Factors determining electron-transfer rates in cytochrome c oxidase: Studies of the FQ(I-391) mutant of the Rhodobacter sphaeroides enzyme

Pia Ädelroth, David M. Mitchell, Robert B Gennis, Peter Brzezinski

Research output: Contribution to journalArticlepeer-review

Abstract

The mechanisms of internal electron transfer and oxygen reduction were investigated in cytochrome c oxidase from Rhodobacter sphaeroides (cytochrome aa3) using site-directed mutagenesis in combination with time-resolved optical absorption spectroscopy. Electron-transfer reactions in the absence of O2 were studied after flash photolysis of CO from the partly-reduced enzyme and the reaction of the fully-reduced enzyme with O2 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 ~2% (~30 s-1) of that of the wild-type enzyme. After flash photolysis of CO from the partly-reduced mutant enzyme ~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 103 s-1, which is significantly slower than in the wild-type enzyme. The redox potentials of hemes a and a3 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 O2 a rapid kinetic phase with a rate constant of 1.2 x 105 s-1, presumably associated with O2 binding, was followed by formation of the P intermediate with electrons from heme a3 and Cu(B) with a rate of ~4 x 103 s-1, and oxidation of the enzyme with a rate of ~30 s-1. The dramatically slower electron transfer between the hemes during O2 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 O2 reduction. The relation between the intrinsic heme a/heme a3 electron-transfer rate and equilibrium constant, and the electron-transfer rate from heme a to the binuclear center during O2 reduction is discussed.

Original languageEnglish (US)
Pages (from-to)11787-11796
Number of pages10
JournalBiochemistry
Volume36
Issue number39
DOIs
StatePublished - Sep 30 1997

ASJC Scopus subject areas

  • Biochemistry

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