aurachin-c and ubiquinol

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.

Topics: Binding Sites; Binding, Competitive; Cytochrome b Group; Cytochromes; Escherichia coli; Escherichia coli Proteins; Hydroxyquinolines; Kinetics; Models, Biological; Molecular Structure; Mutagenesis, Site-Directed; Mutation; Oxidation-Reduction; Oxidoreductases; Oxygen; Protein Binding; Quinolones; Quinones; Substrate Specificity; Ubiquinone

Natural aurachin C is the most potent inhibitor of oxidation of ubiquinols by cytochromes bo and bd from Escherichia coli. To probe the structural properties of the substrate oxidation site in the ubiquinol oxidases, we synthesized a systematic set of aurachin C analogues (N-hydroxy-4-quinolone derivatives) and examined how their structure affects their activity towards cytochromes bo and bd, which are structurally unrelated. We found that the presence of the 3-methyl group of the 2-n-decyl and 2-n-undecyl derivatives increased the inhibitory potency towards both enzymes, probably due to a local steric congestion that allows favorable interaction of the alkyl tail with the enzyme. Increase in the chain length of the 3-alkyl tail of the 2-n-undecyl derivatives decreased the inhibitory potency only in cytochrome bo, indicating that the binding site for the alkyl tails of cytochrome bo is smaller than that of cytochrome bd. Based on these findings, we discuss the differences in the molecular mechanism of substrate oxidation by these two terminal ubiquinol oxidases.

Topics: Binding Sites; Cytochrome b Group; Cytochromes; Electron Transport Chain Complex Proteins; Enzyme Inhibitors; Escherichia coli; Escherichia coli Proteins; Mutation; Oxidation-Reduction; Oxidoreductases; Quinolones; Structure-Activity Relationship; Ubiquinone