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.