Characterization of the redox properties of MtbDsbA. (a) pKa determination of the nucleophilic cysteine of MtbDsbA. This is a representative plot of three independent measurements of the pH-dependent thiolate-specific absorbance of the catalytic cysteine. The pKa was obtained from the nonlinear fit to the Henderson–Hasselbach equation. (b) Determination of the redox equilibria of MtbDsbA with glutathione at pH 7.0 and 298 K. The plot shows the averaged fraction (three replicates) of reduced MtbDsbA at various ratios of reduced:oxidized glutathione. The resulting equilibrium constant Keq (17.37 ± 0.1 µM) from the nonlinear curve fit for a one-site binding equation was used to calculate the redox potential of MtbDsbA relative to the glutathione (GSH/GSSG) standard potential of −240 mV (Gilbert, 1995). (c) Relative thermal stability of oxidized (grey) and reduced (black) MtbDsbA at pH 7.0. The normalized average far-UV CD signal from three measurements was fitted to a two-state unfolding model as described previously (Kurz et al., 2009). The resulting melting temperature (Tm) of MtbDsbA shows that its reduced form (351.7 ± 0.1 K) is more stable than its oxidized form (338.6 ± 0.1 K).