ISOLATION AND CHARACTERIZATIONS OF QUINONE ANALOG-RESISTANT MUTANTS OF BO-TYPE UBIQUINOL OXIDASE FROM ESCHERICHIA-COLI

Cytochrome bo is a member of the heme-copper terminal oxidase superfam ily and serves as a four-subunit ubiquinol oxidase in the aerobic resp iratory chain of Escherichia coli. To probe the location and structura l properties of the ubiquinol oxidation site, we isolated and characte rized five or 10 spontaneous mutants resistant to either 2,6-dimethyl- 1,4-benzoquinone, 2,6-dichloro-4-nitrophenol, or 2,6-dichloro-4-dicyan ovinylphenol, the potent competitive inhibitors for the oxidation of u biquinol-l [Sato-Watanabe, M., Mogi, T., Miyoshi, H., Iwamura, H., Mat sushita, K., Adachi, O., and Anraku, Y. (1994) J. Biol. Chem. 269, 288 99-28907]. Analyses of the growth yields and the ubiquinol-1 oxidase a ctivities of the mutant membranes showed that the mutations increased the degree of the resistance to the selecting compounds. Notably, seve ral mutants showed me cross-resistance. These data indicate that the b inding sites for substrate and the competitive inhibitors are partiall y overlapped in the ubiquinol oxidation site. All the mutations were l inked to the expression vector, and 23 mutations examined were all pre sent in the C-terminal hydrophilic domain (Pro(96)-His(315)) of Subuni t II. Sequencing analysis revealed that seven mutations examined are l ocalized 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 Cu- A Site in subunit II, whereas Ile129Thr, Asn198Thr, and Gln233His are rather scattered in a three-dimensional structure and closer to transm embrane helices of subunit II. Our data suggest that the Qox domain an d the Cu-A end of the cupredoxin fold provide the quinol oxidation sit e and are involved in electron transfer to the metal centers in subuni t I.