The O2 reduction and proton pumping gate mechanism of bovine heart cytochrome c oxidase

X-ray structures of bovine heart cytochrome c oxidase with bound respiratory inhibitors (O2 analogues) have been determined at 1.8–2.05 Å resolution to investigate the function of the O2 reduction site which includes two metal sites (Fea32+ and CuB1+). The X-ray structures of the CO- and NO-bound derivatives indicate that although there are three possible electron donors that can provide electrons to the bound O2, located in the O2 reduction site, the formation of the peroxide intermediate is effectively prevented to provide an O2-bound form as the initial intermediate. The structural change induced upon binding of CN− suggests a non-sequential 3-electoron reduction of the bound O2− for the complete reduction without release of any reactive oxygen species. The X-ray structure of the derivative with CO bound to CuB1+ after photolysis from Fea32+ demonstrates weak side-on binding. This suggests that CuB controls the O2 supply to Fea32+ without electron transfer to provide sufficient time for collection of protons from the negative side of the mitochondrial membrane. The proton-pumping pathway of bovine heart cytochrome c oxidase includes a hydrogen-bond network and a water channel located in tandem between the positive and negative side of the mitochondrial membrane. Binding of a strong ligand to Fea3 induces a conformational change which significantly narrows the water channel and effectively blocks the back-leakage of protons from the hydrogen bond network. The proton pumping mechanism proposed by these X-ray structural analyses has been functionally confirmed by mutagenesis analyses of bovine heart cytochrome c oxidase. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.

► Respiratory inhibitors as probes for mechanism of cytochrome c oxidase function.
► O2 reduction by O2 binding to Fe2+, followed by non-sequential 3-electron reduction
► Positive charges of low spin heme, upon oxidation, pump protons electrostatically.
► X-ray and mutational identification of proton pump system of cytochrome c oxidase.