Probing the ubiquinol-binding site in cytochrome bd by site-directed mutagenesis.
To probe the structure of the quinol oxidation site in loop VI/VII of the Escherichia coli cytochrome bd, we substituted three conserved residues (Gln249, Lys252, and Glu257) in the N-terminal region and three glutamates (Glu278, Glu279, and Glu280) in the first internal repeat. We found that substitutions of Glu257 by Ala or Gln, and Glu279 and Glu280 by Gln, severely reduced the oxidase activity and the expression level of cytochrome bd. In contrast, Lys252 mutations reduced only the oxidase activity. Blue shifts in the 440 and 630 nm peaks of the reduced Lys252 mutants and in the 561 nm peak of the reduced Glu257 mutants indicate the proximity of Lys252 to the heme b(595)-d binuclear center and Glu257 to heme b(558), respectively. Perturbations of reduced heme b(558) upon binding of aurachin D support structural changes in the quinol-binding site of the mutants. Substitutions of Lys252 and Glu257 caused large changes in kinetic parameters for the ubiquinol-1 oxidation. These results indicate that Lys252 and Glu257 in the N-terminal region of the Q-loop are involved in the quinol oxidation by bd-type terminal oxidase.
Mogi
Tatsushi
T
Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8503, Japan. tmogi@res.titech.ac.jp
Akimoto
Satoru
S
Endou
Sachiko
S
Watanabe-Nakayama
Takahiro
T
Mizuochi-Asai
Eri
E
Miyoshi
Hideto
H
D000595:Amino Acid Sequence; D001665:Binding Sites; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D045222:Electron Transport Chain Complex Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015363:Quinolones / Q000737:chemistry; D016415:Sequence Alignment; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000378:metabolism
D003573:Cytochrome b Group; D003580:Cytochromes; D045222:Electron Transport Chain Complex Proteins; D029968:Escherichia coli Proteins; D015363:Quinolones; C052097:aurachin D; D014451:Ubiquinone; D010088:Oxidoreductases; C040077:cytochrome bd terminal oxidase complex, E coli; C003741:ubiquinol