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

Lewin, Allison, Crow, Allister, Hodson, Christopher T. C., Hederstedt, Lars and Le Brun, Nick E. ORCID: https://orcid.org/0000-0001-9780-4061 (2008) Effects of substitutions in the CXXC active-site motif of the extracytoplasmic thioredoxin ResA. Biochemical Journal, 414 (1). 81–91. ISSN 0264-6021

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Abstract

The thiol–disulfide oxidoreductase ResA from Bacillus subtilis fulfils a reductive role in cytochrome c maturation. The pKa values for the CEPC (one-letter code) active-site cysteine residues of ResA are unusual for thioredoxin-like proteins in 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 pKa values of both the active-site cysteine residues: in CPHC (DsbA-type) ResA, ?pKa 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 an increase of +40 mV 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 the 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 in properties. Finally, the in vivo functional properties of CEHC ResA are shown to be significantly affected compared with those of the wild-type protein.

Item Type:	Article
Faculty \ School:	Faculty of Science > School of Chemistry (former - to 2024)
UEA Research Groups:	Faculty of Science > Research Groups > Biophysical Chemistry (former - to 2017) Faculty of Science > Research Groups > Centre for Ocean and Atmospheric Sciences Faculty of Science > Research Centres > Centre for Molecular and Structural Biochemistry Faculty of Science > Research Groups > Chemistry of Life Processes
Depositing User:	Rachel Smith
Date Deposited:	07 Apr 2011 14:14
Last Modified:	24 Sep 2024 09:33
URI:	https://ueaeprints.uea.ac.uk/id/eprint/28487
DOI:	10.1042/BJ20080356

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