IDENTIFICATION OF THE UBIQUINOL-BINDING SITE IN THE CYTOCHROME BO(3)-UBIQUINOL OXIDASE OF ESCHERICHIA-COLI

The cytochrome bo(3)-ubiquinol oxidase, one of two ubiquinol oxidases in Escherichia coli, is a member of the heme copper oxidase superfamil y. The enzyme contains four protein subunits (I-IV) with apparent mole cular masses of 58, 33, 22, and 17 kDa, respectively. Cytochrome bo(3) catalyzes the 2-electron oxidation of ubiquinol and the reduction of molecular oxygen to water. Although the primary structures of all four subunits have been determined, the ubiquinol-binding site has not bee n investigated. The photoreactive radiolabeled azidoubiquinone derivat ive zido-2-methyl-5-methoxy-6-geranyl-1,4-benzoquinone (azido-Q), whic h has been widely used in locating the ubiquinone-binding sites of oth er enzymes, was used to identify the subunit(s) involved in the bindin g of quinol to cytochrome bo(3). When reduced by dithioerythritol, the azido-Q derivative functioned as a substrate with partial effectivene ss, suggesting that azido-Q interacts with a legitimate quinol-binding site. When cytochrome bo(3) was incubated with an 8-fold molar excess of azido-Q, illumination by UV light for 10 min resulted in a 50% los s of activity. The uptake of radiolabeled azido-Q by the oxidase compl ex upon illumination correlated with the photoinactivation. In the pre sence of the competitive inhibitor 2-heptyl-4-hydroxyquinoline or ubiq uinol, the rate of azido-Q uptake and the loss of enzyme activity upon illumination decreased. Analysis of the distribution of radioactivity among the subunits after separation by SDS-polyacrylamide gel electro phoresis showed that subunit II was heavily labeled by azido-Q, but th at the other subunits were not. This suggests that the ubiquinol-bindi ng site of the cytochrome bo(3) complex is located at least partially on subunit II.