The influence of energy-transfer inhibitors on proton permeability and photophosphorylation in normal and preilluminated Rhodospirillum rubrum chromatophores

(1) Chromatophores were preilluminated in the presence of phenazine methosulphate or diaminodurene, and without phosphorylation substrates; next they were transferred to fresh medium and assayed for light-induced proton uptake, light-induced 9-aminoacridin fluorescence quenching, and photophosphorylation.(2) Preillumination in the presence of phenazine methosulphate or diaminodurene causes an inhibition of the photophosphorylation rate. The presence of ADP + MgCl₂ + phosphate, or ADP + MgCl₂ + arsenate during preillumination provides full protection against this effect.(3) Preilluminated chromatophores are leaky for protons. The leak is expressed as an accelerated dark decay, and a diminished extent of succinate-supported, light-induced proton uptake. The extent of light-induced 9-aminoacridin fluorescence quenching is also diminished.(4) The proton leak can be closed by oligomycin and by dicyclohexyl carbodiimide (at concentrations similar to those used to inhibit photophosphorylation), but not by aurovertin. Closure of the proton leak results in partial restoration of the photophosphorylation rate.(5) The inhibition of phosphorylation by oligomycin or dicyclohexyl carbodiimide is time-dependent. In untreated chromatophores, the time-dependence is determined by the extent of membrane energization. In preilluminated chromatophores, the time-dependence is determined in addition by the extent to which the proton leaks have been closed. The reasons for this are briefly discussed.     

Photoinactivation of photophosphorylation and dark ATPase in Rhodospirillum rubrum chromatophores.

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 about 70%) 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 m-chlorophenyl hydrazone is present or when phenazine methosulfate is absent during the preillumination, indicating that the reaction occurs only when the membrane is energized. Phosphorylation conditions offer a practically complete protection against the photoinactivation. Inorganic phosphate, Mg2+ or ADP do not provide a significant protection against the photoinactivation, nor does ATP. The pH-dependence of the reaction(s) leading to photoinactivation may indicate that a partial reaction of the photophosphorylation process (perhaps only a conformational change of the coupling factor) precedes the photoinactivation.     

Size distribution and photophosphorylation of chromatophores from t Rhodospirillum rubrum

Morphology and photophosphorylation of chromatophores from t Rhodospirillum rubrum have been investigated by dynamic light scattering (DLS) and in situ ³¹P-NMR measurement. Two components, designated as light and heavy fractions, with different average sizes and size distributions were detected by the DLS and can be separated by sucrose density gradient centrifugation. The light fraction has an average size of about 140 nm in diameter with a narrow distribution and shows a high activity of photophosphorylation. About 70 of ADP were found to be converted to ATP purely by the photophosphorylative reaction. In contrast, the heavy fraction has a broad size distribution centered around 350 nm and a low activity of photophosphorylation. Only about 50 of ADP was converted into ATP and AMP with a ratio of 7:3, indicating that most membrane-bound adenylate kinase are attached on the particles of the heavy fraction. Effect of physical disruption on the structural integrity of chromatophores has been examined by using sonication with various oscillating strengths. The result shows that the morphology of chromatophores for both light and heavy fractions is relatively stable to the disruption, while the photophosphorylative activity of the light fraction is very sensitive to the disrupting strength, suggesting that the internal structure of the purified chromatophores could be partially damaged by the disruption.     

Flash-induced photophosphorylation in Rhodospirillum rubrum chromatophores. I. The relationship between cytochrome c-420 content and photophosphorylation.

The content of cytochrome c-420 in Rhodospirillum rubrum chromatophores prepared by grinding with alumina is 5--10% of that in whole cells, and 20--40% in chromatophores by 'French' pressing. Flash-induced phosphorylation of various chromatophores which varied in cytochrome content from 7 to 40% is proportional to the cytochrome content. Extrapolating the cytochrome c-420 content to that observed in whole cells, a ratio ATP/P+X- near 1 is calculated. At low flash intensity the phosphorylation per flash is proportional to flash energy. Photophosphorylation in flashes given after a time of several minutes is only slightly dependent on the number of flashes. If the flashes are spaced from 0.1 to 10 s, relative phosphorylation in the first flash is about 70% and in the second 90+ of that observed in the following flashes. Proton binding is not affected by the cytochrome c-420 content and a ratio of H+/P+x- of 2.3 was found. These results can be explained by a working hypothesis in which charge separation occurring at one reaction centre and the resulting electron transport mediated amongst others by c-420, results in the injection of two protons into an ATPase, this in contrast to a chemiosmotic mechanism, where the protons are released in the chromatophore inner space.     

Temperature dependency of the Mg-ATPase and photophosphorylation activities in Rhodospirillum rubrum chromatophores

Arrhenius plots for ATP synthesis, coupled to endogenous and Phenazine methosulfate or N,N,N,N,-Tetramenthyl-1,4-Phenylene diamine-mediated photosynthetic election transport and for ATP hydrolysis were studied in Rhodospirillum rubrum chromatophores.Coupled or uncoupler induced Mg-ATPase show no discontinuity in the range tested (30°C-5°C) and they also have the same activation energy. Phenazine methosulfatecatalyzed photophosphorylation has also a single activation energy where as the endogenous reaction shows complex and ageing dependent behaviour, alternating temperature ranges having high (45.2 to 144,4 kJ·mol^-1) and very low (ca 0.0 to 3.3 kJ·mol^-1) activation energy.Abbreviations Bchl Bacteriochlorophyll - Ea Activation energy - FCCP Carbonyl Cyanide p. Trifluoromethoxy henyl Hydrazone - PMS phenazine methosulfate - TMPD N,N,N,N-Tetramethyl-1,4-Phenylene diamine - R Rhodospirillum     

The effect of electron donors and acceptors on light-induced absorbance changes and photophosphorylation in Rhodospirillum rubrum chromatophores.

Light-induced difference spectra between 400 and 640 nm of Rhodospirillum rubrum chromatophores were performed in the presence and absence of exogenous electron donor/acceptor systems and compared with the chemical oxidation spectrum. The results indicate that the component previously defined as P430 is not a unique entity but rather represents different species, or a mixture of species, under various conditions. Under all conditions in which the reaction center bacteriochlorophyll is reversibly photooxidized, as indicated by the bleaching around 600 nm, it is also contributing to the absorbance increase around 430 nm. In one case, in presence of reduced dichloroindophenol and in the absence of oxygen, the photooxidation of reaction center bacteriochlorophyll is fully supressed. Under these conditions an irreversible change around 430 nm is still observed and seems to be due to the Soret band of b-type cytochrome. In the presence of reduced dichloroindophenol and absence of oxygen there is a marked inhibition of photophosphorylation. This inhibition is apparently due to the complete reduction of the cyclic electron carriers. Addition of the low potential dye benzyl viologen facilitates an almost complete recovery of the reversible photooxidation of reaction center bacteriochlorophyll as well as of photophosphorylation. These results indicate that the apparent mid-point potential of the primary electron acceptor in Rhodospirillum rubrum chromatophores is probably in the range of that of benzyl viologen (E'o = - 340 mV).     

Phosphate binding to chromatophores of Rhodospirillum rubrum.

Equilibrium dialysis has been used to determine the binding of phosphate to chromatophores of Rhodospirillum rubrum. Assuming a complete exchange of the added 32Pi with endogenous phosphate, the saturation with phosphate retained in any form by chromatophores was reached at about 20 nmoles Pi per mg of bacteriochlorophyll. The retention of phosphate had a pH optimum at pH 6.5 to 6.8. At pH 8.0 only chromatophores which have not been liberated from DNA and RNA show a considerable retention of phosphate. However, illumination of chromatophores prior to dialysis in the presence of ADP leads to a retention of phosphate at pH 8.0 which persists during dark dialysis in the absence of added magnesium.     

The photo-oxidation of succinate by chromatophores of Rhodospirillum rubrum

1. The stoicheiometry of the photo-oxidation of succinate by chromatophores has been investigated with [2,3-¹⁴C₂]succinate. It was found that there is a stoicheiometric relationship between the amount of succinate oxidized and the NAD reduced, and that fumarate is the only product of succinate oxidation. 2. The possibility of a direct hydrogen transfer from succinate to NAD in this reaction was investigated with tritiated substrates. With tritiated succinate less than 3% of the activity expected if direct hydrogen transfer occurred was recovered in the NADH₂, and this was due to contamination with the substrate. In experiments with tritiated water, NADH₂ was labelled, and had half the specific activity of the water, as expected if water was the source of protons. It was also found that chromatophores catalyse an exchange reaction between NADH₂ and water. 3. It is concluded that the exchange reaction makes it impossible to interpret these results as indicating either a hydrogen-transfer or an electron-transfer mechanism for the photoreduction reaction.     

The effect of equisetin on energy-linked reactions in Rhodospirillum rubrum chromatophores

Light-induced proton uptake, light-induced carotenoid absorbance shift, photophosphorylation, and hydrolysis of Mg-ATP, Ca-ATP, and PPi in Rhodospirillum rubrum chromatophores are shown to be inhibited by the antibiotic equisetin. The Mg- and Ca-ATPase activities of purified F0F1-ATPase are inhibited by equisetin. In contrast, only the Ca-ATPase activity of purified F1-ATPase is decreased by equisetin, whereas the Mg-ATPase is stimulated. Both equisetin and N,N'-dicyclohexylcarbodiimide (DCCD) inhibit the hydrolytic activity of the purified H+-PPase but not the hydrolytic activity of soluble PPase from R. rubrum and yeast. The I50 for the PPi hydrolysis is near 20 microM for both equisetin and DCCD. The action of equisetin on membranes is compared to the effect of Triton X-100 and carbonyl cyanide p-trifluoromethoxyhydrazone. On the basis of these new data, equisetin is proposed to act nonspecifically on membranes and hydrophobic domains of proteins.     

The reconstitution of photophosphorylation and the effect of inorganic phosphate at different pH's on the phenazine methosulfate photoinhibition of Rhodospirillum rubrum chromatophores

Preillumination of R. rubrum membranes in the presence of 50M phenazine methosulfate produces an inhibition of their photophosphorylating capacity.At low pH's phosphate protects against this photo-inhibition, whereas increasing the pH eliminates this protective effect. The inhibition can be produced not only by preillumination at high pH in the presence of phenazine methosulfate, but also by preillumination at low pH (5.0) and then shifting the pH to 8.0 in the dark. We have also measured the effect of preillumination in the presence of an uncoupler such as carbonylcyanide p-trifluoromethoxyphenylhydrazone, and found that at normal pH's while photophosphorylation was protected, the uncoupler activated ATPase was inhibited pointing to a clear difference for both reactions and perhaps different structural requirements for their activities.The purified coupling factor protein isolated from either normal or photoinactivated membrane will reconstitute normal photophosphorylation in previously uncoupled membranes but not in uncoupled membranes which were inactivated by preillumination with phenazine methosulfate prior to uncoupling.     

Studies on photosynthetic inorganic pyrophosphate formation in Rhodospirillum rubrum chromatophores

Photosynthetic formation of inorganic pyrophosphate (PP_i) in Rhodospirillum rubrum chromatophores has been studied utilizing a new and sensitive method for continuous monitoring of PP_i 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 PP_i synthesis is twice the rate of ATP synthesis. Antimycin A, at a concentration which strongly inhibited the photosynthetic ATP formation, inhibited the PP_i synthesis much less. Even at low rates of electron transport a significant rate of PP_i synthesis is obtained. The rate of photosynthetic ATP formation is stimulated up to 20% when PP_i synthesis is inhibited. It is shown that PP_i 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.