Interchangeability of phosphorylation coupling factors in photosynthetic and respiratory energy conversion.

The nonsulfur purple photosynthetic bacterium Rhodopseudomonas capsulata can obtain energy for growth either by anaerobic photophosphorylation or dark oxidative (aerobic) phosphorylation. Successful resolution of phosphorylation coupling factors from energy-converting membranes of this bacterium permitted tests for reciprocal function of such protein factors in oxidative-and photophosphorylation processes. Evidence was obtained for the interchangeability of coupling factor preparations from dark-grown and photosynthetically grown cells in both kinds of energy conversion.

Energy tranduction in photosynthetic bacteria. XI. Further resolution of cytochromes of b type and the nature of the co-sensitive oxidase present in the respiratory chain of Rhodopseudomonas capsulata.

1. In membranes prepared from dark grown cells of Rhodopseudomonas capsulata, five cytochromes of b type (E'0 at pH 7.0 +413+/-5, +270+/-5, +148+/-5, +56+/-5 and -32+/-5 mV) can be detected by redox titrations at different pH values. The midpoint potentials of only three of these cytochromes (b148, b56, and b-32) vary as a function of pH with a slope of 30 mV per pH unit. 2. In the presence of a CO/N2 mixture, the apparent E'0 of cytochrome b270 shifts markedly towards higher potentials (+355mV); a similar but less pronounced shift is apparent also for cytochrome b150. The effect of CO on the midpoint potential of cytochrome b270 is absent in the respiration deficient mutant M6 which possesses a specific lesion in the CO-sensitive segment of the branched respiratory chain present in the wild type strain. 3. Preparations of spheroplasts with lysozyme digestion lead to the release of a large amount of cytochrome c2 and of virtually all cytochrome cc'. These preparations show a respiratory chain impaired in the electron pathway sensitive to low KCN concentration, in agreement with the proposed role of cytochrome c2 in this branch; on the contrary, the activity of the CO-sensitive branch remains unaffected, indicating that neither cytochrome c2 nor the CO-binding cytochrome cc' are involved in this pathway. 4. Membranes prepared from spheroplasts still possess a CO-binding pigment characterized by maxima at 420.5, 543 and 574 nm and minima at 431, 560 nm in C0-difference spectra and with an alpha band at 562.5 nm in reduced minus oxidized difference spectra. This membrane-bound cytochrome, which is coincident with cytochrome b270, can be classified as a typical cytochrome "0" and considered the alternative CO-sensitive oxidase.

Energy transduction in photosynthetic bacteria. X. Composition and function of the branched oxidase system in wild type and respiration deficient mutants of Rhodopseudomonas capsulata.

The respiratory chain of Rhodopseudomonas capsulata, strain St. Louis and of two respiration deficient mutants (M6 and M7) has been investigated by examining the redox and spectral characteristics of the cytochromes and their response to substrates and to specific respiratory inhibitors. Since the specific lesions of M6 and M7 have been localized on two different branches of the multiple oxidase system of the wild type strain, the capability for aerobic growth of these mutants can be considered as a proof of the physiological significance of both branched systems "in vivo". Using M6 and M7 mutants the response of the branched chain to respiratory inhibitors could be established. Cytochrome oxidase activity, a specific function of an high potential cytochrome b (E'0 = +413 mV) is sensitive to low concentrations of KCN (5-10(-5) M); CO is a specific inhibitor of an alternative oxidase, which is also inhibited by high concentrations of KCN (10(-3) M). Antimycin A inhibits preferentially the branch of the chain affected by low concentrations of cyanide. Redox titrations and spectral data indicate the presence in the membrane of three cytochromes of b type (E'0 = +413, +260, +47 vM) and two cytochromes of c type (E'0 = +342, +94 mV). A clear indication of the involvement in respiration of cytochrome b413, cytochrome c342 and cytochrome b47 has been obtained. Only 50% of the dithionite reducible cytochrome b can be reduced by respiratory substrates also in the presence of high concentrations of KCN or in anaerobiosis. The presence and function of quinones in the respiratory electron transport system has been clearly demonstrated. Quinones, which are reducible by NADH and succinate to about the same extent can be reoxidized through both branches of the respiratory chain, as shown by the response of their redox state to KCN. The possible site of the branching of the electron transport chain has been investigated comparing the per cent level of reduction of quinones and of cytochromes b and c as a function of KCN concentrations in membranes from wild type and M6 mutants cells. The site of the branching has been localized at the level of quinones-cytochrome b47. A tentative scheme of the respiratory chains operating in Rhodopseudomonas capsulata, St. Louis and in the two respiration deficient mutants, M6 and M7 is presented.

The induction kinetics of bacterial photophosphorylation. Threshold effects by the phosphate potential and correlation with the amplitude of the carotenoid absorption band shift.

1. ATP synthesis (monitored by luciferin-luciferase) can be elicited by a single turnover flash of saturating intensity in chromatophores from Rhodopseudomonas capsulata, Kb1. The ATP yield from the first to the fourth turnover is strongly influenced by the phosphate potential: at high phosphate potential (-11.5 kcal/mol) no ATP is formed in the first three turnovers while at lower phosphate potential (-8.2 kcal/mol) and the yield in the first flash is already one half of the maximum, which is reached after 2-3 turnovers. 2. The response to ionophores indicates that the driving force for ATP synthesis in the first 20 turnovers is mainly given by a membrane potential. The amplitude of the carotenoid band shift shows that during a train of flashes an increasing delta psi is built up, which reaches a stationary level after a few turnovers; at high phosphate potential, therefore, more turnovers of the same photosynthetic unit are required to overcome an energetic threshold. 3. After several (six to seven) flashes the ATP yield becomes constant, independently from the phosphate potential; the yield varies, however, as a function of dark time (td) between flashes, with an optimum for td = 160-320 ms. 4. The decay kinetics of the high energy state generated by a long (125 ms) flash have been studied directly measuring the ATP yield produced in post-illumination by one single turnover flash, under conditions of phosphate potential (-10 kcal/mol), which will not allow ATP formation by one single turnover. The high energy state decays within 20 s after the illumination. The decay rate is strongly accelerated by 10(-8) M valinomycin. 5. Under all the experimental conditions described, the amplitude of the carotenoid signal correlates univocally with the ATP yield per flash, demonstrating that this signal monitores accurately an energetic state of the membrane directly involved in ATP synthesis. 6. Although values of the carotenoid signal much larger than the minimal threshold are present, relax slowly, and contribute to the energy input for phosphorylation, no ATP is formed unless electron flow is induced by a single turnover flash. 7. The conclusions drawn are independent from the assumption that a delta psi between bulk phases is evaluable from the carotenoid signal.

Photosynthetic control and estimation of the optimal ATP: electron stoichiometry during flash activation of chromatophores from Rhodopseudomonas capsulata.

(1) When chromatophores from Rhodopseudomonas capsulata Ala pho+ are exposed to a train of high-frequency, saturating flashes the kinetics of the reaction centre bacteriochlorophyll absorption change enter a pseudo steady-state in which the extent of oxidation during the flashes is equal to the extent of reduction in between the flashes. The level of the pseudo steady-state is lowered by the presence of a phosphate acceptor system, raised by further addition of oligomycin, lowered by a combination of nigericin and valinomycin and raised by antimycin A. (2) In the pseudo steady-state, the extent of reaction centre bacteriochlorophyll oxidation taking place during the flash may be estimated by subtraction from the total concentration of reaction centre bacteriochlorophyll. This value is equated with the amount of electrons transported through the photosynthetic chain. Comparison with the measured ATP yield per flash in the pseudo steady-state permits calculation of the ATP: two electron ratio. The value of the ratio is 1.1 for flash frequencies between 3 and 12.5 Hz and declines at lower and higher frequencies. The ATP: two electron ratio is approximately halved in the presence of antimycin A. (3) An alternative estimate of the ATP: two electron ratio, based on the assumption that high-frequency flashes approximate to the condition of continuous illumination, was approx. 0.8.

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.

The stimulation of photophosphorylation and ATPase by artificial redox mediators in chromatophores of Rhodopseudomonas capsulata at different redox potentials.

(1) Inhibition of cyclic phosphorylation in chromatophores of Rhodopseudomonas capsulata by antimycin A can be fully reversed by artificial redox mediators, provided the ambient redox potential is maintained around 200 mV. The redox mediator need not be a hydrogen carrier in its reduced form, N-methyl-phenazonium methosulfate and N,N,N',N'-tetramethyl-p-phenylenediamine being equally effective. However, the mediator needs to be lipophilic. Endogenous cyclic phosphorylation is fastest around 130 mV. A shift to 200 mV can also be observed if high concentrations of artificial redox mediator are present in the absence of antimycin. (2) ATPase activity of Rhodopseudomonas capsulata, in the light as well as in the dark, activated or not activated by inorganic phosphate, can also be stimulated by N-methylphenazonium methosulfate. This stimulation is highest at redox potentials between 60 to 80 mV and is sensitive to antimycin A. In this case N,N,N',N-tetramethyl-p-phenylenediamine is much less effective.

The reconstitution of oxidative phosphorylation in mitochondria isolated from a ubiquinone-deficient mutant of Saccharomyces cerevisiae.

Mitochondria, isolated from the ubiquinone-deficient nuclear mutant of Saccharomyces cerevisiae E3-24, are practically unable to oxidize exogenous substrates. Respiratory activity, coupled to ATP synthesis, can, however, be reconstituted by the simple addition of ethanolic solutions of ubiquinones. A minimal length of the isoprenoid side chain (greater than or equal to 3) was required for the restoration. Saturation of the reconstitution required a large amount of exogeneous ubiquinone, in excess over the normal content present in the mitochondria of the wild type strain. A similar pattern of reconstituted activities could be also obtained using sonicated inverted particles. Mitochondria and sonicated particles are also able to carry out a dye-mediated electron flow coupled to ATP synthesis in the absence of added ubiquinone, using ascorbate or succinate as electron donor. This demonstrates that the energy conserving mechanism at the third coupling site of the respiratory chain is fully independent of the presence of the large mobile pool of ubiquinone in the membrane.

Structural requirements of quinone coenzymes for endogenous and dye-mediated coupled electron transport in bacterial photosynthesis.

Electron transport in continuous light has been investigated in chromatophores of Rhodopseudomonas capsulata. Ala pho+, depleted in ubiquinone-10 and subsequently reconstituted with various ubiquinone homologs and analogs. In addition the restoration of electron transport in depleted chromatophores by the artificial redox compounds N-methylphenazonium methosulfate and N,N,N',N'-tetramethyl-p-phenylenediamine was studied. The following pattern of activities was obtained: (1) Reconstitution of cyclic photophosphorylation with ubiquinone-10 was saturated at about 40 ubiquinone molecules per reaction center. (2) Reconstitution by ubiquinone homologs was dependent on the length of the isoprenoid side chain and the amount of residual ubiquinone in the extracted chromatophores. If two or more molecules of ubiquinone-10 per reaction center were retained, all homologs with a side chain longer than two isoprene units were as active as ubiquinone-10 in reconstitution, and the double bonds in the side chain were not required. If less than two molecules per reaction center remained, an unsaturated side chain longer than five units was necessary for full activity. Plastoquinone, alpha-tocopherol, and naphthoquinones of the vitamin K series were relatively inactive in both cases. (3) All ubiquinone homologs, also ubiquinone-1 and -2, could be reduced equally well by the photosynthetic reaction center, as measured by light-induced proton binding in the presence of antimycin A and uncoupler. Plastoquinone was found to be a poor electron acceptor. (4) Photophosphorylation could be reconstituted by N-methylphenazonium methosulfate as well as by N,N,N',N'-tetramethyl-p-phenylenediamine in an antimycin-insensitive way, if more than two ubiquinones per reaction center remained. These compounds were active also in more extensively extracted particles reconstituted with ubiquinone-1, which itself was inactive.

Role of phosphorylation coupling factor in light-dependent proton translocation by Rhodopseudomonas capsulata membrane preparations.

The activity of the light-dependent proton pump (in the absence of phosphorylation substrates) of Rhodopseudomonas capsulata "membrane vesicles," in contrast to that of chloroplasts, is not appreciably affected by detachment of phosphorylation coupling factor(s). Proton uptake by such "uncoupled" (low phosphorylation activity) preparations is also unaffected by the addition of phosphorylation substrates (ADP + arsenate + Mg(2+)). The H(+) pump of "coupled" preparations, however, is stimulated when all of the substrates are present simultaneously. Oligomycin appears to affect both photophosphorylation and the H(+) pump by interaction with a component of the energy transfer sequence that is distinct from coupling factor. The results obtained suggest that in photosynthetic bacteria light-dependent cyclic electron flow (the driving force for photophosphorylation) is accelerated by active phosphorylation which, in turn, is dependent on the presence of protein coupling factors in the energy-converting membrane.