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 (O-2 analogues) have been determined at 1.8-2.05 angstrom resolution to investigate the function of the O-2 reduction site which includes two metal sites (Fe-a3(2+) and Cu-B(1+)). 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 O-2, located in the O-2 reduction site, the formation of the peroxide intermediate is effectively prevented to provide an O-2-bound form as the initial intermediate. The structural change induced upon binding of CN- suggests a non-sequential 3-electoron reduction of the bound O-2(-) for the complete reduction without release of any reactive oxygen species. The X-ray structure of the derivative with CO bound to Cu-B(1+) after photolysis from Fe-a3(2+) demonstrates weak side-on binding. This suggests that Cu-B controls the O-2 supply to Fe-a3(2+) 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 Fe-a3 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. (C) 2011 Published by Elsevier B.V.