The active site cysteinyls and hydrophobic cavity residues of ResA are important for cytochrome c maturation in Bacillus subtilis

From the Centre for Molecular and Structural Biochemistry, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, UK; and the Department of Cell and Organism Biology, Lund University, Lund SE-22362, Sweden

^* To whom correspondence should be addressed. Email: n.le-brun{at}uea.ac.uk.

	Abstract

ResA is an extra-cytoplasmic 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 oxidised 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 analyse the cellular cytochrome c content, we demonstrate: (i) that the N-terminal trans-membrane 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 apo-cytochrome is distinct from the other three endogenous c-type cytochromes of B. subtilis.