STUDIES ON A STABILIZATION OF UBISEMIQUINONE BY ESCHERICHIA-COLI QUINOL OXIDASE, CYTOCHROME BO

The Escherichia coli quinol oxidase, cytochrome bo, is closely related to the cytochrome c oxidase, cytochrome aa(3) in all aspects of its structure and function except for the replacement of the cytochrome-c-binding site and its attendant Cu-A 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 E(m7) of approximately 70 mV. Below pH 9, the pH dependence of the two-electron couple is -60 mV/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)).