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Journal of Biochemistry Advance Access published online on March 2, 2009
Journal of Biochemistry, doi:10.1093/jb/mvp033
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© The authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Probing the heme d-binding site in cytochrome bd quinol oxidase by site-directed mutagenesis

Tatsushi Mogi1,2,*

1Department of Biomedical Chemistry, Graduate School of Medicine, the University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033; 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 microaerophilic 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 heme d through a proton channel. Electrons are transferred from quinols to heme d through heme b558 and heme b595. All three hemes are bound to subunit I but only the axial ligand of heme d remains to be determined. Hemes b595 and d form a heme-heme binuclear center and substitutions of either His19 in helix I (heme b595 ligand) and Glu99 in helix III eliminated or severely reduced both hemes. To probe the location of the heme 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 heme d-binding site. Our data are consistent with the proposal that Glu99 serves as a heme d ligand of cytochrome bd.


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