The redox state of cytochrome c, cytochrome a and the mitochondrial NAD couple, and the phosphorylation state of the adenine nucleotides, were measured in suspensions of isolated rat liver cells. 2. The DeltaG for the transfer of two electrons from the mitochondrial NAD to the cytochrome c couple is calculated to be 104kJ (24.8kcal). 3. The DeltaG associated with the synthesis of ATP at the measured phosphorylation state is calculated to be 95kJ (22.7kcal)/2mol of ATP. 4. The near equality of DeltaG of the electron-transport process and DeltaG required for ATP synthesis indicates near-equilibrium between the mitochondrial respiratory chain and the extramitochondrial phosphorylation state. 5. The existence of near-equilibrium in the coupled reactions implies that the respiratory activity depends on the ratio [ATP]/[ADP][P(i)] and not on the concentrations of the individual reactants. 6. If the overall system of oxidative phosphorylation is at near-equilibrium, all intermediary reactions must also be at equilibrium. Hence if the intramitochondrial and extramitochondrial phosphorylation states are indeed different, it follows that any differences in the activities of ATP, ADP and P(i) must be coupled to ion gradients and/or potentials across the inner mitochondrial membrane in such a way that translocation occurs without loss of free energy. 7. The metabolic state of the mitochondria in the cell can be defined by the turnover number of the cytochromes, the cytoplasmic phosphorylation state, and the oxidation-reduction potential of the NAD couple, rather than by the availability of ADP, substrate and O(2).
