Time-resolved generation of membrane potential by ba cytochrome c oxidase from Thermus thermophilus coupled to single electron injection into the O and O states.

Two electrogenic phases with characteristic times of 14μs and ~290μs are resolved in the kinetics of membrane potential generation coupled to single-electron reduction of the oxidized "relaxed" O state of ba oxidase from T. thermophilus (O→E transition). The rapid phase reflects electron redistribution between Cu and heme b. The slow phase includes electron redistribution from both Cu and heme b to heme a, and electrogenic proton transfer coupled to reduction of heme a. The distance of proton translocation corresponds to uptake of a proton from the inner water phase into the binuclear center where heme a is reduced, but there is no proton pumping and no reduction of Cu. Single-electron reduction of the oxidized "unrelaxed" state (O→E transition) is accompanied by electrogenic reduction of the heme b/heme a pair by Cu in a "fast" phase (22μs) and transfer of protons in "middle" and "slow" electrogenic phases (~0.185ms and ~0.78ms) coupled to electron redistribution from the heme b/heme a pair to the Cu site. The "middle" and "slow" electrogenic phases seem to be associated with transfer of protons to the proton-loading site (PLS) of the proton pump, but when all injected electrons reach Cu the electronic charge appears to be compensated by back-leakage of the protons from the PLS into the binuclear site. Thus proton pumping occurs only to the extent of ~0.1 H/e, probably due to the formed membrane potential in the experiment.