Proton uptake controls electron transfer in cytochrome c oxidase

Proton uptake controls electron transfer in cytochrome c oxidase

^*Department of Biochemistry and Biophysics, Göteborg University, (Medicinaregatan 9C) P.O. Box 462, SE-405 30 Göteborg, Sweden, and ^†Department of Biochemistry, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden; and ^‡Department of Biochemistry, Michigan State University, East Lansing, MI 48824

Communicated by Rudolph A. Marcus, California Institute of Technology, Pasadena, CA (received for review June 17, 1998)

Abstract

In cytochrome c oxidase, a requirement for proton pumping is a tight coupling between electron and proton transfer, which could be accomplished if internal electron-transfer rates were controlled by uptake of protons. During reaction of the fully reduced enzyme with oxygen, concomitant with the “peroxy” to “oxoferryl” transition, internal transfer of the fourth electron from Cu_A to heme a has the same rate as proton uptake from the bulk solution (8,000 s⁻¹). The question was therefore raised whether the proton uptake controls electron transfer or vice versa. To resolve this question, we have studied a site-specific mutant of the Rhodobacter sphaeroides enzyme in which methionine 263 (SU II), a Cu_A ligand, was replaced by leucine, which resulted in an increased redox potential of Cu_A. During reaction of the reduced mutant enzyme with O₂, a proton was taken up at the same rate as in the wild-type enzyme (8,000 s⁻¹), whereas electron transfer from Cu_A to heme a was impaired. Together with results from studies of the EQ(I-286) mutant enzyme, in which both proton uptake and electron transfer from Cu_A to heme a were blocked, the results from this study show that the Cu_A → heme a electron transfer is controlled by the proton uptake and not vice versa. This mechanism prevents further electron transfer to heme a ₃–Cu_B before a proton is taken up, which assures a tight coupling of electron transfer to proton pumping.

flow–flash/proton pumping/cytochrome aa3/flash photolysis/gating

Footnotes

↵ § To whom reprint requests should be addressed. e-mail: peter{at}bcbp.gu.se.
ABBREVIATIONS:

PR,

peroxy intermediate formed in the fully reduced enzyme;

F,

oxoferryl intermediate;

O,

fully oxidized enzyme;

τ,

time constant [exp(-t/τ)];

WT,

wild type;

CuA,

copper A;

CuB,

copper B;

mixed-valence state,

a state in which heme a3/CuB are reduced and heme a/CuA are oxidized;

mutant-enzyme nomenclature,

ML(II-263) denotes a replacement of methionine-263 of subunit II by leucine. If not otherwise indicated, amino acid residues are numbered according to the R. sphaeroides sequence