The fifth electron in the fully reduced caa₃ from Thermus thermophilus is competent in proton pumping

The time-resolved kinetics of membrane potential generation coupled to oxidation of the fully reduced (five-electron) caa₃ cytochrome oxidase from Thermus thermophilus by oxygen was studied in a single-turnover regime. In order to calibrate the number of charges that move across the vesicle membrane in the different reaction steps, the reverse electron transfer from heme a₃ to heme a and further to the cytochrome c/Cu_A has been resolved upon photodissociation of CO from the mixed valence enzyme in the absence of oxygen. The reverse electron transfer from heme a₃ to heme a and further to the cytochrome c/Cu_A pair is resolved as a single transition with τ ∼ 40 μs. In the reaction of the fully reduced cytochrome caa₃ with oxygen, the first electrogenic phase (τ ∼ 30 μs) is linked to OO bond cleavage and generation of the P _R state. The next electrogenic component (τ ∼ 50 μs) is associated with the P_R → F transition and together with the previous reaction step it is coupled to translocation of about two charges across the membrane. The three subsequent electrogenic phases, with time constants of ∼ 0.25 ms, ∼ 1.4 ms and ∼ 4 ms, are linked to the conversion of the binuclear center through the F → O_H → E_H transitions, and result in additional transfer of four charges through the membrane dielectric. This indicates that the delivery of the fifth electron from heme c to the binuclear center is coupled to pumping of an additional proton across the membrane.