Redox-Controlled Proton Gating in Bovine Cytochrome C Oxidase

Cytochrome c oxidase is the terminal enzyme in the electron transfer chain of essentially all organisms that utilize oxygen to generate energy. It reduces oxygen to water and harnesses the energy to pump protons across the mitochondrial membrane in eukaryotes and the plasma membrane in prokaryotes. The mechanism by which the oxygen reduction reaction is coupled to proton pumping remains unresolved, owing to the difficulty of visualizing proton movement within the massive membrane-associated protein matrix. Here, with a novel hydrogen/deuterium exchange resonance Raman spectroscopy method (1), we have identified two critical elements of the proton pump: a proton loading site near the propionate A group of heme a, which is capable of transiently storing protons uploaded from the negative-side of the membrane prior to their release into the positive-side of the membrane and a conformational gate that controls proton translocation in response to the change in the redox state of heme a. These findings form the basis for a new molecular model describing the mechanism, by which unidirectional proton translocation is coupled to electron transfer from heme a to heme a3 associated with oxygen chemistry occurring in the heme a3 site during enzymatic turnover.