title: Proton-transport mechanisms in cytochrome c oxidase revealed by studies of kinetic isotope effects.
abstract: Cytochrome c oxidase (CytcO) is a membrane-bound enzyme, which catalyzes the reduction of di-oxygen to water and uses a major part of the free energy released in this reaction to pump protons across the membrane. In the Rhodobacter sphaeroides aa₃ CytcO all protons that are pumped across the membrane, as well as one half of the protons that are used for O₂ reduction, are transferred through one specific intraprotein proton pathway, which holds a highly conserved Glu286 residue. Key questions that need to be addressed in order to understand the function of CytcO at a molecular level are related to the timing of proton transfers from Glu286 to a "pump site" and the catalytic site, respectively. Here, we have investigated the temperature dependencies of the H/D kinetic-isotope effects of intramolecular proton-transfer reactions in the wild-type CytcO as well as in two structural CytcO variants, one in which proton uptake from solution is delayed and one in which proton pumping is uncoupled from O₂ reduction. These processes were studied for two specific reaction steps linked to transmembrane proton pumping, one that involves only proton transfer (peroxy-ferryl, P→F, transition) and one in which the same sequence of proton transfers is also linked to electron transfer to the catalytic site (ferryl-oxidized, F→O, transition). An analysis of these reactions in the framework of theory indicates that that the simpler, P→F reaction is rate-limited by proton transfer from Glu286 to the catalytic site. When the same proton-transfer events are also linked to electron transfer to the catalytic site (F→O), the proton-transfer reactions might well be gated by a protein structural change, which presumably ensures that the proton-pumping stoichiometry is maintained also in the presence of a transmembrane electrochemical gradient. Furthermore, the present study indicates that a careful analysis of the temperature dependence of the isotope effect should help us in gaining mechanistic insights about CytcO.
authors.affiliation: Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, Sweden.
authors.forename: Ann-Louise
authors.initials: AL
authors.lastname: Johansson
authors.forename: Suman
authors.initials: S
authors.lastname: Chakrabarty
authors.forename: Catrine L
authors.initials: CL
authors.lastname: Berthold
authors.forename: Martin
authors.initials: M
authors.lastname: Högbom
authors.forename: Arieh
authors.initials: A
authors.lastname: Warshel
authors.forename: Peter
authors.initials: P
authors.lastname: Brzezinski
mesh_terms: D003576:Electron Transport Complex IV / Q000737:chemistry / Q000378:metabolism*; D017136:Ion Transport; D007554:Isotopes; D007700:Kinetics; D008958:Models, Molecular; D011522:Protons; D012242:Rhodobacter sphaeroides / Q000201:enzymology; D013696:Temperature
chemical_list: D007554:Isotopes; D011522:Protons; D003576:Electron Transport Complex IV