Mechanism of substrate specificity in Bacillus subtilis ResA, a thioredoxin-like protein involved in cytochrome c maturation
- Christopher L. Colbert*,
- Qiong Wu*,†,
- Paul J. A. Erbel*,†,
- Kevin H. Gardner*,†, and
- Johann Deisenhofer*,‡,§
- ‡Howard Hughes Medical Institute and Departments of
- *Biochemistry and
- †Pharmacology, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX 75390
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Contributed by Johann Deisenhofer, January 20, 2006
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 thiol-disulfide oxidoreductase and enables us to outline a structural mechanism of regulating the usage of electrons in a thiol-disulfide oxidoreductase system.
Footnotes
- §To whom correspondence should be addressed. E-mail: johann.deisenhofer{at}utsouthwestern.edu
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Author contributions: C.L.C. designed research; C.L.C., Q.W., P.J.A.E., and K.H.G. performed research; C.L.C., Q.W., P.J.A.E., K.H.G., and J.D. analyzed data; and C.L.C., Q.W., and K.H.G. wrote the paper.
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Conflict of interest statement: No conflicts declared.
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Data deposition: The x-ray crystallographic structure factors and coordinates for the refined model have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 2F9S).
- Abbreviations:
- CCM,
- cytochrome c maturation;
- GSSG,
- oxidized glutathione;
- HSQC,
- heteronuclear single quantum coherence.
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Freely available online through the PNAS open access option.
- © 2006 by The National Academy of Sciences of the USA
