A mechanistic principle for proton pumping by cytochrome c oxidase.

In aerobic organisms, cellular respiration involves electron transfer to oxygen through a series of membrane-bound protein complexes. The process maintains a transmembrane electrochemical proton gradient that is used, for example, in the synthesis of ATP. In mitochondria and many bacteria, the last enzyme complex in the electron transfer chain is cytochrome c oxidase (CytcO), which catalyses the four-electron reduction of O2 to H2O using electrons delivered by a water-soluble donor, cytochrome c. The electron transfer through CytcO, accompanied by proton uptake to form H2O drives the physical movement (pumping) of four protons across the membrane per reduced O2. So far, the molecular mechanism of such proton pumping driven by electron transfer has not been determined in any biological system. Here we show that proton pumping in CytcO is mechanistically coupled to proton transfer to O2 at the catalytic site, rather than to internal electron transfer. This scenario suggests a principle by which redox-driven proton pumps might operate and puts considerable constraints on possible molecular mechanisms by which CytcO translocates protons.

Subunit III of cytochrome c oxidase of Rhodobacter sphaeroides is required to maintain rapid proton uptake through the D pathway at physiologic pH.

The catalytic core of cytochrome c oxidase is composed of three subunits where subunits I and II contain all of the redox-active metal centers and subunit III is a seven transmembrane helix protein that binds to subunit I. The N-terminal region of subunit III is adjacent to D132 of subunit I, the initial proton acceptor of the D pathway that transfers protons from the protein surface to the buried active site approximately 30 A distant. The absence of subunit III only slightly alters the initial steady-state activity of the oxidase at pH 6.5, but activity declines sharply with increasing pH, yielding an apparent pK(a) of 7.2 for steady-state O(2) reduction. When subunit III is present, cytochrome oxidase is more active at higher pH, and the apparent pK(a) of steady-state O(2) reduction is 8.5. Single-turnover experiments show that proton uptake through the D pathway at pH 8 slows from >10000 s(-1) in the presence of subunit III to 350 s(-1) in its absence. At low pH (5.5) the D pathway of the oxidase lacking subunit III regains its capacity for rapid proton uptake. Analysis of the F --> O transition indicates that the apparent pK(a) of the D pathway in the absence of subunit III is 6.8, similar to that of steady-state O(2) reduction (7.2). The pK(a) of D132 itself may decline in the absence of subunit III since its carboxylate group will be more exposed to solvent water. Alternatively, part of a proton antenna for the D pathway may be lost upon removal of subunit III. It is proposed that one role of subunit III in the normal oxidase is to maintain rapid proton uptake through the D pathway at physiologic pH.

Relocation of an internal proton donor in cytochrome c oxidase results in an altered pK(a) and a non-integer pumping stoichiometry.

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 (approximately 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 O(2), the rates of the two first transitions (up to formation of the 'peroxy' intermediate, P(r)) 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 approximately 30 and approximately 400, respectively. Thus, in spite of the dramatically reduced transition rates, the proton-pumping stoichiometry is reduced only by approximately 40%. The apparent pK(a) values in the pH-dependencies of the rates of the P(R)-->F and F-->O transitions were >3 and approximately 2 units lower than those of the corresponding transitions in the wild-type enzyme, respectively. The relation between the modified pK(a)s, the transition rates between oxygen intermediates and the pumping stoichiometry is discussed.