Upper and lower limits of the charge translocation stoichiometry of cytochrome c oxidase

The mechanistic stoichiometry of charge separation coupled to the flow of electrons through cytochrome c oxidase has remained a center of controversy since it was first demonstrated that cytochrome oxidase is an H+ pump. Currently the major dispute is whether the q+/O ratio for this segment is 4 or 6. One cause of the controversy is incomplete coupling between electron flow, electrogenic H+ ejection, and electrophoretic cation uptake, which is usually attributed to finite rates of H+ leakage and/or slippage of the H+ pumps. To minimize the uncertainty which incomplete coupling introduces into estimates of the mechanistic stoichiometry, a new approach (Beavis, A. D., and Lehninger, A. L. (1986) Eur. J. Biochem. 158, 307-314) has been used to determine the upper and lower limits of the mechanistic q+/O translocation stoichiometry of cytochrome oxidase. In this approach, the relationship between the rate of valinomycin-dependent K+ uptake, JK, and rate of O2 consumption, JO, is determined as the rates are modulated by two distinct means. When the rates are modulated by the rate of electron flow (i.e. rate of energy supply) the slope of JK versus JO must at all points be less than the mechanistic K+/O ratio. On the other hand, when the rates are modulated by varying the concentration of valinomycin (i.e. the rate of energy utilization) the slope of JK versus JO must at all points be greater than the mechanistic K+/O ratio. The results indicate that the q+/O ratio lies between 4.3 and 5.5. These data are inconsistent with both currently favored stoichiometries, and it is suggested that the true mechanistic stoichiometry of charge separation coupled to electron flow through cytochrome oxidase may be 5 q+/O.