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 (cytochrome aa(3)) using site-directed mutagenesis in combination with time-resolved optical absorption spectroscopy. Electron-transfer reactions in the absence of O-2 were studied after flash photolysis of CO from the partly-reduced enzyme and the reaction of the fully-reduced enzyme with O-2 was studied using the so-called flow-flash technique. Results from studies of the wild-type and mutant enzyme in which phenylalanine-391 of subunit I was replaced by glutamine (FQ(I-391)) were compared. The turnover activity of the mutant enzyme was similar to 2% (similar to 30 s(-1)) of that of the wild-type enzyme. After flash photolysis 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 enzyme, and the rate of this electron transfer was 3.2 x 10(3) s(-1), which is significantly slower than in the wild-type enzyme. The redox potentials of hemes a and a(3) in the mutant enzyme were found to be shifted by about +30 and -70 mV, respectively, as compared to the wild-type enzyme. During the reaction of the fully-reduced FQ(I-391) mutant enzyme 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 formation of the P intermediate with electrons from heme a(3) and Cu-B with a rate of similar to 4 x 10(3) s(-1), and oxidation of the enzyme with a rate of similar to 30 s(-1). The dramatically slower electron transfer between the hemes during O-2 reduction in the mutant enzyme is not 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 electron transfer from heme a to the binuclear center during O-2 reduction. The relation between the intrinsic heme a/heme a(3) electron-transfer rate and equilibrium constant, and the electron-transfer rate from heme a to the binuclear center during O-2 reduction is discussed.