Cytochrome bo is a member of the heme-copper terminal oxidase superfamily and serves as a four-subunit ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli. To probe the location and structural properties of the ubiquinol oxidation site, we isolated and characterized five or 10 spontaneous mutants resistant to either 2,6-dimethyl-1,4-benzoquinone, 2,6-dichloro-4-nitrophenol, or 2,6-dichloro-4-dicyanovinylphenol, the potent competitive inhibitors for the oxidation of ubiquinol-1 [Sato-Watanabe, M., Mogi, T., Miyoshi, H., Iwamura, H., Matsushita, K., Adachi, O., and Anraku, Y. (1994) J. Biol. Chem. 269, 28899-28907]. Analyses of the growth yields and the ubiquinol-1 oxidase activities of the mutant membranes showed that the mutations increased the degree of the resistance to the selecting compounds. Notably, several mutants showed the cross-resistance. These data indicate that the binding sites for substrate and the competitive inhibitors are partially overlapped in the ubiquinol oxidation site. All the mutations were linked to the expression vector, and 23 mutations examined were all present in the C-terminal hydrophilic domain (Pro96-His315) of subunit II. Sequencing analysis revealed that seven mutations examined are localized near both ends of the cupredoxin fold. Met248Ile, Ser258Asn, Phe281Ser, and His284Pro are present in a quinol oxidase-specific (Qox) domain and proximal to low-spin heme b in subunit I and the lost CuA site in subunit II, whereas Ile129Thr, Asn198Thr, and Gln233His are rather scattered in a three-dimensional structure and closer to transmembrane helices of subunit II. Our data suggest that the Qox domain and the CuA end of the cupredoxin fold provide the quinol oxidation site and are involved in electron transfer to the metal centers in subunit I.