Impaired proton pumping in cytochrome c oxidase upon structural alteration of the D pathway

Cytochrome c oxidase is a membrane-bound enzyme, which catalyses the one-electron oxidation of four molecules of cytochrome c and the four-electron reduction of O₂ to water. Electron transfer through the enzyme is coupled to proton pumping across the membrane. Protons that are pumped as well as those that are used for O₂ reduction are transferred though a specific intraprotein (D) pathway. Results from earlier studies have shown that replacement of residue Asn139 by an Asp, at the beginning of the D pathway, results in blocking proton pumping without slowing uptake of substrate protons used for O₂ reduction. Furthermore, introduction of the acidic residue results in an increase of the apparent pK_a of E286, an internal proton donor to the catalytic site, from 9.4 to ~ 11. In this study we have investigated intramolecular electron and proton transfer in a mutant cytochrome c oxidase in which a neutral residue, Thr, was introduced at the 139 site. The mutation results in uncoupling of proton pumping from O₂ reduction, but a decrease in the apparent pK_a of E286 from 9.4 to 7.6. The data provide insights into the mechanism by which cytochrome c oxidase pumps protons and the structural elements involved in this process.