The active-site cysteinyls and hydrophobic cavity residues of ResA are important for cytochrome c maturation in Bacillus subtilis.
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 caa(3), indicating that this apocytochrome is distinct from the other three endogenous c-type cytochromes of B. subtilis.
Hodson
Christopher T C
CT
Centre for Molecular and Structural Biochemistry, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, United Kingdon.
Lewin
Allison
A
Hederstedt
Lars
L
Le Brun
Nick E
NE
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D045304:Cytochromes c / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D045304:Cytochromes c