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

The thiol-disulfide oxidoreductase ResA from Bacillus subtilis fulfils a reductive role in cytochrome c maturation. The pKa values of the CEPC active site cysteines of ResA are unusual for thioredoxin-like proteins, in that they are both high (> 8), and within half a 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 to 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 cysteines: in CPHC (DsbA-type) ResA, ΔpKa 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 to 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 oxidised 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 to those of the wild-type protein.