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/CuA 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/CuA pair is resolved as a single transition with τ~40μs. In the reaction of the fully reduced cytochrome caa3 with oxygen, the first electrogenic phase (τ~30μs) is linked to OO bond cleavage and generation of the PR state. The next electrogenic component (τ~50μs) is associated with the PR→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 ~0.25ms, ~1.4ms and ~4ms, are linked to the conversion of the binuclear center through the F→OH→EH 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.