Energy-liked reactions in photosynthetic bacteria. X. Solubilization of the membrane-bound energy-linked inorganic pyrophosphatase of Rhodospirillum rubrum.

The energy-linked membrane-bound inorganic pyrophosphatase of $\text{Rhodospirollum}$$\text{rubrum}$, G-9, has been solubilized with good yield from chromatophores using cholate in the presence of MgCl₂. The enzyme has been partially purified using ammonium sulfate fractionation and gel chromatography. After fractionation the enzyme requires phospholipid for activity. The solubilized enzyme is specific for PP_i and requires Mg²⁺ for activity as has been found for other PP_iases.     

Purification of the membrane-bound proton-translocating inorganic pyrophosphatase from Rhodospirillum rubrum

The proton translocating membrane-bound inorganic pyrophosphatase of Rhodospirillum rubrum S1, has been solubilized with good yield from chromatophores using Triton X-100 (9–10 oxyethylene groups) in the presence of high concentrations of MgCl₂ and ethyleneglycol. The enzyme has been purified 80-fold by hydroxylapatite column chromatography, to a state of near homogeneity, according to polyacrylamide-gelelectrophoresis. The enzyme appears to be a very hydrophobic integrally bound membrane protein. Phospholipids or Triton X-100 reconstitutes the enzyme activity after solubilization and purification. The purified enzyme preparation has a specific activity of 24 units. Both the purified and the chromatophore-bound enzyme are inhibited by N-ethylmaleimide, 4-chloro-7-nitrobenzo-2-oxo-1,3-diazol (NBF-Cl), sodium fluoride, imidodiphosphate, methylenediphosphonate and the antibiotic Dio-9 (energy-transfer inhibitor). In the solubilized state the purified enzyme is not stimulated by uncouplers or inhibited by dicyclohexylcarbodiimide in contrast to the chromatophore-bound pyrophosphatase. When reconstituted into liposomes the purified enzyme regains the stimulation by uncouplers.     

Energy-linked reactions catalyzed by the purified ATPase complex (F0F1) from Rhodospirillum rubrum chromatophores

1. The isolation of F0F1-ATPase complex from Rhodospirillum rubrum chromatophores by the use of taurodeoxycholate is described. 2. The enzyme preparation contains about 12 polypeptides; five are subunits of the F1 moiety. 3. The ATPase activity of the purified enzyme is dependent on the addition of phospholipids. 4. Km-vales for Mg2+-ATP and Ca2+-ATP are similar to the values obtained for the membrane-bound enzyme. 5. The F0F1-ATPase complex is more than 70% inhibited by oligomycin and N,N'-dicyclohexylcarbodiimide. 6. The F0F1-ATPase complex was integrated into liposomes. The reconstituted proteoliposomes catalyzed energy transduction as shown by ATP-dependent quenching of acridine dye fluorescence and ATP-32Pi exchange.     

Inorganic pyrophosphate-driven ATP-synthesis in liposomes containing membrane-bound inorganic pyrophosphatase and F0-F1 complex from Rhodospirillum rubrum

PPi driven ATP synthesis has been reconstituted in a liposomal system containing the membrane-bound energy-linked PPiase and coupling factor complex, both highly purified from Rhodospirillum rubrum. This energy converting model system was made by mixing both enzyme preparations with an aqueous suspension of sonicated soybean phospholipids and subjecting to a freeze-thaw procedure. In the presence of ADP, Mg2+, Pi and PPi the system catalyzed phosphorylation by up to 25 nmol ATP formed X mg protein-1 X min-1, at 20 degrees C, which was sensitive to uncouplers and inhibitors of phosphorylation such as oligomycin, efrapeptin and N,N'-dicyclohexylcarbodiimide.     

Kinetics of the membrane-bound inorganic pyrophosphatase from Rhodospirillum rubrum chromatophores: Effect of the transmembrane electrical potential on the rate constants

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_i 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.     

Properties of the solubilized membrane-bound hydrogenase from the photosynthetic bacterium Rhodospirillum rubrum.

The membrane-bound hydrogenase of the photosynthetic bacterium Rhodospirillum rubrum has been purified 490-fold with a yield of 5.8%. The enzyme was homogeneous by disc gel electrophoresis. A method for the permanent, oxygen-insensitive, staining of hydrogenase on polyacrylamide gels is described. The enzyme is a monomer of molecular weight about 66,000 containing four iron and four acid-labile sulfur atoms per molecule. The electron paramagnetic resonance spectrum at 20 °K exhibits a strong signal in the oxidized state only with g > 2—this is characteristic of high potential iron-sulfur protein. The hydrogenase is thermostable and also resistant to both denaturation agents and oxygen inactivation. Carbon monoxide reversibly inhibits the enzyme but metal-complexing and thiol-blocking reagents have little effect on activity. The enzyme will catalyze both H₂ evolution and H₂ uptake in the presence of many artificial electron carriers but the two activities differ in their pH optima. There is a correlation between H₂ evolution activity and the redox potential of the mediator dye. Ferredoxins and pyridine nucleotides do not readily interact with the hydrogenase. We have shown that irradiation of a solution containing methyl viologen, EDTA, proflavin, and R. rubrum hydrogenase will evolve hydrogen continuously for over 9 h. However, the enzyme evolves hydrogen at only very low rates from in vitro chloroplast-ferredoxin and chloroplast-methyl viologen systems. R. rubrum hydrogenase has a number of properties in common with the hydrogenases purified from two other photosynthetic bacteria, Chromatium and Thiocapsa, but is distinct from the hydrogenases of nonphotosynthetic bacteria.     

The phosphate-pyrophosphate exchange and hydrolytic reactions of the membrane-bound pyrophosphatase ofRhodospirillum rubrum: Effects of pH and divalent cations

The relation that exist between the Pi-PPi exchange reaction and pyrophosphate hydrolysis by the membrane-bound pyrophosphatase of chromatophores ofRhodospirillum rubrum was studied. The two reactions have a markedly different requirement for pH. The optimal pH for hydrolysis was 6.5 while the Pi-PPi exchange reaction was at 7.5; the pH affects mainly theK _m of Mg²⁺ or Pi for the enzyme; Mn²⁺ and Co²⁺ support the Pi-PPi exchange reaction partially (50%), but the reaction is slower than with Mg²⁺; other divalent cations like Zn²⁺ or Ca²⁺ do not support the exchange reaction. In the hydrolytic reaction, Zn²⁺, at low concentration, substitutes for Mg²⁺ as substrate, and Co²⁺ also substitutes in limited amount (50%). Other cations (Ca²⁺, Cu²⁺, Fe²⁺, etc.) do not act as substrates in complex with PPi. The Zn²⁺ at high concentrations inhibited the hydrolytic reaction, probably due to uncomplexed free Zn²⁺. In the presence of high concentration of substrate for the hydrolysis (Mg-PPi) the divalent cations are inhibitory in the following order: Zn²⁺>Mn²⁺>Ca²⁺Co²⁺>Fe²⁺>Cu²⁺>Mg²⁺. The data in this work suggest that H⁺ and divalent cations in their free form induced changes in the kinetic properties of the enzyme.     

Regulation of divalent cations of the membrane-bound pyrophosphatase of Rhodospirillum rubrum,as shown by the hydrolysis of tripositive-pyrophosphate complexes

Tripositive-pyrophosphate [M(III)-PPi] complexes were used to investigate the role of free divalent cations on the membrane-bound pyrophosphatase. Divalent cations remain free and the M(III)-PPi complexes were employed as substrates. Formation of a La-PPi complex was studied by fluorescence, and the fact that Zn²⁺ and Mg²⁺ remain free in the solution was validated. Hydrolysis of La-PPi is stimulated by the presence of fixed concentrations of free Mg²⁺ or Zn²⁺ and this stimulation depends on the concentration of the cations when the La-PPi complex is fixed. The divalent cation stimulation order is Zn²⁺ > Co²⁺ > Mg²⁺ > Mn²⁺ > Ca²⁺ (at 0.5 mm of free cation). With different M(III)-PPi complexes, Zn²⁺ produces the same K_m, for all the complexes and Mg²⁺ stimulates with a different K_m. The results suggest that both Mg²⁺ and Zn²⁺ activate the membrane-bound pyrophosphatase but through different mechanisms.     

Isolation and characterization of a membrane-bound, low-potential c-type cytochrome from purple photosynthetic bacteria, with special reference to Rhodospirillum rubrum.

Other investigators have isolated soluble, low-potential, c-type cytochromes (cytochrome c3) from a few photosynthetic procaryotes, i.e., a cyanobacterium and two species of purple nonsulfur bacteria. However, such cytochromes appeared to be absent from other purple bacteria, including Rhodospirillum rubrum and Chromatium vinosum. We now report evidence for the presence of low-potential c-type cytochromes in these two species, in which they were found to be bound to the photosynthetic membranes. Evidence for a membrane-bound, low-potential c-type cytochrome was also found in Rhodopseudomonas sphaeoides. The low-potential c-type cytochrome of R. rubrum was solubilized by a Triton X-100 treatment of chromatophores and was partly purified. It was found to have a molecular weight of about 17,000, a midpoint oxidation-reduction potential of -192 mV, and an alpha-absorption peak at 552 nm. It appears that low-potential c-type cytochromes may be present in all purple photosynthetic bacteria, of both the sulfur and the nonsulfur types.     

Synthesis of pyrophosphate by chromatophores of Rhodospirillum rubrum in the light and by soluble yeast inorganic pyrophosphatase in water-organic solvent mixtures

Chromatophores of Rhodospirillum rubrum contain a membrane-bound pyrophosphatase that synthesizes pyrophosphate when an electrochemical H+ gradient is formed across the chromatophore membrane upon illumination. In this report it is shown that MgCl2 and Pi have different effects on the synthesis of pyrophosphate in the light depending on whether initial velocities or steady-state levels are examined. When the water activity of the medium is reduced by the addition of organic solvents, soluble yeast inorganic pyrophosphatase (no H+ gradient present) synthesizes pyrophosphate in amounts similar to those synthesized by the chromatophores in totally aqueous medium during illumination, (H+ gradient present). The pH, MgCl2 and Pi dependence for the synthesis of pyrophosphate by the chromatophores at steady-state is similar to that observed at equilibrium with the soluble enzyme in the presence of organic solvents. The possibility is raised that a decrease in water activity may play a role in the mechanism by which the energy derived from the electrochemical H+ gradient is used for the synthesis of pyrophosphate in chromatophores of R. rubrum.     

Theoretical analysis of distribution of [18O]Pi species during exchange with water. Application to exchanges catalyzed by yeast inorganic pyrophosphatase

A theoretical analysis has been derived which allows the analytical calculation of the complete distribution of 18O-labeled Pi species expected to occur during medium Pi equilibrium HOH exchange of [18O]Pi and to be produced by intermediate Pi equilibrium HOH exchange during net hydrolysis of [18O]PPi or other labeled phosphate compounds. The observed distributions with catalysis by yeast inorganic pyrophosphatase are found to agree closely with the theoretical values indicating that the exchange reaction can be adequately described by a unique value of the partitioning of bound Pi between release from the enzyme versus formation of bound PPi with loss of an oxygen to the water. The limitations on the exclusion of other mechanisms are discussed. The extent of this partitioning does change, however, under some experimental conditions. At low pH, with activation by Mg2+ or Mn2+, the relative rate of release of Pi is found to increase. The extent of exchange is also dependent on the nature of the activating metal, being greatest with Co2+. During PPi hydrolysis with PPi in excess over Mg2+, a shift to lower extents of exchange is observed.