TY - JOUR
T1 - Mechanism of substrate specificity in Bacillus subtilis ResA, a thioredoxin-like protein involved in cytochrome c maturation
AU - Colbert, Christopher L.
AU - Wu, Qiong
AU - Erbel, Paul J A
AU - Gardner, Kevin H.
AU - Deisenhofer, Johann
PY - 2006/3/21
Y1 - 2006/3/21
N2 - 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.
AB - 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.
KW - NMR
KW - Thiol-disulfide oxidoreductase
KW - X-ray crystallography
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U2 - 10.1073/pnas.0600552103
DO - 10.1073/pnas.0600552103
M3 - Article
C2 - 16537372
AN - SCOPUS:33645212086
SN - 0027-8424
VL - 103
SP - 4410
EP - 4415
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 12
ER -