The quinone-binding sites of the cytochrome bo3 ubiquinol oxidase from Escherichia coli.
Cytochrome bo(3) is the major respiratory oxidase located in the cytoplasmic membrane of Escherichia coli when grown under high oxygen tension. The enzyme catalyzes the 2-electron oxidation of ubiquinol-8 and the 4-electron reduction of dioxygen to water. When solubilized and isolated using dodecylmaltoside, the enzyme contains one equivalent of ubiquinone-8, bound at a high affinity site (Q(H)). The quinone bound at the Q(H) site can form a stable semiquinone, and the amino acid residues which hydrogen bond to the semiquinone have been identified. In the current work, it is shown that the tightly bound ubiquinone-8 at the Q(H) site is not displaced by ubiquinol-1 even during enzyme turnover. Furthermore, the presence of high affinity inhibitors, HQNO and aurachin C1-10, does not displace ubiquinone-8 from the Q(H) site. The data clearly support the existence of a second binding site for ubiquinone, the Q(L) site, which can rapidly exchange with the substrate pool. HQNO is shown to bind to a single site on the enzyme and to prevent formation of the stable ubisemiquinone, though without displacing the bound quinone. Inhibition of the steady state kinetics of the enzyme indicates that aurachin C1-10 may compete for binding with quinol at the Q(L) site while, at the same time, preventing formation of the ubisemiquinone at the Q(H) site. It is suggested that the two quinone binding sites may be adjacent to each other or partially overlap.
Yap
Lai Lai
LL
Department of Biochemistry, University of Illinois, 600 S. Goodwin Avenue, Urbana, IL 61801, USA.
Lin
Myat T
MT
Ouyang
Hanlin
H
Samoilova
Rimma I
RI
Dikanov
Sergei A
SA
Gennis
Robert B
RB
D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive / Q000187:drug effects; D003573:Cytochrome b Group; D003580:Cytochromes / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D006912:Hydroxyquinolines / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008954:Models, Biological; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction / Q000187:drug effects; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D015363:Quinolones / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011809:Quinones / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D014451:Ubiquinone / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism
D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D006912:Hydroxyquinolines; D015363:Quinolones; D011809:Quinones; C083850:cytochrome bo3, E coli; C052096:aurachin C; D014451:Ubiquinone; C001333:2-(n-heptyl)-4-hydroxyquinoline N-oxide; D010088:Oxidoreductases; C432586:ubiquinol oxidase; C003741:ubiquinol; D010100:Oxygen