Proton pumping in cytochrome c oxidase: the coupling between proton and electron gating.

C ytochrome c oxidase is the terminal oxidase in cellular respiration. This membrane protein accepts electrons from ferrocytochrome c in the periplasmic space of the mitochondrion, one electron at a time, and transfers the reducing equivalents to the binuclear heme-iron copper site (the socalled Fea3, CuB site), where dioxygen binds and the O·O bond is subsequently cleaved (1). In this manner, the binuclear center is activated by the dioxygen, and the heme iron is oxidized by two oxidizing equivalents to form Fea3–oxyferryl, namely, Fea3 4+ = O, and the CuB site by two oxidizing equivalents to form the CuB 2+ –OH/tyrosyl radical species (2). Subsequent inputs of additional reducing equivalents from reduced cytochrome c to the low-potential CuA and Fea centers and the transfers of these electrons to the activated binuclear center are linked to proton pumping (3). In a high-resolution X-ray structural analysis of the CO,NO, andCN derivatives of bovine heart cytochrome c oxidase reported in PNAS, Yoshikawa et al. (2) have attempted to derive insights into the possible structural changes that might occur in the enzyme near the active site upon the activation of the binuclear center by molecular oxygen. It is conjectured that dioxygen binding to the Fea3 2+ first generates a Fea3 -superoxo species (Fea3 -O2 ), mimicking a CN species bound to the oxidized Fea3 3+ (Fea3 −CN). Subsequent transfer of two reducing equivalents from the CuB 1+ and Tyr244, the tyrosine that is covalently linked to the CuB histidine ligand His240, cleaves the O·O bond, creating the oxylferryl species (Fea3 4+ = O) and the oxidized CuB site (CuB −OH/tyrosyl radical) mentioned earlier. The authors argue that this mechanism of dioxygen reduction minimizes the likelihood of releasing active oxygen species, which is undoubtedly true. More importantly, however, the authors have inferred from these structural results a set of conformational changes at the binuclear site that might be linked to the gating of the protons poised for proton pumping, an obligatory step in the overall mechanism of proton translocation mediated by this enzyme. A redox-linked proton pump is a complex molecular machine (3–6). First, there must be sufficient redox energy to drive the protons uphill: in the case of cytochrome c oxidase, against the protomotive force that exists across the inner membrane of the mitochrondrion. To accomplish the process with reasonable rates, there must be some compromise of the thermodynamic efficiency in the conversion of the redox energy to chemical work. As a kinetically driven process, some fraction of the redox energy driving the proton-pumping reaction mediated by cytochrome c oxidase must be dissipated as heat. Second, there must be a molecular mechanism coupling the redox energy driving the process to the protons that are being pumped. In other words, there must be a mechanism of redox linkage (3, 4). This mechanism could be an electrostatic one, in which the coulomb interaction between the electron and proton somehow drives the coupled vectorial proton and electron transfer in opposite directions (Fig. 1). In this simple scenario, the protons must first be gated by dioxygen activation of the binuclear site, which ultimately accepts the electrons linked to the vectorial proton translocation. Alternatively (4), with the binuclear site already activated by dioxygen, the redox linkage could occur at CuA or Fea, the site(s) receiving the reducing equivalents from the cytochrome c. Here, part of the redox energy between the low-potential centers and the dioxygen-activated binuclear center could be transferred nonadiabatically to the protein, changing its conformation irreversibly, albeit slightly, but in a manner that gates the electron flow as well as the protons to be pumped (Fig. 2). This gating of the electron flow would allow the enzyme to Ferrocytochrome c