title: Proton transfer from glutamate 286 determines the transition rates between oxygen intermediates in cytochrome c oxidase.
abstract: We have investigated the electron-proton coupling during the peroxy (P(R)) to oxo-ferryl (F) and F to oxidised (O) transitions in cytochrome c oxidase from Rhodobacter sphaeroides. The kinetics of these reactions were investigated in two different mutant enzymes: (1) ED(I-286), in which one of the key residues in the D-pathway, E(I-286), was replaced by an aspartate which has a shorter side chain than that of the glutamate and, (2) ML(II-263), in which the redox potential of Cu(A) is increased by approximately 100 mV, which slows electron transfer to the binuclear centre during the F-->O transition by a factor of approximately 200. In ED(I-286) proton uptake during P(R)-->F was slowed by a factor of approximately 5, which indicates that E(I-286) is the proton donor to P(R). In addition, in the mutant enzyme the F-->O transition rate displayed a deuterium isotope effect of approximately 2.5 as compared with approximately 7 in the wild-type enzyme. Since the entire deuterium isotope effect was shown to be associated with a single proton-transfer reaction in which the proton donor and acceptor must approach each other (M. Karpefors, P. Adelroth, P. Brzezinski, Biochemistry 39 (2000) 6850), the smaller deuterium isotope effect in ED(I-286) indicates that proton transfer from E(I-286) determines the rate also of the F-->O transition. In ML(II-263) the electron-transfer to the binuclear centre is slower than the intrinsic proton-transfer rate through the D-pathway. Nevertheless, both electron and proton transfer to the binuclear centre displayed a deuterium isotope effect of approximately 8, i.e., about the same as in the wild-type enzyme, which shows that these reactions are intimately coupled.
authors.affiliation: Department of Biochemistry, The Arrhenius Laboratories for Natural Sciences, Stockholm University, Sweden.
authors.forename: P
authors.initials: P
authors.lastname: Adelroth
authors.forename: M
authors.initials: M
authors.lastname: Karpefors
authors.forename: G
authors.initials: G
authors.lastname: Gilderson
authors.forename: F L
authors.initials: FL
authors.lastname: Tomson
authors.forename: R B
authors.initials: RB
authors.lastname: Gennis
authors.forename: P
authors.initials: P
authors.lastname: Brzezinski
mesh_terms: D003903:Deuterium; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry*; D009154:Mutation; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry*; D010782:Photolysis; D011522:Protons; D012242:Rhodobacter sphaeroides
chemical_list: D011522:Protons; D018698:Glutamic Acid; D003903:Deuterium; D003576:Electron Transport Complex IV; D010100:Oxygen