Proton pumping by cytochrome oxidase: progress, problems and postulates

The current status of our knowledge about the mechanism of proton pumping b y cytochrome oxidase is discussed. Significant progress has resulted from t he study of site-directed mutants within the proton-conducting pathways of the bacterial oxidases. There appear to be two channels to facilitate proto n translocation within the enzyme and they are important at different parts of the catalytic cycle. The use of hydrogen peroxide as an alternative sub strate provides a very useful experimental tool to explore the enzymology o f this system, and insights gained from this approach are described. Proton transfer is coupled to and appears to regulate the rate of electron transf er steps during turnover. It is proposed that the initial step in the react ion involves a proton transfer to the active site that is important to conv ert metal-ligated hydroxide to water, which can more rapidly dissociate fro m the metals and allow the reaction with dioxygen which, we propose, can bi nd; the one-electron reduced heme-copper center. Coordinated movement of pr otons and electrons over both short and long distances within the enzyme ap pear to be important at different parts of the catalytic cycle. During the initial reduction of dioxygen, direct hydrogen transfer to form a tyrosyl r adical at the active site seems likely. Subsequent steps can be effectively blocked by mutation of a residue at the surface of the protein, apparently preventing the entry of protons. (C) 2000 Elsevier Science B.V. All rights reserved.