Background: In the membrane-bound enzyme cytochrome c oxidase, electron transfer from cytochrome c to O2is linked to proton uptake from solution to form H2O, resulting in a charge separation across the membrane. In addition, the reaction drives pumping of protons across the membrane. Methods: In this study we have measured voltage changes as a function of pH during reaction of the four-electron reduced cytochrome c oxidase from Rhodobacter sphaeroides with O2. These electrogenic events were measured across membranes containing purified enzyme reconstituted into lipid vesicles. Results: The results show that the pH dependence of voltage changes (primarily associated with proton transfer) during O2reduction does not match that of the previously studied absorbance changes (primarily associated with electron transfer). Furthermore, the voltage changes decrease with increasing pH. Conclusions: The data indicate that cytochrome c oxidase does not pump protons at high pH (10.5) (or protons are taken from the "wrong" side of the membrane) and that at this pH the net proton-uptake stoichiometry is ∼ 1/2 of that at pH 8. Furthermore, the results provide a basis for interpretation of results from studies of mutant forms of the enzyme. General significance: These results provide new insights into the function of cytochrome c oxidase. © 2009 Elsevier B.V. All rights reserved.
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