Relocation of an internal proton donor in cytochrome c oxidase results in an altered pKa and a non-integer pumping stoichiometry Original Research Article

Cytochrome c oxidase from Rhodobacter sphaeroides has two proton-input pathways leading from the protein surface towards the catalytic site, located within the membrane-spanning part of the enzyme. One of these pathways, the D-pathway, contains a highly conserved Glu residue [E(I-286)], which plays an important role in proton transfer through the pathway. In a recent study, we showed that a mutant enzyme in which E(I-286) was re-located to the opposite side of the D-pathway [EA(I-286)/IE(I-112) double mutant enzyme] was able to pump protons, although with a stoichiometry that was lower than that of the wild-type enzyme (∼0.6 H+/e−) (Aagaard et al. (2000) Biochemistry 39, 15847–15850). These results showed that the residue must not necessarily be located at a specific place in the amino-acid sequence, but rather at a specific location in space. In this study, we have investigated the effect of moving E(I-286) on the kinetics of specific reaction steps of the catalytic cycle in the pH range 6–11. Our results show that during the reaction of the four-electron reduced enzyme with O2, the rates of the two first transitions (up to formation of the ‘peroxy’ intermediate, Pr) are the same for the double mutant as for the wild-type enzyme, but formation of the oxo-ferryl (F) and fully oxidized (O) states, associated with proton uptake from the bulk solution, are slowed by factors of ∼30 and ∼400, respectively. Thus, in spite of the dramatically reduced transition rates, the proton-pumping stoichiometry is reduced only by ∼40%. The apparent pKa values in the pH-dependencies of the rates of the PR→F and F→O transitions were >3 and ∼2 units lower than those of the corresponding transitions in the wild-type enzyme, respectively. The relation between the modified pKas, the transition rates between oxygen intermediates and the pumping stoichiometry is discussed.