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, Johann Deisenhofer

Research output: Contribution to journalArticle

31 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)4410-4415
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume103
Issue number12
DOIs
StatePublished - Mar 21 2006

Fingerprint

Protein Disulfide Reductase (Glutathione)
Thioredoxins
Substrate Specificity
Cytochromes c
Bacillus subtilis
Proteins
Heme
Peptides
Oxidation-Reduction
Cytochrome c Group
Electrons
Glutathione Disulfide
Sulfides
Oxidoreductases

Keywords

  • NMR
  • Thiol-disulfide oxidoreductase
  • X-ray crystallography

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

@article{962c38944712459d86cacc1f7a566a9a,
title = "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.",
keywords = "NMR, Thiol-disulfide oxidoreductase, X-ray crystallography",
author = "Colbert, {Christopher L.} and Qiong Wu and Erbel, {Paul J A} and Gardner, {Kevin H.} and Johann Deisenhofer",
year = "2006",
month = "3",
day = "21",
doi = "10.1073/pnas.0600552103",
language = "English (US)",
volume = "103",
pages = "4410--4415",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "12",

}

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

UR - http://www.scopus.com/inward/record.url?scp=33645212086&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33645212086&partnerID=8YFLogxK

U2 - 10.1073/pnas.0600552103

DO - 10.1073/pnas.0600552103

M3 - Article

C2 - 16537372

AN - SCOPUS:33645212086

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

SN - 0027-8424

IS - 12

ER -