Single electron reduction of cytochrome c oxidase compound F: Resolution of partial steps by transient spectroscopy

by Zaslavsky, D.; Sadoski, R. C.; Wang, K. F.; Durham, B.; Gennis, R. B.; Millett, F.

The final step of the catalytic cycle of cytochrome oxide, the reduction of oxyferryl heme a(3) 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 Cu-A 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 Cu-A 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 k(1) = 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 k(1) is the same in compound F where heme a(3) is in the oxyferryl form as in the resting enzyme where heme a(3) is ferric. Reduction of heme a in compound F is followed by electron transfer from heme a to oxyferryl heme a(3) 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 a(3) reduction is observed, showing that no electron-transfer 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, and found to decrease upon titration of a group with a pK(a) 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 a(3) 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 a(3) 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.

Start Page
1520-4995; 0006-2960