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

Abstract

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.

Structure and Functional Properties of Bacillus subtilis Endospore Biogenesis Factor StoA

“…Substitution of Glu-80 in ResA has been shown previously to have a significant effect on the active site properties of the enzyme; for example, the pK a values of both active site cysteines were significantly lowered in an E80Q variant (13), and a B. subtilis strain containing E80Q ResA was also severely impaired in its ability to mature c-type cytochromes (14). Thus, it may be that these buried polar residues are also important in StoA function.…”

“…This led us to propose that Glu-80 is important for the binding of apocytochrome substrates to ResA (12)(13)(14)42). This residue is conserved in StoA (Glu-71), and we have shown here that it is also functionally important in StoA (Table 2).…”

“…It has been demonstrated that Glu-80 of ResA plays a key role in vivo (14), and in vitro studies showed that it is important for the elevated pK a values of the active site cysteines and that it is capable of hydrogen bonding to amino acid side chain residues bound in the cavity (13,21). This led us to propose that Glu-80 is important for the binding of apocytochrome substrates to ResA (12)(13)(14)42).…”

“…In contrast to most other TDORs that have been characterized, both thiols of the ResA active site are reactive to thiol-modifying reagents. In addition, a glutamate (Glu-80) in the vicinity of the active site has been shown to play a key role in controlling the reactivity of the enzyme (13,14). StoA is not involved in cytochrome c synthesis and cannot functionally replace ResA.…”

Co-factor Insertion and Disulfide Bond Requirements for Twin-arginine Translocase-dependent Export of the Bacillus subtilis Rieske Protein QcrA

“…Notably, cytochrome bc 1 is a membrane protein complex that faces the extracellular side of the cytoplasmic membrane. The Sec pathway individually translocates the cytochrome b and cytochrome c 1 components across the membrane before they are processed, matured, and combined (32,(35)(36)(37)(38)(39). It is believed that cytochrome b and cytochrome c 1 form a primary protease-resistant complex, where the final step in complex maturation is the incorporation of the Rieske protein (32).…”