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Journal of Bacteriology, July 2008, p. 4697-4705, Vol. 190, No. 13
0021-9193/08/$08.00+0     doi:10.1128/JB.00145-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

The Active-Site Cysteinyls and Hydrophobic Cavity Residues of ResA Are Important for Cytochrome c Maturation in Bacillus subtilis ^,

Christopher T. C. Hodson,¹ Allison Lewin,¹ Lars Hederstedt,² and Nick E. Le Brun^1*

Centre for Molecular and Structural Biochemistry, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, United Kingdon,¹ Department of Cell and Organism Biology, Lund University, Lund SE-22362, Sweden²

Received 28 January 2008/ Accepted 22 April 2008

ResA is an extracytoplasmic membrane-bound thiol-disulfide oxidoreductase required for cytochrome c maturation in Bacillus subtilis. Previous biochemical and structural studies have revealed that the active-site cysteinyls cycle between oxidized and reduced states with a low reduction potential and that, upon reduction, a hydrophobic cavity forms close to the active site. Here we report in vivo studies of ResA-deficient B. subtilis complemented with a series of ResA variants. Using a range of methods to analyze the cellular cytochrome c content, we demonstrated (i) that the N-terminal transmembrane segment of ResA serves principally to anchor the protein to the cytoplasmic membrane but also plays a role in mediating the activity of the protein; (ii) that the active-site cysteines are important for cytochrome c maturation activity; (iii) that Pro141, which forms part of the hydrophobic cavity and which adopts a cis conformation, plays an important role in protein stability; (iv) that Glu80, which lies at the base of the hydrophobic cavity, is important for cytochrome c maturation activity; and, finally, (v) that Pro141 and Glu80 ResA mutant variants promote selective maturation of low levels of one c-type cytochrome, subunit II of the cytochrome c oxidase caa₃, indicating that this apocytochrome is distinct from the other three endogenous c-type cytochromes of B. subtilis.

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* Corresponding author. Mailing address: School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, United Kingdom. Phone: 44 1603 592699. Fax:. 44 1603 592003. E-mail: n.le-brun{at}uea.ac.uk

Published ahead of print on 2 May 2008.

Supplemental material for this article may be found at http://jb.asm.org/.

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Journal of Bacteriology, July 2008, p. 4697-4705, Vol. 190, No. 13
0021-9193/08/$08.00+0     doi:10.1128/JB.00145-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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