclische Photophosphorylierung durch O₂ stark gehemmt. Maximale Phosphorylierungsraten werden unter H₂-Atmosphäre erzielt. Cytochrom c und Rinderserumalbumin haben keinen deutlichen Effekt auf die Reaktion. Demgegenüber haben Dichlorphenolindophenol und Phenazinmethosulfat eine hemmende Wirkung auf die cyclische Photophosphorylierung. Die cyclische Photophosphorylierung wird durch Antimycin A stark gehemmt, ist aber gegenüber Heptyl-hydroxy-chinolin-N-oxyd auffallend resistent. Weder Phenazinmethosulfat noch Dichlorphenolindophenol oder Tetramethyl-p-phenylendiamin bewirken eine antimycin-resistente Phosphorylierung. Oligomycin hemmt die Photophosphorylierung stark. Eine durch Ascorbat-Dichlorphenolindophenol verursachte Überreduktion wirkt sich stark hemmend auf die Phosphorylierung aus. In Gegenwart von Fumarat ist die durch Überreduktion bedingte Hemmung stark verringert. Diese vom Fumarat abhängige Photophosphorylierung wird jedoch durch Antimycin A beinahe vollständig gehemmt. Atebrin ist ein starker Hemmstoff für die cyclische Photophosphorylierung. Demgegenüber ist die durch Dinitrophenol bewirkte Hemmung der cyclischen Photophosphorylierung gering.

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Abbreviations ADP adenosine diphosphate - ATP adenosine triphosphate - BChl bacteriochlorophyll - DNP 2,4-dinitrophenol - DCPIP dichlorophenolindophenol - FAD flavinadenine dinucleotide - FMN flavin mononucleotide - G-6-P glucose-6-phosphate - HOQNO heptylhydroxy-quinoline-N-oxide - NAD(P) nicotinamid-adenine-dinucleotide (phosphate) - PMS phenazonium methosulfate - Rh. Rhodospirillum - Rhps. Rhodopseudomonas - TMPD tetramethyl-p-phenylenediamine     

Influence of thylakoid protein phosphorylation on photosynthetic electron transport and photophosphorylation

Data are reported which show that thylakoid protein phosphorylation decreases photosystem II fluorescence yield and enhances the photosystem I dependent photophosphorylation catalyzed by phenazinemethosulphate in the presence of DCMU. The stimulation is larger at low light intensity, but is still observed at high intensity. These observations are interpreted to demonstrate that thylakoid protein phosphorylation causes a transfer of excitation energy from PS II to PS I, but may also have an independent stimulatory effect on PS I dependent photophosphorylation.     

Inorganic nitrogen assimilation by the photosynthetic bacterium Rhodopseudomonas capsulata.

The photosynthetic bacterium Rhodopseudomonas capsulata lacks glutamate dehydrogenase and normally uses the glutamine synthetase/glutamate synthase sequence of reactions for assimilation of N2 and ammonia. The glutamine synthetase in cell-free extracts of the organism is completely sedimented by centrifugation at 140,000 X g for 2 h, is inhibited by L-alanine but not by adenosine 5'-monophosphate, and exhibits two apparent Km values for ammonia (ca. 13 muM and 1 mM).     

Occurence of thioredoxin m activity in the photosynthetic bacteria Rhodopseudomonas capsulata.

Thioredoxin capable of activating NADP-Malate dehydrogenase from Spinach has been isolated from a cell free extract of Rhodopseudomonas capsulata. These activating factors, approximately 12,000 Daltons in molecular weight, were partially purified after thermal treatment by using gel filtration, ion-exchange and adsorption chromatographic techniques. The occurence of thioredoxin f activity, though more difficult to obtain, was also evidenced in these bacteria. The present results have been tentatively discussed by a comparison between the thioredoxinspresent in higher plants and bacterial fields.     

Asymmetry of an energy transducing membrane the location of cytochrome c2 in Rhodopseudomonas spheroides and Rhodopseudomonas capsulata.

Monospecific antibodies have been prepared against cytochrome c2 from Rhodopseudomonas spheroides and Rhodopseudomonas capsulata, and against cytochrome c' from Rps. capsulata. These antibodies precipitated their respective antigens, but did not cross react with a wide range of procaryotic or eucaryotic cytochromes, or with other bacterial proteins. The cytochromes produced during aerobic growth were immunologically indistinguishable from those produced during photosynthetic growth. Cytochrome c2 is located in vivo in the periplasmic space between the cell was and the cell membrane, and when chromatophores are prepared from whole cells the cytochrome becomes trapped inside these vesicles. The implications of these results to energy coupling in the photosynthetic bacteria are discussed.     

H2 metabolism in the photosynthetic bacterium Rhodopseudomonas capsulata: H2 production by growing cultures.

Purple photosynthetic bacteria produce H2 from organic compounds by an anaerobic light-dependent electron transfer process in which nitrogenase functions as the terminal catalyst. It has been established that the H2-evolving function of nitrogenase is inhibited by N2 and ammonium salts, and is maximally expressed in cells growing photoheterotrophically with certain amino acids as sources of nitrogen. In the present studies with Rhodopseudomonas capsulata, nutritional factors affecting the rate and magnitude of H2 photoproduction in cultures growing with amino acid nitrogen sources were examined. The highest H2 yields and rates of formation were observed with the organic acids: lactate, pyruvate, malate, and succinate in media containing glutamate as the N source; under optimal conditions with excess lactate, H2 was produced at rates of ca. 130 ml/h per g(dry weight) of cells. Hydrogen production is significantly influenced by the N/C ratio in the growth substrates; when this ratio exceeds a critical value, free ammonia appears in the medium and H2 is not evolved. In the "standard" lactate + glutamate system, both H2 production and growth are "saturated" at a light intesity of ca. 600 ft-c (6,500 lux). Evolution of H2, however, occurs during growth at lithe intensities as low as 50 to 100 ft-c (540 to 1,080 lux), i.e., under conditions of energy limitation. In circumstances in which energy conversion rate and supplies of reducing power exceed the capacity of the biosynthetic machinery, energy-dependent H2 production presumably represents a regulatory device that facilitates "energy-idling." It appears that even when light intensity (energy) is limiting, a significant fraction of the available reducing power and adenosine 5'-triphosphate is diverted to nitrogenase, resulting in H2 formation and a bioenergetic burden to the cell.