SINGLE-ELECTRON REDUCTION OF CYTOCHROME-C-OXIDASE COMPOUND-F - RESOLUTION OF PARTIAL STEPS BY TRANSIENT SPECTROSCOPY

The final step of the catalytic cycle of cytochrome oxide, the reducti on of oxyferryl heme a(3) in compound F, was investigated using a binu clear polypyridine ruthenium complex (Ru2C) as a photoactive reducing agent. The net charge of +4 on Ru2C allows it to bind electrostaticall y near Cu-A in subunit II of cytochrome oxidase. Photoexcitation of Ru 2C with a laser flash results in formation of a metal-to-ligand charge -transfer excited state, Ru2C, which rapidly transfers an electron to Cu-A of cytochrome oxidase from either beef heart or Rhodobacter spha eroides. This is followed by reversible electron transfer from CuA to heme a with forward and reverse rate constants of k(1) = 9.3 x 10(4) s (-1) and k(-1) = 1.7 x 10(4) s(-1) for R. sphaeroides cytochrome oxida se in the resting state. Compound F was prepared by treating the resti ng enzyme with excess hydrogen peroxide. The value of the rate constan t k(1) is the same in compound F where heme a(3) is in the oxyferryl f orm as in the resting enzyme where heme a(3) is ferric. Reduction of h eme a in compound F is followed by electron transfer from heme a to ox yferryl heme a(3) with a rate constant of 700 s(-1), as indicated by t ransients at 605 and 580 nm. No delay between heme a reoxidation and o xyferryl heme a(3) reduction is observed, showing that no electron-tra nsfer intermediates, such as reduced Cu-B, accumulate in this process. The rate constant for electron transfer from heme a to oxyferryl heme a(3) was measured in beef cytochrome oxidase from pH 7.0 to pH 9.5, a nd found to decrease upon titration of a group with a pK(a) of 9.0. Th e rate constant is slower in D2O than in H2O by a factor of 4.3, indic ating that the electron-transfer reaction is rate-limited by a proton- transfer step. The pH dependence and deuterium isotope effect for redu ction of isolated compound F are comparable to that observed during re action 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 a(3) is not controlled by conformational effects impos ed by the initial redox state of the enzyme. The rate constant for ele ctron transfer from heme a to oxyferryl heme a(3) is the same in the R . sphaeroides K362M CcO mutant as in wild-type CcO, indicating that th e K-channel is not involved in proton uptake during reduction of compo und F.