Reaction of ubiquinone in the high-affinity quinone-binding site (QH) in bo-type ubiquinol oxidase from Escherichia coli was revealed by EPR and optical studies. In the QH site, ubiquinol was shown to be oxidized to ubisemiquinone and to ubiquinone, while no semiquinone signal was detected in the oxidase isolated from mutant cells that cannot synthesize ubiquinone. The QH site highly stabilized ubisemiquinone radical with a stability constant of 1–4 at pH 8.5 and the stability became lower at the lower pH. Midpoint potential of QH2/Q couple was −2 mV at pH 8.5 and showed −60 mV/pH dependence indicative of 2H+/2e− reaction. The E m was more negative than that of low-spin heme b above pH 7.0. We conclude that the QH mediates intramolecular electron transfer from ubiquinol in the low-affinity quinol oxidation site (QL) to low-spin heme b. Unique roles of the quinone-binding sites in the bacterial ubiquinol oxidase are discussed.
