Proton-transfer pathways in the mitochondrial S. cerevisiae cytochrome c oxidase

Abstract

Abstract In cytochrome c oxidase (Cyt c O) reduction of O 2 to water is linked to uptake of eight protons from the negative side of the membrane: four are substrate protons used to form water and four are pumped across the membrane. In bacterial oxidases, the substrate protons are taken up through the K and the D proton pathways, while the pumped protons are transferred through the D pathway. On the basis of studies with Cyt c O isolated from bovine heart mitochondria, it was suggested that in mitochondrial Cyt c Os the pumped protons are transferred though a third proton pathway, the H pathway, rather than through the D pathway. Here, we studied these reactions in S. cerevisiae Cyt c O, which serves as a model of the mammalian counterpart. We analyzed the effect of mutations in the D (Asn99Asp and Ile67Asn) and H pathways (Ser382Ala and Ser458Ala) and investigated the kinetics of electron and proton transfer during the reaction of the reduced Cyt c O with O 2 . No effects were observed with the H pathway variants while in the D pathway variants the functional effects were similar to those observed with the R. sphaeroides Cyt c O. The data indicate that the S. cerevisiae Cyt c O uses the D pathway for proton uptake and presumably also for proton pumping.