Identification of the ubiquinol-binding site in the cytochrome bo3-ubiquinol oxidase of Escherichia coli.
The cytochrome bo3-ubiquinol oxidase, one of two ubiquinol oxidases in Escherichia coli, is a member of the heme-copper oxidase superfamily. The enzyme contains four protein subunits (I-IV) with apparent molecular masses of 58, 33, 22, and 17 kDa, respectively. Cytochrome bo3 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 been investigated. The photoreactive radiolabeled azidoubiquinone derivative 3-[3H]azido-2-methyl-5-methoxy-6-geranyl-1,4-benzoquinone (azido-Q), which has been widely used in locating the ubiquinone-binding sites of other enzymes, was used to identify the subunit(s) involved in the binding of quinol to cytochrome bo3. When reduced by dithioerythritol, the azido-Q derivative functioned as a substrate with partial effectiveness, suggesting that azido-Q interacts with a legitimate quinol-binding site. When cytochrome bo3 was incubated with an 8-fold molar excess of azido-Q, illumination by UV light for 10 min resulted in a 50% loss of activity. The uptake of radiolabeled azido-Q by the oxidase complex upon illumination correlated with the photoinactivation. In the presence of the competitive inhibitor 2-heptyl-4-hydroxyquinoline or ubiquinol, 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 electrophoresis showed that subunit II was heavily labeled by azido-Q, but that the other subunits were not. This suggests that the ubiquinol-binding site of the cytochrome bo3 complex is located at least partially on subunit II.
Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater 74078.
R
R
Welter
L Q
LQ
Gu
L
L
Yu
C A
CA
Yu
J
J
Rumbley
R B
RB
Gennis
D000345:Affinity Labels; D001386:Azides / Q000378:metabolism; D001665:Binding Sites; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D011809:Quinones / Q000737:chemistry / Q000494:pharmacology; D011868:Radioisotopes; D013379:Substrate Specificity; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D011809:Quinones; D011868:Radioisotopes; C083850:cytochrome bo3, E coli; D014451:Ubiquinone; C048123:3-azido-2-methyl-5-methoxy-6-(3,7-dimethyloctyl)-1,4-benzoquinone; C038862:cytochrome o oxidase; D003576:Electron Transport Complex IV; C003741:ubiquinol