Studies on the light-dependent synthesis of inorganic pyrophosphate by Rhodospirillum rubrum chromatophores (original) (raw)

Studies on photosynthetic inorganic pyrophosphate formation in Rhodospirillum rubrum chromatophores

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1986

Photosynthetic formation of inorganic pyrophosphate (PPi) in RhodospiriUum rubrum chromatophores has been studied utilizing a new and sensitive method for continuous monitoring of PPm synthesis. Studies of the reaction kinetics under a variety of conditions, e.g., at different substrate concentrations and different electron-transport rates, have been performed. At very low light intensities the rate of PPi synthesis is twice the rate of ATP synthesis. Antimycin A, at a concentration which strongly inhibited the photosynthetic ATP formation, inhibited the PPi synthesis much less. Even at low rates of electron transport a significant rate of PPi synthesis is obtained. The rate of photosynthetic ATP formation is stimulated up to 20% when PPI synthesis is inhibited. It is shown that PP~ synthesis and ATP synthesis compete with each other. No inhibition of pyrophosphatase activity is observed at high carbonyl cyanide p-trifluoromethoxyhydrazone concentration while ATPase activity is strongly inhibited under the same conditions.

Demonstration of ΔpH- and Δψ-induced synthesis of inorganic pyrophosphate in chromatophores from Rhodospirillum rubrum

FEBS Letters, 1987

It is possible to obtain synthesis of PPi by artifical ion potentials in Rhodospirillum rubrum chromatophores. PPi can be formed by K +-diffusion gradients (A ~,), H + gradients (ApH) or a combination of both. In contrast, ATP can only be synthesized by imposed A~u or d~,+dpH. For ATP formation there is also a threshold value of K + concentration below which synthesis of ATP is not possible. Such a threshold is not found for PP~ formation. Both PPi and ATP syntheses are abolished by addition of FCCP or nigericin and only marginally affected by electron transport inhibitors. The synthesis of PPi can be monitored for several minutes before it ceases, while ATP production stops within 30 s. As a result the maximal yield of PPi is 200 nmol PPi//tmol BChl, while that of ATP is no more than 25 nmol ATP/Itmol BChl. The initial rates of syntheses were 0.50/tmol PPd/lmol BChl per min and 2.0/tmol ATP/~tmol per min, respectively. These rates are approx. 50 and 20% of the respective photophosphorylation rates under saturating illumination.

Synthesis of pyrophosphate coupled to the reverse energy-linked transhydrogenase reaction inRhodospirillum rubrumchromatophores

FEBS Letters, 1986

Chromatopho~s from the photosynthetic bacterium Rhodospiril~~ IBM contain a membrane-bound transhydrogenase catalyzing the transfer of a hydride ion between NADH and NADP+. The reverse reaction, i.e. reduction of NAD+ by NADPH, can furnish sufficient energy (d$H+) to drive the phosphorylation of inorganic orthophosphate (P,) to pyrophosphate (PP,). The rate of PP, synthesis is 50 nmol PP, formed/ min per pmol Bchl which is 5% of the rate of light-induced PP, synthesis. PP, synthesis is inhibited by both the II+-PPase inhibitor fluoride and the specific transhydrogenase inhibitor palmitoyl-CoA. The effects of both DCCD and uncouplers on the system provide additional evidence that the @H+ generated by the reverse transhydrogenase reaction drives PP, synthesis. The rate of PP, synthesis can be partially inhibited by the addition of NADP+, a substrate of the forward energy-consuming reaction. The dfiH+ generated can also be used to drive ATP synthesis by the H+-ATPase, but at a lower rate than the PP, synthesis.

Kinetics of the membrane-bound inorganic pyrophosphatase from Rhodospirillum rubrum chromatophores

FEBS Letters, 1986

The behaviour of the membrane-bound proton-translocating pyrophosphatase (H+-PPase) in Rhodospirillum rubrum chromatophores upon application of an electrochemical potential is studied. The rate constants are shown to be affected in an asymmetric fashion. The forward rate constant (PP, synthesis) is shown to be at least 45-times larger during illumination than when there is no proton-motive force. The hydrolysis rate is increased maximally 8-times when the potential is dissipated. The effect of the electrical field gradient is thus mainly to increase the forward rate of the reaction. The H+-PPase also seems to be a functionally simpler enzyme than the H+-ATPase, lacking the hydrolysis activation step during energization found in the latter.

Membrane-bound phosphate as driving force for ATP synthesis in chromatophores ofRhodospirillum rubrum

Journal of Bioenergetics, 1975

Chromatophores of Rhodospirillum rubrum preilluminated in the presence of unlabelled phosphate form labelled ATP in the dark after being separated from the preincubation mixture by gel-filtration and incubated with ADP and 32P i. The driving force for the synthesis of this labelled ATP was previously shown to be ATP firmly bound to the membrane. The amount of labelled ATP produced is determined by measuring the incorporated 32P i and extrapolation of the values towards zero time incubation and is shown to correlate with the phosphoryIation activity of the chromatophores used. 2.5 nmoles ATP correspond to a phosphorylation activity of 100 Ltmoles ATP/mg Bchl. h. The results were compared with those from assays in which chromatophores were preincubated with pyrophosphate in the dark. The results strongly support the hypothesis that the X~ P formed in the light does not transfer its P to ADP in solution and therefore give further evidence to a double sited coupling factor as proposed previously.

Photosynthetic formation of inorganic pyrophosphate in phototrophic bacteria

Photosynthesis Research, 1990

In this paper we report studies on photosynthetic formation of inorganic pyrophosphate (PP0 in three phototrophic bacteria. Formation of PP~ was found in chromatophores from Rhodopseudomonas vMdis but not in chromatophores from Rhodopseudomonas blastica and Rhodobacter capsufatus. The maximal rate-of PPi synthesis in Rps. viridis was 0.15#tool PPi formed/(min,/~mol Bacteriochlorophytl) at 23°C. The synthesis of PP~ was inhibited by electron transport inhibitors, uncouplers and fluoride, but was insensitive to oligomycin and venturicidin. The steady state rate of PP~ synthesis under continuous illumination was about 15% of the steady-state rate of ATP synthesis. The synthesis of PP~ after short light flashes was also studied. The yield of PPi after a single lms flash was equivalent to approximately 1 #tool PPi/500#mol Bacteriochlorophyll. In Rps. vMdis chromatophores, PPi was also found to induce a membrane potential, which was sensitive to carbonyl cyanide p-trifluoromethoxyphenylhydrazone and NaF.

Photoinactivation of photophosphorylation and dark ATPase in Rhodospirillum rubrum chromatophores

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1976

Preillumination of Rhodospirillum rubrum chromatophores with strong, far-red light in the presence of phenazine methosulfate under non-phosphorylation conditions results in a selective, irreversible inactivation (typically aboutT0 f) of photophosphorylation and of uncoupler-stimulated dark ATPase. The time course of the photoinactivation is similar to the light-on kinetics of the light-induced proton uptake in the absence of ADP. Only little photoinactivation occurs when the uncoupler carbonyl cyanide z-chlorophenyl hydrazone is present or when phenazine methosulfate is absent during the preillumination, indicating that the reaction occurs only when the membrane is energized.