© 2006 The Japanese Biochemical Society.
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Kinetic Mechanism of Quinol Oxidation by Cytochrome bd Studied with Ubiquinone-2 Analogs
1 Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8503; 2 Department of Physics, Chuo University, Kasuga, Bunkyo-ku, Tokyo 112-8551; 3 Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502; 4 Department of Biomedical Chemistry, Graduate School of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033; 5 ATP System Project, Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Organization (JST), Nagatsuta 5800-2, Midori-ku, Yokohama 226-0026; and 6 Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033
* To whom correspondence should be addressed. Tel: +81-45-922-5238, Fax: +81-45-922-5239, E-mail: tmogi{at}res.titech.ac.jp
Cytochrome bd is a heterodimeric terminal ubiquinol oxidase of Escherichia coli under microaerophilic growth conditions. The oxidase activity shows sigmoidal concentration-dependence with low concentrations of ubiquinols, and a marked substrate inhibition with high concentrations of ubiquinol-2 analogs [Sakamoto, K., Miyoshi, H., Takegami, K., Mogi, T., Anraku, Y., and Iwamura H. (1996) J. Biol. Chem. 271, 29897–29902]. Kinetic analysis of the oxidation of the ubiquinol-2 analogs, where the 2- or 3-methoxy group has been substituted with an azido or ethoxy group, suggested that its peculiar enzyme kinetics can be explained by a modified ping-pong bi-bi mechanism with the formation of inactive binary complex FS in the one-electron reduced oxygenated state and inactive ternary complex (E2S)Sn on the oxidation of the second quinol molecule. Structure-function studies on the ubiquinol-2 analogs suggested that the 6-diprenyl group and the 3-methoxy group on the quinone ring are involved in the substrate inhibition. We also found that oxidized forms of ubiquinone-2 analogs served as weak noncompetitive inhibitors. These results indicate that the mechanism for the substrate oxidation by cytochrome bd is different from that of the heme-copper terminal quinol oxidase and is tightly coupled to dioxygen reduction chemistry.
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