Timing of electron and proton transfer in the ba₃ cytochrome c oxidase from Thermus thermophilus

Heme-copper oxidases are membrane-bound proteins that catalyze the reduction of O₂ to H₂O, a highly exergonic reaction. Part of the free energy of this reaction is used for pumping of protons across the membrane. The ba₃ oxidase from Thermus thermophilus presumably uses a single proton pathway for the transfer of substrate protons used during O ₂ reduction as well as for the transfer of the protons that are pumped across the membrane. The pumping stoichiometry (0.5 H⁺/ electron) is lower than that of most other (mitochondrial-like) oxidases characterized to date (1 H⁺/electron). We studied the pH dependence and deuterium isotope effect of the kinetics of electron and proton transfer reactions in the ba₃ oxidase. The results from these studies suggest that the movement of protons to the catalytic site and movement to a site located some distance from the catalytic site [proposed to be a "proton-loading site" (PLS) for pumped protons] are separated in time, which allows individual investigation of these reactions. A scenario in which the uptake and release of a pumped proton occurs upon every second transfer of an electron to the catalytic site would explain the decreased proton pumping stoichiometry compared to that of mitochondrial-like oxidases. (Graph Presented).