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Journal of Biochemistry Advance Access originally published online on January 27, 2009
Journal of Biochemistry 2009 145(5):625-633; doi:10.1093/jb/mvp017
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© The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Probing Structure of Heme A Synthase from Bacillus subtilis 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; and 2ATP System Project, ERATO, JST, Nagatsuta, Midori-ku, Yokohama 226-0026, Japan

*To whom correspondence should be addressed. Tel: +81-3-5841-8202, Fax: +81-3-5841-3444, E-mail: tmogi{at}m.u-tokyo.ac.jp

Received December 20, 2008; Accepted January 19, 2009


  Abstract

Biosynthesis of heme A from heme B is catalysed by two enzymes, heme O and heme A synthases, in the membrane. Heme A synthase in Bacillus subtilis (CtaA) has eight transmembrane helices and oxidizes a methyl group on pyrrole ring D of heme O to an aldehyde. In this study, to explore structure of heme binding site(s) in heme A synthase, we overproduced the B. subtilis His6-CtaA in Escherichia coli and characterized spectroscopic properties of the purified CtaA. On the contrary to a previous report (Svensson, B., Andersson, K.K., and Hederstedt, L. (1996) Low-spin heme A in the heme A biosynthetic protein CtaA from Bacillus subtilis. Eur. J. Biochem. 238, 287–295), we found that two molecules of heme B were bound to CtaA. Further, we demonstrated that substitutions of His60 and His126 did not affect heme binding while His216 and His278 in the carboxy-halves are essential in heme binding. And we found that Ala substitutions of Cys191 and Cys197 in loop 5/6 reduced heme content to a half of the wild-type level. On the basis of our findings, we proposed a helical-wheel-projection model of CtaA.

Key Words: Bacillus subtilis, heme A synthase, terminal oxidase, dioxygen reduction, mitochondria

Abbreviations: HPLC, high-performance liquid chromatography; SML, sucrose monolaurate


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