LAUSR

Significance We present a comprehensive investigation of mitochondrial DNA-encoded variants of cytochrome c oxidase (CcO) that harbor mutations within their core catalytic subunit I, designed to interrogate the presently disputed functions of the three putative proton channels. We assess overall respiratory competence, specific CcO catalytic activity, and, most importantly, proton/electron (H+/e−) stoichiometry from adenosine diphosphate to oxygen ratio measurements on preparations of intact mitochondria. We unequivocally show that yeast mitochondrial CcO uses the D-channel to translocate protons across its hydrophilic core, providing direct evidence in support of a common proton pumping mechanism across all members of the A-type heme-copper oxidase superfamily, independent of their bacterial or mitochondrial origin. Mitochondria metabolize almost all the oxygen that we consume, reducing it to water by cytochrome c oxidase (CcO). CcO maximizes energy capture into the protonmotive force by pumping protons across the mitochondrial inner membrane. Forty years after the H+/e− stoichiometry was established, a consensus has yet to be reached on the route taken by pumped protons to traverse CcO’s hydrophobic core and on whether bacterial and mitochondrial CcOs operate via the same coupling mechanism. To resolve this, we exploited the unique amenability to mitochondrial DNA mutagenesis of the yeast Saccharomyces cerevisiae to introduce single point mutations in the hydrophilic pathways of CcO to test function. From adenosine diphosphate to oxygen ratio measurements on preparations of intact mitochondria, we definitely established that the D-channel, and not the H-channel, is the proton pump of the yeast mitochondrial enzyme, supporting an identical coupling mechanism in all forms of the enzyme.