Bruno Andrea Melandri

Energy transduction in photosynthetic bacteria VI. Respiratory sites of energy conservation in membranes from dark-grown cells of Rhodopseudomonas capsulata

Abstract Membranes prepared from Rhodopseudomonas capsulata grown heterotrophically in the dark perform phosphorylation linked to oxidation of NADH and succinate, with P 2e − ratios of about 0.5 and 0.15, respectively. The localization of the sites of energy conservation was investigated by observing the respiration-induced quenching of the fluorescence of atebrine. Energization of the membrane can be demonstrated when NADH is oxidized by O2, ferricyanide or Q1, when succinate is oxidized by O2 or by oxidized diaminodurene, and during the oxidation of reduced diaminodurene. Antimycin A completely inhibits energization between succinate and O2 or succinate and diaminodurene; however, it only inhibits partially NADH or succinate oxidases and energization between NADH and O2. KCN inhibits NADH oxidase in a biphasic way: the first level of inhibition is observed at concentrations which block the oxidation of exogenous cytochrome c or of diaminodurene and energization between succinate or ascorbate-diaminodurene and O2. The second level corresponds to the inhibition of the antimycin-insensitive oxidase. The results are interpreted as evidence of the presence in these bacteria of a respiratory chain branching after the dehydrogenase system, one arm of the chain being sensitive to antimycin A and low concentrations of KCN and capable of energy conservation, the other being represented by a completely uncoupled system.

[50] Partial resolution of the photophosphorylating system of Rhodopseudomonas capsulata

Publisher Summary Pigmented membrane particles prepared from the facultative phototroph Rhodopseudomonas capsulata catalyze light-induced phosphorylation of ADP coupled to cyclic electron transport. This system can be resolved by sonication, in the presence of EDTA, into two components: nonphotophosphorylating particles and a protein factor present in the sonic extract fluid (or in chromatophore acetonic powder) that can restore completely the photophosphorylating activity of the resolved particles. Rhodopseudomonas capsulata is grown anaerobically in a synthetic medium (initial pH 6.8) containing 0.4% DL-malic acid, 0.1% (NH 4 ) 2 SO 4 , 0.0001% thiamine hydrochloride, and additional inorganic salts. The medium is inoculated with about a 1% (v/v) inoculum of a culture in early stationary phase. Cultures are incubated at 34 ° C and illuminated with banks of 60-W tubular incandescent lamps (light intensity 500 foot candles). This chapter describes the procedure for the purification of the coupling factor. To achieve maximum recoupling, a 20-minute contact time is required between nonphosphorylating particles and coupling factor in the presence of Mg 2+ . The preincubation is performed in the dark at the temperature of the assay (usually 30 ° C) and in the presence of the reagents and buffer required, except the ones used to start the reaction.

The localized coupling of bacterial photophosphorylation: Effect of antimycin a and N,N-dicyclohexylcarbodiimide) in chromatophores from Rhodopseudomonas sphaeroides, Ga, studied by single turnover event analysis

Abstract 1. The inhibition by antimycin A of the cyclic electron transfer has been studied in chromatophores from Rhodopseudomonas sphaeroides Ga following an approach based on the analysis of the relaxation kinetics of the reaction center optical changes in pulsed light. The recovery kinetics of the bacteriochlorophyll redox state have been found to be clearly biphasic. The half-times of the fast phase (13 ms) and slow phase (about 400 ms) were not modified by antimycin in a range of concentrations from 0.1 to 9 μM. On the other hand the percentage extent of the fast phase, which reflects the rate of the cyclic electron transfer, was monotonically decreased by increasing concentrations of the inhibitor. This indicates that antimycin decreases progressively the fraction of the photosynthetic units, active in cyclic electron transfer. 2. The ATP yield per flash observed under conditions of controlled inhibition of electron flow was strongly dependent upon the amount of active redox cycles. On the other hand, the amplitude of the carotenoid band shift, which has been demonstrated unequivocally to be correlated to the ATP yield per flash in uninhibited chromatophores, was not affected by antimycin up to a 40% inhibition of electron flow. 3. The effect of a progressive limitation by DCCD in the number of active ATP synthetase complexes on flash-induced phosphorylation has been examined. The decrease in ATP yield observed over a wide range of flash frequencies is related simply to the ATPase activity and to phosphorylation in continuous light, irrespective of the value of the membrane potential, which appears to be stabilized by this inhibitor. 4. As a whole, the results obtained at low concentrations of antimycin and under conditions of partial inhibition by DCCD evidence a localized coupling between the redox reactions and phosphorylation.

Limited cooperativity in the coupling between electron flow and photosynthetic ATP synthesis A comparative study in chromatophores phosphorylating at very different rates

1,2] and correl- ated with the value of the protonmotive force estab- lished in the steady state under continous illumination in a variety of experimental conditions. In these studies the value of Ap was monitored by spectro- scopic methods (electrochromic band shift of caro- tenoids for the evaluation of the membrane potential (A

Localized coupling in oxidative phosphorylation by mitochondria from Jerusalem artichoke (Helianthus tuberosus)

) and light-induced quenching of the fluorescence of 9aminoacridine for an estimate of transmem- brane ApH) and related to the rates of photophos- phorylation measured concurrently. An unexpected conclusion of this work was that the rate of photophosphorylation was not unequivo- cally related to the value of Ap (nor to that of A

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

or ApH), as expected if a chemiosmotic mecha- nism of coupling [3] between the redox reactions of photosynthetic electron flow and ATP synthesis operates. In fact, a different relation linked the rate of

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

Abstract Delocalized chemiosmotic coupling of oxidative phosphorylation requires that a single-value correlation exists between the extent of Δ \ gm H + and the kinetic parameters of respiration and ATP synthesis. This expectation was tested experimentally in nigericin-treated plant mitochondria in single combined experiments, in which simultaneously respiration (in State 3 and in State 4) was measured polarographically, FΔψ (which under these conditions was equivalent to Δ \ gm H + ) was evaluated potentiometrically from the uptake of tetraphenylphosphonium+ and the rate of phosphorylation was estimated from the transient depolarization of mitochondria during State 4-State 3-State 4 transitions. The steady-state rates of the different biochemical reactions were progressively inhibited by specific inhibitors active with different modalities on various steps of the energy-transducing process: succinate respiration was inhibited competitively with malonate or noncompetitively with antimycin A, or by limiting the rate of transport into the mitochondria of the respiratory substrate with phenylsuccinate; Δ \ gm H + was dissipated by uncoupling with increasing concentrations of valinomycin; ADP phosphorylation was limited with oligomycin. The results indicate generally that when the rate of respiratory electron flow is decreased, a parallel inhibition of the rate of phosphorylation is also observed, while very limited effects can be detected on the extent of Δ \ gm H + . This behavior is in marked contrast to the effect of uncoupling where the decreased rate of ATP synthesis is clearly due to energy limitation. Extending previous observations in bacterial photosynthesis and in respiration by animal mitochondria and submitochondrial particles the results indicate, therefore, that respiration tightly controls the rate of ATP synthesis, with a mechanism largely independent of Δ \ gm H + . These data cannot be reconciled with a delocalized chemiosmotic coupling model.

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

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.

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

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.

Electrochemical proton gradient and phosphate potential in bacterial chromatophores

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