Single Electron Reduction of Cytochrome c Oxidase Compound F:  Resolution of Partial Steps by Transient Spectroscopy

• Published in: Biochemistry (Easton) 1998-10, Vol.37 (42), p.14910-14916
• Main Authors: Zaslavsky, Dmitry, Sadoski, Robert C, Wang, Kefei, Durham, Bill, Gennis, Robert B, Millett, Francis
• Format: Article
• Language: English
• Subjects: 2,2'-Dipyridyl - analogs & derivatives; 2,2'-Dipyridyl - chemistry; Animals; Cattle; Electron Transport; Electron Transport Complex IV - chemistry; Electron Transport Complex IV - genetics; Electron Transport Complex IV - metabolism; Heme - analogs & derivatives; Heme - chemistry; Heme - metabolism; Indicators and Reagents; Kinetics; Lysine - genetics; Methionine - genetics; Oxidation-Reduction; Photolysis; Proton Pumps - chemistry; Rhodobacter sphaeroides - enzymology; Rhodobacter sphaeroides - genetics; Spectrophotometry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi981490z

The final step of the catalytic cycle of cytochrome oxidase, 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 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 k 1 = 9.3 × 104 s-1 and k - 1 = 1.7 × 104 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 CuB, 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.