Selective N-bromosuccinimide oxidation of the nonfluorescent tryptophan-31 in the active center of thioredoxin from Escherichia coli.

The two tryptophan residues (Trp-28 and Trp-31) of thioredoxin-S2 from Escherichia coli were selectively tritiated with trifluoroacetic [3H]acid. The 3H label was introduced to permit quantitative amino acid sequence analyses of the result of N-bromosuccinimide oxidation of tryptophan to oxindolylalanine. Addition of 3-fold molar excess of N-bromosuccinimide at pH 4 modifies a tryptophan in thioredoxin-S2 that is nonessential for enzyme activity with thioredoxin reductase and has a strongly quenched fluorescence in both oxidized and reduced thioredoxin. This residue was shown to be Trp-31 by amino acid sequence analyses of 3H-labeled chymotryptic peptides from the modified protein. The results demonstrate that the second tryptophan residue, Trp-28, signals a conformational change on reduction of the active-center disulfide to a dithiol by increasing its fluorescence quantum yield about 6-fold at pH 7. The differential reactivity of the tryptophan residues agrees with the known three-dimensional structure of thioredoxin-S2.