Mechanism of substrate specificity in Bacillus subtilis ResA, a thioredoxin-like protein involved in cytochrome c maturation

Abstract

The covalent attachment of heme cofactors to the apo-polypeptides via thioether bonds is unique to the maturation of c-type cytochromes. A number of thiol-disulfide oxidoreductases prepare the apocytochrome for heme insertion in system I and II cytochrome c maturation. Although most thiol-disulfide oxidoreductases are nonspecific, the less common, specific thiol-disulfide oxidoreductases may be key to directing the usage of electrons. Here we demonstrate that unlike other thiol-disulfide oxidoreductases, the protein responsible for reducing oxidized apocytochrome c in Bacillus subtilis, ResA, is specific for cytochrome c550 and utilizes alternate conformations to recognize redox partners. We report solution NMR evidence that ResA undergoes a redox-dependent conformational change between oxidation states, as well as data showing that ResA utilizes a surface cavity present only in the reduced state to recognize a peptide derived from cytochrome c550. Finally, we confirm that ResA is a specific thiol-disulfide oxidoreductase by comparing its reactivity to our mimetic peptide with its reactivity to oxidized glutathione, a nonspecific substrate. This study biochemically demonstrates the specificity of this thioldisulfide oxidoreductase and enables us to outline a structural mechanism of regulating the usage of electrons in a thiol-disulfide oxidoreductase system. NMR ͉ thiol-disulfide oxidoreductase ͉ x-ray crystallography

2-Nitrobenzoate 2-Nitroreductase (NbaA) Switches Its Substrate Specificity from 2-Nitrobenzoic Acid to 2,4-Dinitrobenzoic Acid under Oxidizing Conditions

“…The common mechanism underlying cysteine oxidation is exerted via conformational changes through the formation of disulfide or cyclic sulfenamide covalent bonds or sulfenic or sulfonic acids. For example, Bacillus subtilis ResA, a thioredoxin-like protein, is able to undergo a redox-induced conformational change between the reduced and oxidized states and uses alternative conformations to select the substrates (39). We indicate that alanine substitution of the four cysteine residues in NbaA at positions 39, 103, 141, and 194 alters the catalytic properties and substrate specificity of the enzyme for 2-NBA and 2,4-DNBA.…”

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

“…ResA is located on the outside of the cytoplasmic membrane and is tethered to it by an N-terminal transmembrane segment, such that the N terminus of the protein is located in the cytoplasm. A soluble form of ResA has been structurally characterized (in both oxidized and reduced states), and the redox and pK a properties of the protein have been determined (8)(9)(10)27). While it is unlikely that the membrane anchor significantly affects the physicochemical properties of the soluble domain, it is not clear whether the membrane anchor serves only to localize ResA to the correct cellular compartment.…”

Strategic Roles of Axial Histidines in Structure Formation and Redox Regulation of Tetraheme Cytochrome c3

“…A mechanism for the His recognition has been proposed on the basis of the crystal structure of B. subtilis ResA (a component of a type II Ccm) (38), where a special cavity called "a histidine clamp" was assumed to recognize His in the heme-binding motif (38). ResA exhibits a high degree of homology to CcmG of E. coli in terms of amino acid sequence (30% identical) (39). Thus, the involvement of the fifth axial His in heme linking would be a general mechanism in Ccms.…”