Probing the haem d-binding site in cytochrome bd quinol oxidase by site-directed mutagenesis.
Cytochrome bd is a cyanide-resistant terminal quinol oxidase under micro-aerophilic growth conditions and generates a proton motive force via scalar protolytic reactions. Protons used for dioxygen reduction are taken up from the cytoplasm and delivered to haem d through a proton channel. Electrons are transferred from quinols to haem d through haem b558 and haem b595. All three haems are bound to subunit I but only the axial ligand of haem d remains to be determined. Haems b595 and d form a haem-haem binuclear centre and substitutions of either His19 in helix I (haem b595 ligand) and Glu99 in helix III eliminated or severely reduced both haems. To probe the location of the haem d ligand, we introduced mutations around His19 and Glu99 and examined the cyanide-resistance of the oxidase activity and spectroscopic properties. In contrast to mutations around His19, I98F and L101T reduced the IC50 for cyanide to 0.18 and 0.41 mM, respectively, from 1.4 mM of the wild-type. Blue shifts in the alpha peak of I98F suggest that Ile98 is in the vicinity of the haem d-binding site. Our data are consistent with the proposal that Glu99 serves as a haem d ligand of cytochrome bd.
Mogi
Tatsushi
T
Department of Biomedical Chemistry, Graduate School of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. tmogi@m.u-tokyo.ac.jp
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D003486:Cyanides / Q000494:pharmacology; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D045222:Electron Transport Chain Complex Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009153:Mutagens / Q000494:pharmacology; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid
D003486:Cyanides; D003573:Cytochrome b Group; D003580:Cytochromes; D045222:Electron Transport Chain Complex Proteins; D029968:Escherichia coli Proteins; D009153:Mutagens; D006418:Heme; D010088:Oxidoreductases; C040077:cytochrome bd terminal oxidase complex, E coli