FACTORS DETERMINING ELECTRON-TRANSFER RATES IN CYTOCHROME-C-OXIDASE -STUDIES OF THE FQ(I-391) MUTANT OF THE RHODOBACTER-SPHAEROIDES ENZYME

The mechanisms of internal electron transfer and oxygen reduction were investigated in cytochrome c oxidase from Rhodobacter sphaeroides (cy tochrome aa(3)) using site-directed mutagenesis in combination with ti me-resolved optical absorption spectroscopy. Electron-transfer reactio ns in the absence of O-2 were studied after flash photolysis of CO fro m the partly-reduced enzyme and the reaction of the fully-reduced enzy me with O-2 was studied using the so-called flow-flash technique. Resu lts from studies of the wild-type and mutant enzyme in which phenylala nine-391 of subunit I was replaced by glutamine (FQ(I-391)) were compa red. The turnover activity of the mutant enzyme was similar to 2% (sim ilar to 30 s(-1)) of that of the wild-type enzyme. After flash photoly sis of CO from the partly-reduced mutant enzyme similar to 80% of Cu-A was reduced, which is a much larger fraction than in the wild-type en zyme, and the rate of this electron transfer was 3.2 x 10(3) s(-1), wh ich is significantly slower than in the wild-type enzyme. The redox po tentials of hemes a and a(3) in the mutant enzyme were found to be shi fted by about +30 and -70 mV, respectively, as compared to the wild-ty pe enzyme. During the reaction of the fully-reduced FQ(I-391) mutant e nzyme with O-2 a rapid kinetic phase with a rate constant of 1.2 x 10( 5) s(-1), presumably associated with O-2 binding, was followed by form ation of the P intermediate with electrons from heme a(3) and Cu-B wit h a rate of similar to 4 x 10(3) s(-1), and oxidation of the enzyme wi th a rate of similar to 30 s(-1). The dramatically slower electron tra nsfer between the hemes during O-2 reduction in the mutant enzyme is n ot only due to the slower intrinsic electron transfer, but also due to the altered redox potentials. In addition, the results show that the reduced overall activity of the mutant enzyme is due to the slower ele ctron transfer from heme a to the binuclear center during O-2 reductio n. The relation between the intrinsic heme a/heme a(3) electron-transf er rate and equilibrium constant, and the electron-transfer rate from heme a to the binuclear center during O-2 reduction is discussed.