title: Studies on a stabilisation of ubisemiquinone by Escherichia coli quinol oxidase, cytochrome bo.
abstract: The Escherichia coli quinol oxidase, cytochrome bo, is closely related to the cytochrome c oxidase, cytochrome aa3 in all aspects of its structure and function except for the replacement of the cytochrome-c-binding site and its attendant CuA prosthetic group with a quinone-binding site. The putative oxidation of quinol by ferrihaem (cytochrome b) at this site in sequential one-electron steps requires the stabilisation of semiquinone. We have observed, by electron paramagnetic resonance, the properties of a ubisemiquinone radical in appropriately poised samples of purified enzyme reconstituted with excess ubiquinone. The ubisemiquinone is highly stabilised with respect to free ubisemiquinone; significant free radical can be observed even at pH 7.0, while at pH 9.0 the stability constant is 5-10. The pH dependence of the stability constant indicates that the anionic form of the semiquinone predominates above pH 7.5. The two-electron couple has an Em7 of approximately 70 mV. Below pH 9, the pH dependence of the two-electron couple is -60mV/pH, indicative of a 2H+/2e- reaction. The line width of the EPR spectrum is approximately 0.9 mT, which is consistent with a ubisemiquinone anion. In comparison with other respiratory chain Q.- species that have been described, the relaxation rate in the presence of reduced haems appears comparable to magnetically isolated Q.- radicals. Partially resolved splittings of approximately 0.4 mT can be observed in the spectrum of Q.-bo (QH.bo).
authors.affiliation: School of Biological and Medical Sciences, University of St. Andrews, Scotland.
authors.forename: W J
authors.initials: WJ
authors.lastname: Ingledew
authors.forename: T
authors.initials: T
authors.lastname: Ohnishi
authors.forename: J C
authors.initials: JC
authors.lastname: Salerno
mesh_terms: D001665:Binding Sites; D003067:Coenzymes; D003573:Cytochrome b Group; D003580:Cytochromes / Q000378:metabolism*; D004355:Drug Stability; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D004734:Energy Metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005609:Free Radicals; D006863:Hydrogen-Ion Concentration; D010084:Oxidation-Reduction; D011199:Potentiometry; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
chemical_list: D003067:Coenzymes; D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D005609:Free Radicals; D014451:Ubiquinone; C017168:cytochrome bo, E coli; C024989:coenzyme Q10