A. Baccarini-Melandri

Cytochrome c2 — an electron carrier shared by the respiratory and photosynthetic electron transport chain of Rhodopseudomonas capsulata

The function of cytochrome c2 as the primary electron donor to the reaction center in photosynthetic electron transport of Rhodospirillaceae has been clearly established [ 1,2]. However, its role in the respiratory pathways on which these bacteria depend under heterotrophic growth conditions has been a matter of debate since many years [3-81. The study of respiration in these organisms is often complicated by the presence of branched electron transport chains [7,9,19] and by possible regulatory phenomena in the activity of the terminal oxidases dependent upon physical parameters of growth, such as oxygen concentration and light intensity [ 11 ,121. The Rhodopseudomonas capsulata cytochrome c2 is thought to function in only one of the branches as electron donor for a cytochrome b-type oxidase (cyt. b& [6,13]. This suggestion rests o,n experimental evidence, such as the observed redox changes of cytochrome c2 upon addition of antimycin A or during transition to anaerobiosis, and the lack of reoxidation of cytochrome c2 in membranes from a respiration-deficient mutant (M7) in which cytochrome beI is absent [6]. In this communication we present evidence for a role of cytochrome c2 in respiration of Rps. capsulata; definite proof has been obtained by an immunological ahproach taking into account both the vectorial orientation of the membrane and the complexity of the respiratory chain. These two difficulties have been overcome using spheroplast preparations of a respiratory mutant (M6) endowed with a linear respiratory chain where cytochrome c oxidase (cyt. b,& is the only terminal enzyme present [6,10]. The data demonstrate conclusively the role of this cytochrome in the respiratory as well as in the photosynthetic chain of Rps. capsulata.

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.

d-Glyceraldehyde-3-phosphate dehydrogenase in photosynthetic cells: I. The reversible light-induced activation in vivo of NADP-dependent enzyme and its relationship to NAD-dependent activities

1. 1. The possibility of the existence in photosynthetic tissues of a single d-glyceraldehyde-3-phosphate dehydrogenase enzyme, active with NAD and NADP, was investigated. By (NH2)4SO4 precipitation a fraction active only with NAD and containing 40% of the total units was isolated from another fraction active with both coenzymes. Repetitive salt fractionation of this latter fraction failed to achieve further resolution of the two activities. 2. 2. The lack of additivity of NAD- and NADP-dependent activities, always verified in pea leaves crude extracts, supports the idea that at least one glyceraldehyde-3-P dehydrogenase non-specific with respect to pyridine nucleotides is present in leaves. 3. 3. The reversible light-induced activation in vivo of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase was clearly shown to be independent from net protein synthesis. 4. 4. The kinetic parameters of this activation phenomenon were examined both for the oxidative and reductive reaction. The activation was found to correspond to a reversible increase of the υmax of the NADP- and NADPH-dependent activities, expressed on a protein basis. Neither the υmax of NAD+ and NADH linked activities, nor any of the apparent Km values of substrates are significantly affected.

Energy transduction in photosynthetic bacteria. The nature of cytochrome C oxidase in the respiratory chain of rhodopseudomonas capsulata

In a previous paper [l] it has been demonstrated that in dark grown cells of Rhodopseudomonas capsulata, respiration proceeds to oxygen through a branched chain, in which only one arm contains the site of inhibition by antimycin A and the enzymes involved in the oxidation of exogenous cytochrome c or of the ascorbate-diaminodurene couple. These conclusions were based mainly on ‘in vitro’ experiments which showed a lower sensitivity to KCN inhibition of NADHor succinate-oxidase activities (half inhibition at 10e4 M and 5 . lo-’ M) compared with that of cyt. c or ascorbate-DAD oxidase (both showing half inhibition at 2 . lo-’ M). The possibility of a branched chain was proposed at the same time also by Marrs and Gest [2] on the basis of the growth characteristics of several respiratory mutants and of the activities present in membranes prepared therefrom. One of these mutant strains, denominated M7, appears to be of particular interest since it lacks completely cyt. c oxidase, although it is able to grow heterotrophically on malate. The results presented in this paper elucidate the nature of the lesion present in this mutant and allow

Energy transduction in photosynthetic bacteria: IV. Light-dependent ATPase in photosynthetic membranes from Rhodopseudomonas capsulata

Abstract ATPase activity of photosynthetic membrane fragments from the bacterium Rhodopseudomonas capsulata can be stimulated by continuous illumination under conditions of active cyclic electron flow. The activation corresponds to an increase in the maximum velocity of the reaction and does not affect the apparent K m for ATP (0.11 mM). No stimulation in the light is observed in the presence of classical uncouplers or oxidized 2,6-dichlorophenolindophenol (DCIP), which, per se , stimulate ATPase in the dark. It is demonstrated, however, that oxidized DCIP acts as an uncoupler of bacterial photophosphorylation. The effect of light is elicited after a few minutes of preillumination, or in a much shorter time if an ADP trapping system is supplied. Activation does not occur if ADP is added during the preillumination (apparent K m for inhibition by ADP = 1 μ M). The effect of ADP is not related to competitive inhibition with ATP, which can be observed at higher concentrations (apparent K i = 0.26 mM). ADP, however, is not effective if added after some minutes of preillumination.

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 ofRhodopseudomonas 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 compoundsN-methylphenazonium methosulfate andN,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, α-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 byN-methylphenazonium methosulfate as well as byN,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.

Energy transduction in photosynthetic bacteria V. Role of coupling factor ATPase in energy conversion as revealed by light or ATP-induced quenching of atebrine fluorescence.

quenching of atebrine fluorescence has been shown to change with changes in the energetic state of phosphorylating membrane systems [l-3] . More recently it has been suggested that this phenomenon is related to the distribution of atebrine across the membrane, in response to the formation of a trans- membrane pH gradient [4], and that the mechanism of atebrine uptake is similar to that previously pro- posed for uptake of other amines by illuminated chloroplasts [5]. In recent work on chromatophores from

The Stimulation of Photophosphorylation and ATPase by Artificial Redox Mediators in Chromatophores of Rhodopseudomonas capsulata at Different Redox Potentials

Abstract(1) Inhibition of cyclic phosphorylation in chromatophores ofRhodopseudomonas 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 A. (2) ATPase activity ofRhodopseudomonas 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.

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.

Energy transduction in photosynthetic bacteria III. Coincidence of coupling factor of photosynthesis and respiration inRhodopseudomonas capsulata

Recent work in our laboratory has dealt with the enzyme catalyzing ATP synthesis in membranes of facultative photosynthetic bacteria (specifically Rhodopseudomonas capsulata). The aim pursued by these studies, beyond the mere extension of previous researches on coupling factor proteins to prokaryotic photosynthetic organisms, was a comparison of the mechanism of photosynthetic and oxidative phosphorylation. In fact non-sulfur purple bacteria offer a very suitable model system for this purpose, since membrane preparations from these microorganisms can carry out either cyclic photophosphorylation or oxidative phosphorylation at rates depending on the conditions of growth (i.e. photosynthetic or aerobic). Previously we reported about the reversible resolution of the photophosphorylating system [l] , the purification and properties of a coupling factor protein from photosynthetic membranes [2] and its involvement in energy conservation [3]. Subsequently we have described a similar resolution of the respiratory system and the successful restoration of oxidative phosphorylation by coupling factor prepared from photosynthetic cells [4]. These results led us to suggest that in non-sulfur purple bacteria functional interchangeability between coupling factors of photosynthesis and respiration exists. These conclusions were also supported [4] by the demonstration that an antibody against photosynthetic coupling factor could inhibit indifferently photophosphorylation in pigmented membranes and oxidative phosphorylation in aerobic membranes. In this paper we present de-