Proton transfer from glutamate 286 determines the transition rates between oxygen intermediates in cytochrome c oxidase

• Published in: Biochimica et biophysica acta 2000-08, Vol.1459 (2), p.533-539
• Main Authors: Ådelroth, Pia, Karpefors, Martin, Gilderson, Gwen, Tomson, Farol L., Gennis, Robert B., Brzezinski, Peter
• Format: Article
• Language: English
• Subjects: Cytochrome aa3; Deuterium; Electron transfer; Electron Transport; Electron Transport Complex IV - chemistry; Electron Transport Complex IV - genetics; Flash photolysis; Flow flash; Glutamic Acid - chemistry; Mutation; Oxidation-Reduction; Oxygen - chemistry; Photolysis; Proton pumping; Proton transfer; Protons; Rhodobacter sphaeroides
• ISSN: 0005-2728; 0006-3002; 1879-2650
• DOI: 10.1016/S0005-2728(00)00194-8

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 ∼100 mV, which slows electron transfer to the binuclear centre during the F→O transition by a factor of ∼200. In ED(I-286) proton uptake during P R→F was slowed by a factor of ∼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 ∼2.5 as compared with ∼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. Ådelroth, 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 ∼8, i.e., about the same as in the wild-type enzyme, which shows that these reactions are intimately coupled.