Proton translocation by cytochrome c oxidase

Cell respiration in mitochondria and some bacteria is catalysed by cytochrome c oxidase, which reduces O 2 to water, coupled with translocation of four protons across the mitochondrial or bacterial membrane 1 , 2 , 3 . The enzyme's catalytic cycle consists of a reductive phase, in which the oxidized enzyme receives electrons from cytochrome c , and an oxidative phase, in which the 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 liposomes, we report time-resolved measurements of membrane potential, which show that half of the electrical charges due to proton-pumping actually cross the membrane during reduction after a preceding oxidative phase. pH measurements 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 oxidation, but the other half is unlatched for this purpose only during re-reduction of the enzyme.