Influence of the transmembrane electrochemical proton gradient on catalysis and regulation of the H+-ATP synthase from Rhodobacter capsulatus

For a possible elucidation of the single steps of the reaction cycle it is essential to distinguish between regulatory and catalytic events. In this work we have investigated the effect of ΔμH+ on the activation and the rate of ATP synthesis of the H+-ATP synthase from Rhodobacter capsulatus. A gradient ΔμH+ was generated across the membrane of the chromatophores by subjecting them to acid-base transitions and to K+/valinomycin-induced diffusion potentials. The osmotic component ΔpH was varied by varying the pH of the acidic stage (internal pH) by constant external pH, and the electrical component ΔΦ was varied by varying the internal concentration of K+. The diffusion potentials generated were evaluated using the Goldman equation. The main advantages of this technique are the quantitative definition of ΔμH+ and the possibility of varying its chemical and electrical components independently. A rapid-mixing quenched-flow apparatus was used to measure the initial rate of ATP synthesis. The relative increase in the uncoupled rate of ATP hydrolysis after a ΔμH+ pulse was considered proportional to the degree of activation of the enzyme. The two components of ΔμH+ were found to be kinetically equivalent to driving ATP synthesis. However, in the same range of values, the electrical component was much more effective than the osmotic component in activating the enzyme. Taken together, these results raise the interesting possibility that well-tuned regulatory mechanisms may intervene to compensate a different kinetic effect of ΔΦ and ΔpH on the reaction cycle. © 1994.