Relationship of Transmembrane pH and Electrical Gradients with Respiration and Adenosine 5′-Triphosphate Synthesis in Mitochondria

The mechanism of mitochondrial oxidative phosphorylation and its regulation have been studied by using suspensions of isolated rat liver mitochondria. Parallel measurements were made of mitochondrial volume, respiration, transmembrane pH and electrical gradients, and adenosine 5′-triphosphate (ATP), adenosine 5′-diphosphate (ADP), and inorganic phosphate (P_i) concentrations under various experimental conditions. The transmembrane electrical gradients were calculated from the equilibrium distributions of [³H]-triphenylmethylphosphonium (TPMP⁺), [³H]tribenzylmethylammonium (TBMA⁺), and K⁺(plus valinomycin). The transmembrane distributions of labeled acetate, methylamine, and 5, 5-dimethyloxazolidine-2, 4-dione were used for the calculation of pH gradients. Evaluation of the data shows that the respiratory rate is strictly correlated with [ATP]/([ADP] [P_i]) (free energy of ATP synthesis), whereas thereis no consistent correlation between the transmembrane electrical potential, the pH gradient, or the total “ protonmotive ” force (Δμ_H⁺) and the respiratory rate. Thermodynamic analysis indicates that, in order for the proton electrochemical gradient to serve as an intermediate in ATP synthesis, from three to seven H⁺would have to be transported per each ATP synthesized, depending on the experimental conditions. These results suggest that the proton electrochemical gradient may not serve as a primary intermediate in oxidative phosphorylation.