The current status of our knowledge about the mechanism of proton pumping by cytochrome oxidase is discussed. Significant progress has resulted from the study of site-directed mutants within the proton-conducting pathways of the bacterial oxidases. There appear to be two channels to facilitate proton translocation within the enzyme and they are important at different parts of the catalytic cycle. The use of hydrogen peroxide as an alternative substrate provides a very useful experimental tool to explore the enzymology of this system, and insights gained from this approach are described. Proton transfer is coupled to and appears to regulate the rate of electron transfer steps during turnover. It is proposed that the initial step in the reaction involves a proton transfer to the active site that is important to convert metal-ligated hydroxide to water, which can more rapidly dissociate from the metals and allow the reaction with dioxygen which, we propose, can bind the one-electron reduced heme-copper center. Coordinated movement of protons and electrons over both short and long distances within the enzyme appear to be important at different parts of the catalytic cycle. During the initial reduction of dioxygen, direct hydrogen transfer to form a tyrosyl radical at the active site seems likely. Subsequent steps can be effectively blocked by mutation of a residue at the surface of the protein, apparently preventing the entry of protons.

Flash‐induced single‐electron reduction of cytochrome c oxidase. Compound F (oxoferryl state) by RuII(2,2'‐bipyridyl)2+ 3 [Nilsson (1992) Proc. Natl. Acad. Sci. USA 89, 6497‐6501] gives rise to three phases of membrane potential generation in proteoliposomes with τ values and contributions of ca. 45 μs (20%), 1 ms (20%) and 5 ms (60%). The rapid phase is not sensitive to the binuclear centre ligands, such as cyanide or peroxide, and is assigned to vectorial electron transfer from CuA to heme a. The two slow phases kinetically match reoxidation of heme a, require added H2O2 or methyl peroxide for full development, and are completely inhibited by cyanide; evidently, they are associated with the reduction of Compound F to the Ox state by heme a. The charge transfer steps associated with the F to Ox conversion are likely to comprise (i) electrogenic uptake of a ‘chemical’ proton from the N phase required for protonation of the reduced oxygen atom and (ii) electrogenic H+ pumping across the membrane linked to the F to Ox transition. Assuming heme a ‘electrical location’ in the middle of the dielectric barrier, the ratio of the rapid to slow electrogenic phase amplitudes indicates that the F to Ox transition is linked to transmembrane translocation of 1.5 charges (protons) in addition to an electrogenic uptake of one ‘chemical’ proton required to form Fe3+‐OH− from Fe4+ = O2−. The shortfall in the number of pumped protons and the biphasic kinetics of the millisecond part of the electric response matching biphasic reoxidation of heme a may indicate the presence of 2 forms of Compound F, reduction of only one of which being linked to full proton pumping.

The final step of the catalytic cycle of cytochrome oxidase, the reduction of oxyferryl heme a3 in compound F, was investigated using a binuclear polypyridine ruthenium complex (Ru2C) as a photoactive reducing agent. The net charge of +4 on Ru2C allows it to bind electrostatically near CuA in subunit II of cytochrome oxidase. Photoexcitation of Ru2C with a laser flash results in formation of a metal-to-ligand charge-transfer excited state, Ru2C, which rapidly transfers an electron to CuA of cytochrome oxidase from either beef heart or Rhodobacter sphaeroides. This is followed by reversible electron transfer from CuA to heme a with forward and reverse rate constants of k1 = 9.3 x 10(4) s-1 and k-1 = 1.7 x 10(4) s-1 for R. sphaeroides cytochrome oxidase in the resting state. Compound F was prepared by treating the resting enzyme with excess hydrogen peroxide. The value of the rate constant k1 is the same in compound F where heme a3 is in the oxyferryl form as in the resting enzyme where heme a3 is ferric. Reduction of heme a in compound F is followed by electron transfer from heme a to oxyferryl heme a3 with a rate constant of 700 s-1, as indicated by transients at 605 and 580 nm. No delay between heme a reoxidation and oxyferryl heme a3 reduction is observed, showing that no electron-transfer intermediates, such as reduced CuB, accumulate in this process. The rate constant for electron transfer from heme a to oxyferryl heme a3 was measured in beef cytochrome oxidase from pH 7.0 to pH 9.5, and found to decrease upon titration of a group with a pKa of 9.0. The rate constant is slower in D2O than in H2O by a factor of 4.3, indicating that the electron-transfer reaction is rate-limited by a proton-transfer step. The pH dependence and deuterium isotope effect for reduction of isolated compound F are comparable to that observed during reaction of the reduced, CO-inhibited CcO with oxygen by the flow-flash technique. This result indicates that electron transfer from heme a to oxyferryl heme a3 is not controlled by conformational effects imposed by the initial redox state of the enzyme. The rate constant for electron transfer from heme a to oxyferryl heme a3 is the same in the R. sphaeroides K362M CcO mutant as in wild-type CcO, indicating that the K-channel is not involved in proton uptake during reduction of compound F.

The ba 3 -type cytochrome c oxidase from T. thermophilus is phylogenetically very distant from the aa 3 -type cytochrome c oxidases. Nevertheless, both types of oxidases have the same number of redox-active metal sites and the reduction of O 2 to water is catalysed at a haem a 3 -Cu B catalytic site. The three-dimensional structure of the ba 3 oxidase reveals three possible proton-conducting pathways showing very low homology compared to those of the mitochondrial, R. sphaeroides and P. denitrificans aa 3 oxidases. In this study we investigated the oxidative part of the catalytic cycle of the ba 3 -cytochrome c oxidase using the flow-flash method. After flash-induced dissociation of CO from the fully reduced enzyme in the presence of oxygen we observed rapid oxidation of cytochrome b (k ≅ 6.8*10 4 s −1 ) and formation of the peroxy (P R ) intermediate. In the next step a proton was taken up from solution with a rate constant of ~1.7*10 4 s −1 , associated with formation of the ferryl (F) intermediate, simultaneous with transient reduction of haem b. Finally, the enzyme was oxidized with a rate constant of ~1100 s −1 , accompanied by additional proton uptake. The total proton uptake stoichiometry in the oxidative part of the catalytic cycle was ~1.5 protons per enzyme molecule. The results support the earlier proposal that the P R and F intermediate spectra are similar (Siletsky et al. (2007 and show that even though the architecture of the proton-conducting pathways is different in the ba 3 oxidases, the proton-uptake reactions occur over the same time scales as in the aa 3 -type oxidases.