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Abstract

Several amino acid residues that have been shown to be essential for proton transfer in most cytochrome c oxidases are not conserved in the ba₃-type cytochrome c oxidase from the thermophilic eubacterium Thermus thermophilus. So far, it has been unclear whether the Th. thermophilus ba₃-type cytochrome c oxidase can nevertheless function as an electrogenic proton pump. In this study, we have combined charge translocation measurements on a lipid bilayer with two independent methods of proton pumping measurements to show that enzymatic turnover of the Th. thermophilus cytochrome c oxidase is indeed coupled to the generation of an electrocurrent and proton pumping across the membrane. In addition to a 'vectorial' consumption of 1.0 H⁺/e^- for water formation, proton pumping with a stoichiometry of 0.4-0.5 H⁺/e^- was observed. The implications of these findings for the mechanism of redox-coupled proton transfer in this unusual cytochrome c oxidase are discussed.