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Journal of Bacteriology, December 2005, p. 8361-8369, Vol. 187, No. 24
0021-9193/05/$08.00+0     doi:10.1128/JB.187.24.8361-8369.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Heme A Synthase Enzyme Functions Dissected by Mutagenesis of Bacillus subtilis CtaA

Lars Hederstedt,* Anna Lewin, and Mimmi Throne-Holst{dagger}

Department of Cell & Organism Biology, Lund University, Sölvegatan 35, SE-223 62 Lund, Sweden

Received 15 August 2005/ Accepted 13 September 2005

Heme A, as a prosthetic group, is found exclusively in respiratory oxidases of mitochondria and aerobic bacteria. Bacillus subtilis CtaA and other heme A synthases catalyze the conversion of a methyl side group on heme O into a formyl group. The catalytic mechanism of heme A synthase is not understood, and little is known about the composition and structure of the enzyme. In this work, we have: (i) constructed a ctaA deletion mutant and a system for overproduction of mutant variants of the CtaA protein in B. subtilis, (ii) developed anaffinity purification procedure for isolation of preparative amounts of CtaA, and (iii) investigated the functional roles of four invariant histidine residues in heme A synthase by in vivo and in vitro analyses of the properties of mutant variants of CtaA. Our results show an important function of three histidine residues for heme A synthase activity. Several of the purified mutant enzyme proteins contained tightly bound heme O. One variant also contained trapped hydroxylated heme O, which is a postulated enzyme reaction intermediate. The findings indicate functional roles for the invariant histidine residues and provide strong evidence that the heme A synthase enzyme reaction includes two consecutive monooxygenations.

* Corresponding author. Mailing address: Department of Cell & Organism Biology, Lund University, Sölvegatan 35, SE-223 62 Lund, Sweden. Phone: 46 (46) 2228622. Fax: 46 (46) 2224113. E-mail: Lars.Hederstedt{at}cob.lu.se.

{dagger} Present address: Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway.

Journal of Bacteriology, December 2005, p. 8361-8369, Vol. 187, No. 24
0021-9193/05/$08.00+0     doi:10.1128/JB.187.24.8361-8369.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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  • Mogi, T. (2009). Probing Structure of Heme A Synthase from Bacillus subtilis by Site-Directed Mutagenesis. J Biochem 145: 625-633 [Abstract] [Full Text]  
  • Mogi, T. (2009). Over-Expression and Characterization of Bacillus subtilis Heme O Synthase. J Biochem 145: 669-675 [Abstract] [Full Text]  


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