Effects of substitutions in the CXXC active-site motif of the extracytoplasmic thioredoxin ResA

Allison Lewin, Allister Crow, Christopher T. C. Hodson, Lars Hederstedt, Nick E. Le Brun

Research output: Contribution to journalArticlepeer-review

Abstract

The thiol-disulfide oxidoreductase ResA from Bacillus subtilis fulfils a reductive role in cytochrome c maturation. The pK(a) values for the CEPC (one-letter code) active-site cysteine residues of Ill are unusual for thioredoxin-like proteins ill that they are both high (> 8) and within 0.5 unit of each other. To determine the contribution of the inter-cysteine dipeptide of ResA to its redox and acid-base properties, three variants (CPPC, CEHC and CPHC) were generated representing a stepwise conversion into the active-site sequence of the high-potential DsbA protein from Escherichia coli. The substitutions resulted in large decreases in the pK(a) values of both the active-site cysteine residues: in CPHC (DsbA-type) ResA, Delta pK(a) values of -2.5 were measured for both cysteine residues. Increases in midpoint reduction potentials were also observed, although these were comparatively small: CPHC (DsbA-type) ResA exhibited all increase of +40mV compared with the wild-type protein. Unfolding studies revealed that, despite the observed differences in the properties of the reduced proteins, changes in stability were largely confined to file oxidized state. High-resolution structures of two of the variants (CEHC and CPHC ResA) in their reduced states were determined and are discussed in terms of the observed changes ill properties. Finally, the in vivo functional properties of CEHC RcsA are shown to be significantly affected compared with those of the wild-type protein.
Original languageEnglish
Pages (from-to)81-91
JournalBiochemical Journal
Volume414
DOIs
Publication statusPublished - 2008

Subject classification (UKÄ)

  • Biochemistry and Molecular Biology

Free keywords

  • thioredoxin-like protein
  • oxidoreductase (TDOR)
  • thiol-disulfide
  • reduction potential
  • cytochrome c maturation
  • pK(a)
  • X-ray crystallography

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