Journal of Biochemistry Advance Access originally published online on March 2, 2009
Journal of Biochemistry 2009 145(6):763-770; doi:10.1093/jb/mvp033
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Probing the haem d-binding site in cytochrome bd quinol oxidase by site-directed mutagenesis
1Department of Biomedical Chemistry, Graduate School of Medicine, the University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033; and 2ATP System Project, ERATO, JST, Nagatsuta 5800-2, Midori-ku, Yokohama 226-0026, Japan
*To whom correspondence addressed. Tel: +81-3-5841-8202, Fax: +81-3-5841-3444, E-mail: tmogi{at}m.u-tokyo.ac.jp
Received February 3, 2009; Accepted February 21, 2009
 |
Abstract |
|---|
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 
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.
Key Words: axial ligand, cyanide, Escherichia coli, haem d, quinol oxidase
Abbreviations: IC50, the 50% inhibitory concentration