Proton translocation by cytochrome c oxidase

Cell respiration in mitochondria and some bacteria Ts catalysed by cytochro me c oxidase, which reduces O-2 to water, coupled with translocation of fou r protons across the mitochondrial or bacterial membrane(1-3). The enzyme's catalytic cycle consists of a reductive phase, in which the oxidized enzym e receives electrons from cytochrome c, and an oxidative phase, in which th e reduced enzyme is oxidized by O-2. Previous studies indicated that proton translocation is coupled energetically only to the oxidative phase(4), but this has been challenged(5). Here, with the purified enzyme inlaid in lipo somes, we report time-resolved measurements of membrane potential, which sh ow that hall: of the electrical charges due to proton-pumping actually cros s the membrane during reduction after a preceding oxidative phase. pH measu rements confirm that proton translocation also occurs during reduction, but only when immediately preceded by an oxidative phase. We conclude that all the energy for proton translocation is conserved in the enzyme during its oxidation by O-2. One half of it is utilized for proton-pumping during oxid ation, but fhe other half is unlatched for this purpose only during re-redu ction of the enzyme.