Mechanism of substrate specificity in Bacillus subtilis ResA, a thioredoxin-like protein involved in cytochrome c maturation.
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 thiol-disulfide oxidoreductase and enables us to outline a structural mechanism of regulating the usage of electrons in a thiol-disulfide oxidoreductase system.
Howard Hughes Medical Institute, and Department of Biochemistry, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX 75390, USA.
Christopher L
CL
Colbert
Qiong
Q
Wu
Paul J A
PJ
Erbel
Kevin H
KH
Gardner
Johann
J
Deisenhofer
D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D003574:Cytochrome c Group / Q000737:chemistry / Q000378:metabolism*; D004579:Electron Transport; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011490:Protein Disulfide Reductase (Glutathione) / Q000737:chemistry* / Q000378:metabolism; D012996:Solutions; D013379:Substrate Specificity; D013879:Thioredoxins / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D003574:Cytochrome c Group; D012996:Solutions; D013879:Thioredoxins; C031732:cytochrome C-550; D011490:Protein Disulfide Reductase (Glutathione)