Effects of substitutions in the CXXC active-site motif of the extracytoplasmic thioredoxin ResA.
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 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 pK(a) values of both the active-site cysteine residues: in CPHC (DsbA-type) ResA, DeltapK(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 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.
Lewin
Allison
A
Centre for Molecular and Structural Biochemistry, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, UK.
Crow
Allister
A
Hodson
Christopher T C
CT
Hederstedt
Lars
L
Le Brun
Nick E
NE
D020816:Amino Acid Motifs / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics* / Q000502:physiology; D001412:Bacillus subtilis; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D045604:Extracellular Fluid / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D010084:Oxidation-Reduction; D011490:Protein Disulfide Reductase (Glutathione) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013879:Thioredoxins / Q000235:genetics* / Q000378:metabolism*
D001426:Bacterial Proteins; D013879:Thioredoxins; D011490:Protein Disulfide Reductase (Glutathione)