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 $\text{ATP}\text{P}{}^{\mathrm{+}}\text{X}{}^{\mathrm{?}}$ 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 $\text{H}{}^{\mathrm{+}}\text{P}{}^{\mathrm{+}}\text{X}{}^{\mathrm{?}}$ 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.     

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.     

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     

Factors controlling ATP levels during photophosphorylation catalyzed by Rhodospirillum rubrum chromatophores

Summary Photophosphorylation of ADP catalyzed by chromatophores represents a possible method for regenerating ATP from ADP for use in large-scale enzymatic synthesis of complicated molecules. We found that the extent of conversion of ADP to ATP in the presence of ascorbate and magnesium chloride was influenced by the presence of bubbling gas in contact with the reaction mixture, and by the presence of fumarate: with no fumarate or gas contact, low conversions (usually about 50%) were obtained; however, under the same conditions, but in the presence of fumarate or bubbling gas, 100% conversions were obtained. We discuss mechanistic implications of our findings.     

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.     

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.     

Postillumination adenosine triphosphate synthesis in Rhodospirillum rubrum chromatophores. I. Conditions for maximal yields.

The very low level of postillumination ATP synthesis in chromatophores was markedly stimulated when permeant anions (thiocyanate or perchlorate) or permeant cations (potassium in the presence of valinomycin) were added to the light stage. Although these compounds stimulated also light-induced proton uptake in chromatophores the pH dependence of both photoreactions was different. Proton uptake peaked at pH 6.5 while the amount of postillumination ATP was maximal when the light stage was carried out around pH 7.7. The increased yield of ATP at the more alkaline pH could not be explained by a slower decay of the high energy state at this pH, since the decay rate was faster at pH 7.7 than at pH 6.5. The proton concentration gradient which is maintained across the chromatophore membrane in the light was also found to increase when the external pH was raised from 6.0 to 8.0. Only a minimal amount of postillumination ATP was formed when this gradient was below 2.1 pH units, but above this value the ATP yield rose steeply as a function of the increasing pH gradient. In light of these results it is suggested that in order to obtain a high yield of postillumination ATP synthesis in chromatophores two conditions are required: the particles have to be loaded with a sufficient number of protons and a light-induced pH gradient above a certain threshold value has to be maintained across their membrane. The low yield of postillumination ATP in chromatophores and the increase obtained by adding permeating ions, is thus explained by similar variations in the extent of the pH gradient, which exceeded the threshold value only in the presence of the permeating ions.     

Light-induced reversible pH changes in chromatophores from Rhodospirillum rubrum

Light-induced pH changes with chromatophores from Rhodospirillum rubrum have been studied as a function of pH, chromatophore and salt concentrations, and light intensity. Optimal reaction conditions have been determined. It has been demonstrated that light-induced pH changes may also be obtained at high light intensities by addition of phenzaine methosulfate after a lag period in a system inhibited by 2-n-heptyl-4-hydroxyquinoline-N-oxide.     

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.     

Steady-state kinetics of ADP-arsenate and ATP-synthesis in Rhodospirillum rubrum chromatophores

(1) Rates of ATP synthesis and ADP-arsenate synthesis catalyzed by Rhodospirillum rubrum chromatophores were determined with the firefly luciferase method and by a coupled enzyme assay involving hexokinase and glucose-6-phosphate dehydrogenase. (2) V_m for ADP-arsenate synthesis was about 2-times lower than V_m for ATP-synthesis. With saturating [ADP], K(As_i) was about 20% higher than K(P_i). With saturating [anion], K(ADP) was during arsenylation about 20% lower than during phosphorylation. (3) Plots of $\text{1}\text{v}$ vs. $\text{1}\text{[}\text{substrate}\text{]}$ were non-linear at low concentrations of the fixed substrate. The non-linearity was such as to suggest a positive cooperativity between sites binding the variable substrate, resulting in an increased $\text{V}{}_{\mathrm{<mtext>m</mtext>}}\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ ratio. High concentrations of the fixed substrate cause a similar increase in $\text{V}{}_{\mathrm{<mtext>m</mtext>}}\text{K}{}_{\mathrm{<mtext>m</mtext>}}$, but abolish the cooperativity of the sites binding the variable substrate. (4) Low concentrations of inorganic arsenate (As_i) stimulate ATP synthesis supported by low concentrations of P_i and ADP about 2-fold. (5) At high ADP concentrations, the apparent K_i of As_i for inhibition of ATP-synthesis was 2–3-times higher than the apparent K_m of As_i for arsenylation; the apparent K_i of P_i for inhibition of ADP-arsenate synthesis was about 40% lower than the apparent K_m of P_i for ATP synthesis. (6) The results are discussed in terms of a model in which P_i and As_i compete for binding to a catalytic as well as an allosteric site. The interaction between these sites is modulated by the ADP concentration. At high ADP concentrations, interaction between these sites occurs only when they are occupied with different species of anion.