Time-resolved generation of membrane potential by ba ₃ cytochrome c oxidase from Thermus thermophilus coupled to single electron injection into the O and O _H states

Two electrogenic phases with characteristic times of ~ 14 μs and ~ 290 μs are resolved in the kinetics of membrane potential generation coupled to single-electron reduction of the oxidized “relaxed” O state of ba ₃ oxidase from T. thermophilus (O → E transition). The rapid phase reflects electron redistribution between Cu _A and heme b. The slow phase includes electron redistribution from both Cu _A and heme b to heme a ₃ , and electrogenic proton transfer coupled to reduction of heme a ₃ . The distance of proton translocation corresponds to uptake of a proton from the inner water phase into the binuclear center where heme a ₃ is reduced, but there is no proton pumping and no reduction of Cu _B . Single-electron reduction of the oxidized “unrelaxed” state (O _H → E _H transition) is accompanied by electrogenic reduction of the heme b/heme a ₃ pair by Cu _A in a “fast” phase (~ 22 μs) and transfer of protons in “middle” and “slow” electrogenic phases (~ 0.185 ms and ~ 0.78 ms) coupled to electron redistribution from the heme b/heme a ₃ pair to the Cu _B 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 _B 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.