Figure 4.

TrxS₂-induced oxidation of the Cys⁵⁹-SH/Cys⁶⁴-SH pair in native, (B)CAM-modified, and SeCys⁴⁹⁸ → Cys⁴⁹⁸ mutant TrxR1 enzymes. (B)CAM-labeled enzyme was prepared by labeling NADPH-reduced TrxR1 with 50 μM BIAM alone. E. coli-expressed SeCys⁴⁹⁸ → Cys⁴⁹⁸ mutant TrxR1 was prepared as described in Experimental Procedures. Control, (B)CAM-modified, and mutant enzymes (30 μg) were incubated with three equivalents of Tris(2-carboxyethyl)phosphine per subunit to assure complete reduction of redox centers (9) and then oxidized by exposure for 2 min at 25°C to six equivalents of TrxS₂ per subunit in PBS buffer (pH 7.2) containing 1 mM EDTA. One-half of each TrxS₂-treated enzyme sample was reduced with 200 μM NADPH. Each of the resulting enzyme samples was labeled with IAM, digested with Lys-C, and subjected to HPLC analysis for the Cys⁵⁹/Cys⁶⁴ containing-peptide as described in the legend to Fig. 3.