FIGURE 3.

pK_a determination of all Dtrx ionizable residues. A, 600 MHz two-dimensional CBCACO spectrum of reduced Dtrx at 298 K, pH 5.7, showing the cross-peaks for the C_α-CO and C_β-CO of all residues of the protein. Cross-peaks for the C_α-C_γ and C_β-C_γ of Asn and Asp, and for the C_β-C_δ and C_γ-C_δ of Gln and Glu are also visible and connected by lines. The inset shows a close-up view of the pH-dependent chemical shift variations for the C_β-CO of Asp²¹ (pH 4.9 (pink), 5.6 (red), 6.1 (orange), 6.6 (yellow), 7 (green), 7.8 (blue), and 9.3 (purple)). B, pK_a determination of the nucleophilic cysteine Cys³¹ (black circle) and cysteine Cys³⁴ (white circle) in the reduced form of Dtrx. C, pK_a determination of the histidine His³³ in the oxidized form (white triangle) and reduced form (black circle) of Dtrx. The pH-dependent chemical shift variation of the C_β carbons was measured, normalized, and fitted to one apparent pK_a value using the Henderson-Hasselbach equation.