The Active-Site Cysteinyls and Hydrophobic Cavity Residues of ResA Are Important for CytochromecMaturation inBacillus subtilis

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Abstract
ResA is an extracytoplasmic membrane-bound thiol-disulfide oxidoreductase required for cytochromecmaturation inBacillus 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-deficientB. subtiliscomplemented with a series of ResA variants. Using a range of methods to analyze the cellular cytochromeccontent, 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 cytochromecmaturation activity; (iii) that Pro141, which forms part of the hydrophobic cavity and which adopts acisconformation, plays an important role in protein stability; (iv) that Glu80, which lies at the base of the hydrophobic cavity, is important for cytochromecmaturation activity; and, finally, (v) that Pro141 and Glu80 ResA mutant variants promote selective maturation of low levels of onec-type cytochrome, subunit II of the cytochromecoxidasecaa3, indicating that this apocytochrome is distinct from the other three endogenousc-type cytochromes ofB. subtilis.

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