title: Characterization of the exchangeable protons in the immediate vicinity of the semiquinone radical at the QH site of the cytochrome bo3 from Escherichia coli.
abstract: The cytochrome bo3 ubiquinol oxidase from Escherichia coli resides in the bacterial cytoplasmic membrane and catalyzes the two-electron oxidation of ubiquinol-8 and four-electron reduction of O2 to water. The one-electron reduced semiquinone forms transiently during the reaction, and the enzyme has been demonstrated to stabilize the semiquinone. Two-dimensional electron spin echo envelope modulation has been applied to explore the exchangeable protons involved in hydrogen bonding to the semiquinone by substitution of 1H2O by 2H2O. Three exchangeable protons possessing different isotropic and anisotropic hyperfine couplings were identified. The strength of the hyperfine interaction with one proton suggests a significant covalent O-H binding of carbonyl oxygen O1 that is a characteristic of a neutral radical, an assignment that is also supported by the unusually large hyperfine coupling to the methyl protons. The second proton with a large anisotropic coupling also forms a strong hydrogen bond with a carbonyl oxygen. This second hydrogen bond, which has a significant out-of-plane character, is from an NH2 or NH nitrogen, probably from an arginine (Arg-71) known to be in the quinone binding site. Assignment of the third exchangeable proton with smaller anisotropic coupling is more ambiguous, but it is clearly not involved in a direct hydrogen bond with either of the carbonyl oxygens. The results support a model that the semiquinone is bound to the protein in a very asymmetric manner by two strong hydrogen bonds from Asp-75 and Arg-71 to the O1 carbonyl, while the O4 carbonyl is not hydrogen-bonded to the protein.
authors.affiliation: Department of Biochemistry, University of Illinois, Urbana, Illinois 61801.
authors.forename: Lai Lai
authors.initials: LL
authors.lastname: Yap
authors.affiliation: Institute of Chemical Kinetics and Combustion, Russian Academy of Sciences, Novosibirsk 630090, Russia.
authors.forename: Rimma I
authors.initials: RI
authors.lastname: Samoilova
authors.affiliation: Department of Biochemistry, University of Illinois, Urbana, Illinois 61801. Electronic address: r-gennis@uiuc.edu.
authors.forename: Robert B
authors.initials: RB
authors.lastname: Gennis
authors.affiliation: Department of Veterinary Clinical Medicine, University of Illinois, Urbana, Illinois 61801. Electronic address: dikanov@uiuc.edu.
authors.forename: Sergei A
authors.initials: SA
authors.lastname: Dikanov
mesh_terms: D016880:Anisotropy; D016227:Benzoquinones; D001671:Biochemistry / Q000379:methods; D003573:Cytochrome b Group; D003580:Cytochromes / Q000235:genetics*; D004579:Electron Transport; D045222:Electron Transport Chain Complex Proteins; D004583:Electrons; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006860:Hydrogen Bonding; D007700:Kinetics; D008956:Models, Chemical; D011522:Protons
chemical_list: D016227:Benzoquinones; D003573:Cytochrome b Group; D003580:Cytochromes; D045222:Electron Transport Chain Complex Proteins; D029968:Escherichia coli Proteins; D011522:Protons; C083850:cytochrome bo3, E coli; C025232:semiquinone radicals