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 reaction between cytochrome c (Cc) and Rhodobacter sphaeroides cytochrome c oxidase (CcO) was studied using a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine 55 (Ru-55-Cc).

Electron transfer between the Rieske iron-sulfur protein (Fe(2)S(2)) and cytochrome c(1) was studied using the ruthenium dimer, Ru(2)D, to either photoreduce or photooxidize cytochrome c(1) within 1 micros. Ru(2)D has a charge of +4, which allows it to bind with high affinity to the cytochrome bc(1) complex. Flash photolysis of a solution containing beef cytochrome bc(1), Ru(2)D, and a sacrificial donor resulted in reduction of cytochrome c(1) within 1 micros, followed by electron transfer from cytochrome c(1) to Fe(2)S(2) with a rate constant of 90,000 s(-1). Flash photolysis of reduced beef bc(1), Ru(2)D, and a sacrificial acceptor resulted in oxidation of cytochrome c(1) within 1 micros, followed by electron transfer from Fe(2)S(2) to cytochrome c(1) with a rate constant of 16,000 s(-1). Oxidant-induced reduction of cytochrome b(H) was observed with a rate constant of 250 s(-1) in the presence of antimycin A. Electron transfer from Fe(2)S(2) to cytochrome c(1) within the Rhodobacter sphaeroides cyt bc(1) complex was found to have a rate constant of 60,000 s(-1) at 25 degrees C, while reduction of cytochrome b(H) occurred with a rate constant of 1000 s(-1). Double mutation of Ala-46 and Ala-48 in the neck region of the Rieske protein to prolines resulted in a decrease in the rate constants for both cyt c(1) and cyt b(H) reduction to 25 s(-1), indicating that a conformational change in the Rieske protein has become rate-limiting.

The interaction domain for cytochrome c on the cytochrome bc 1 complex was studied using a series of Rhodobacter sphaeroides cytochrome bc 1 mutants in which acidic residues on the surface of cytochrome c 1 were substituted with neutral or basic residues. Intracomplex electron transfer was studied using a cytochrome c derivative labeled with ruthenium trisbipyri-