In the respiratory chain free energy is conserved by linking the chemical reduction of dioxygen to the electrogenic translocation of protons across a membrane. Cytochrome c oxidase (CcO) is one of the sites where this linkage occurs. Although intensively studied, the molecular mechanism of proton pumping by this enzyme remains unknown. Here, we present data from an investigation of a mutant CcO from Rhodobacter sphaeroides [Asn-139 --> Asp, ND(I-139)] in which proton pumping is completely uncoupled from the catalytic turnover (i.e., reduction of O2). However, in this mutant CcO, the rate by which O2 is reduced to H2O is even slightly higher than that of the wild-type CcO. The data indicate that the disabling of the proton pump is a result of a perturbation of E(I-286), which is located 20 A from N(I-139) and is an internal proton donor to the catalytic site, located in the membrane-spanning part of CcO. The mutation results in raising the effective pKa of E(I-286) by 1.6 pH units. An explanation of how the mutation uncouples catalytic turnover from proton pumping is offered, which suggests a mechanism by which CcO pumps protons.