Time-resolved generation of membrane potential by ba3 cytochrome c oxidase from Thermus thermophilus coupled to single electron injection into the O and OH 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 ba3 oxidase from T. thermophilus (O→E transition). The rapid phase reflects electron redistribution between CuA and heme b. The slow phase includes electron redistribution from both CuA and heme b to heme a3, and electrogenic proton transfer coupled to reduction of heme a3. The distance of proton translocation corresponds to uptake of a proton from the inner water phase into the binuclear center where heme a3 is reduced, but there is no proton pumping and no reduction of CuB. Single-electron reduction of the oxidized "unrelaxed" state (OH→EH transition) is accompanied by electrogenic reduction of the heme b/heme a3 pair by CuA 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 a3 pair to the CuB 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 CuB 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.