Stoichiometry of Adenosine Triphosphate-driven Proton Translocation in Bovine Heart Submitochondrial Particles

Inward-directed proton translocation driven by ATP hydrolysis was investigated in bovine heart submitochondrial particles under conditions where no net pH change occurred upon ATP hydrolysis. A biphasic time course of ATP hydrolysis, due to the presence of adenylate kinase activity, was observed by following the release of 32Pi from [β, γ-32P]ATP under comparable conditions. An equation was derived for calculation of the H+:ATP ratio from the time course of ATP hydrolysis and the transient pH change of the medium due to proton translocation. H+:ATP ratios as high as 1.7 were observed, although a seasonal variation was noted. However, oligomycin raised the H+:O ratios for respiration-driven proton translocation, suggesting that the experimentally observed H+:ATP ratios were suboptimal. The results indicate that the true stoichiometry of ATP-driven proton translocation, characteristic of the ATPase mechanism is 2H+:ATP. The same stoichiometry has been reported previously for outward-directed proton translocation in intact mitochondria.