Characterization and functional role of the QH site of bo-type ubiquinol oxidase from Escherichia coli.
Cytochrome bo is a four-subunit terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli that vectorially translocates protons not only via directed protolytic reactions but also via proton pumping. Previously, we postulated that a bound quinone in the high-affinity quinone binding site (QH) mediates electron transfer from the low-affinity quinol oxidation site (QL) in subunit II to low-spin heme b in subunit I as an electron gate and a transient electron reservoir [Sato-Watanabe, M., Mogi, T., Ogura, T., Kitagawa, T., Miyoshi, H., Iwamura, H., and Anraku, Y. (1994b) J. Biol. Chem. 269, 28908-28912]. In the present study, we carried out screening of ubiquinone analogues using a bound ubiquinone-free enzyme (DeltaUbiA1) that has been isolated from a ubiquinone biosynthesis mutant, and identified PC24 (2-chloro-4, 6-dinitrophenol), PC32 (2,6-dibromo-4-cyanophenol), and PC52 (2-isopropyl-5-methyl-4,6-dinitrophenol) as potent QH site inhibitors. PC15 (2,6-dichloro-4-nitrophenol) and PC16 (2, 6-dichloro-4-dicyanovinylphenol), potent QL site inhibitors, did not exhibit such a selective inhibition of the QH site. Binding studies using the air-oxidized DeltaUbiA enzyme showed that PC32 and PC52 have 4- to 7-fold higher affinity than ubiquinone-1. Reconstitution of the QH site with PC32 and PC52 resulted in a decrease of the apparent Vmax value to 1/7 and 1/3, respectively, of the control activity. These findings suggest that structural features of the QL and QH sites are different, and provide further support for the involvement of the QH site in intramolecular electron transfer and facile oxidation of quinols at the QL site.
Sato-Watanabe
M
M
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Japan.
Mogi
T
T
Miyoshi
H
H
Anraku
Y
Y
D016227:Benzoquinones / Q000378:metabolism*; D001665:Binding Sites; D003576:Electron Transport Complex IV / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010636:Phenols / Q000378:metabolism; D013053:Spectrophotometry; D013329:Structure-Activity Relationship
D016227:Benzoquinones; D004791:Enzyme Inhibitors; D010636:Phenols; C004532:quinone; C038862:cytochrome o oxidase; D003576:Electron Transport Complex IV