The electron distribution in the "activated" state of cytochrome c oxidase.

Cytochrome c oxidase catalyzes reduction of O2 to H2 O at a catalytic site that is composed of a copper ion and heme group. The reaction is linked to translocation of four protons across the membrane for each O2 reduced to water. The free energy associated with electron transfer to the catalytic site is unequal for the four electron-transfer events. Most notably, the free energy associated with reduction of the catalytic site in the oxidized cytochrome c oxidase (state O) is not sufficient for proton pumping across the energized membrane. Yet, this electron transfer is mechanistically linked to proton pumping. To resolve this apparent discrepancy, a high-energy oxidized state (denoted O H ) was postulated and suggested to be populated only during catalytic turnover. The difference between states O and O H was suggested to be manifested in an elevated midpoint potential of CuB in the latter. This proposal predicts that one-electron reduction of cytochrome c oxidase after its oxidation would yield re-reduction of essentially only CuB . Here, we investigated this process and found ~5% and ~6% reduction of heme a3 and CuB , respectively, i.e. the apparent redox potentials for heme a3 and CuB are lower than that of heme a.