Rapid Formation of a Semiquinone Species on Oxidation of Quinol by the Cytochrome bo 3 Oxidase from Escherichia coli

Many bacterial oxidases utilize dihydroquinols, such as ubiquinol or menaquinol, rather than cytochrome c as a substrate. The best-characterized ubiquinol oxidase is cytochrome bo ₃ from Escherichia coli. In this work, the initial oxidation of ubiquinol by this ubiquinol oxidase is examined. Stopped-flow UV-visible spectroscopy and rapid freeze-quench electron paramagnetic resonance (EPR) spectroscopies were used to examine the oxidation of ubiquinol-2 (UQ₂H₂) by cytochrome bo ₃ under multiple turnover conditions. The results show the rapid appearance of the semiquinone radical, coincident with the reduction of the low-spin heme b component of the enzyme. The rate of formation of the semiquinone radical is consistent with the proposition that this is a kinetically relevant intermediate in the reaction sequence. As UQ₂H₂ is depleted, the radical decays and the enzyme forms a "peroxy," or P, complex with dioxygen. No detectable protein radical is associated with the P complex.