Energetics and stoichiometry of oxidative phosphorylation from NADH to cytochrome c in isolated rat liver mitochondria.

The reaction 3-hydroxybutyrate (3-OH-butyrate) + 2 cytochrome c + ~ + 2 ADP + 2 Pi acetoacetate + 2 cytochrome e+’ + 2 ATP was used to study the energy relationships of electron transfer in the forward and reverse directions in suspensions of isolated rat liver mitochondria. Reduction of cytochrome c was monitored continuously at 550-540 nm while aliquots were taken to measure transmembrane pH and electrical gradients, as well as ATP, ADP, Pi, 3-OH-butyrate, and acetoacetate. When oxidation of 3-OH-butyrate was coupled to either O2 or ferricyanide as final electron acceptor and starting [3-OH-butyrate]/[acetoacetate], [ATP], and [Pi] were varied, measurements were made following maximal net ATP synthesis. To study the reverse reaction, mitochondrial suspensions were first incubated with uncoupler to deplete endogenous substrates. The uncoupler was then bound with bovine serum albumin, and the respiratory chain was inhibited by sulfide or cyanide. Ratios of [3-OH-butyrate]/[acetoacetate], ADP, Pi, and ATP were added, resulting in various degrees of reduction of cytochrome c. Samples were taken in the steady states after these and additional amounts of the above reactants were added. For both the forward and reverse reactions, the measured mass action ratios were, within experimental error, equal to each other and to the calculated equilibrium constants (6.17 X lo6 2.08 X lo7 M ~ , depending upon the intramitochondrial pH for the conditions examined); the free energy changes calculated for the overall reaction, therefore, approached zero in all cases. No difference in the free energy available for ATP synthesis was detected in electron transfer to ferricyanide compared with that for transfer to 02, indicating that electron transfer across the mitochondrial membrane from 3-OH-butyrate to cytochrome c is electroneutral. From the similarity of the mass action ratios for the forward and reverse reactions, it can additionally be concluded that the ATP/2estoichiometry from NADH to cytochrome c is 1.97 k 0.06.