Non-equilibrium thermodynamics of oxidative phosphorylation by inverted inner membrane vesicles of rat liver mitochondria.

The relationship of the phosphate potential (delta GP) to the free energy released by the oxidation of NADH (redox potential or delta GR) was studied in suspensions of inverted inner membrane vesicles prepared from rat liver mitochondria. At delta GR values less negative than -52.2 kcal/mol, delta GP was a linear function of delta GR during oxidative phosphorylation at static head. At more negative delta GR, delta GP no longer increased but remained, more or less, at a constant value. At all values of delta GR, delta GP increased as Pi decreased. At high Pi, ATP/ADP was relatively independent of Pi, but at low Pi there was a strong interdependence of ATP/ADP and Pi. The experimental data were analyzed in terms of the theory of non-equilibrium thermodynamics. The degree of coupling, q, averaged 0.8 as estimated from the dependence of respiratory rate on delta GP. From measurements of -delta GR/delta GP at static head and from the estimates of q, an average value of four was calculated for Z, the phenomenological stoichiometry. The results support a 4-proton model of chemiosmotic coupling in which proton stoichiometries are 4H+/site, 3H+/ATPase, and 1H+/translocation of ATP for ADP and Pi. The results further indicate that the site by site reactions of oxidative phosphorylation operate close to thermodynamic equilibrium. This implies that ATP/site ratios are proportional to the redox potentials across each site at static head. Based on the oxidation-reduction potentials of NADH, ubiquinone, and cytochrome c, it follows that the ideal ATP/site ratios of mitochondrial oxidative phosphorylation are 1, 1/2, and 1 1/2, respectively, for sites 1, 2, and 3.