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

Cytochrome c oxidase catalyzes reduction of O₂ to H₂O 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 Cu_B in the latter. This proposal predicts that one-electron reduction of cytochrome c oxidase after its oxidation would yield re-reduction of essentially only Cu_B. Here, we investigated this process and found ~5% and ~6% reduction of heme a₃ and Cu_B, respectively, i.e. the apparent redox potentials for heme a₃ and Cu_B are lower than that of heme a.