Formation and properties of mixed disulfides between thioredoxin reductase from Escherichia coli and thioredoxin: Evidence that cysteine‐138 functions to initiate dithiol‐disulfide interchange and to accept the reducing equivalent from reduced flavin

Abstract

Mutation of one of the cysteine residues in the redox active disulfide of thioredoxin reductase from Escherichia coli results in C135S with CysI3' remaining or C138S with CysI3' remaining. The expression system for the genes encoding thioredoxin reductase, wild-type enzyme, C135S, and C138S has been re-engineered to allow for greater yields of protein. Wild-type enzyme and C135S were found to be as previously reported, whereas discrepancies were detected in the characteristics of C138S. It was shown that the original C138S was a heterogeneous mixture containing C138S and wild-type enzyme and that enzyme obtained from the new expression system is the correct species. C138S obtained from the new expression system having 0.1 % activity and 7% flavin fluorescence of wild-type enzyme was used in this study. Reductive titrations show that, as expected, only 1 mol of sodium dithionite/mol of FAD is required to reduce C138S. The remaining thiol in C135S and C138S has been reacted with 5,5'-dithiobis-(2-nitrobenzoic acid) to form mixed disulfides. The half time of the reaction was <5 s for in C135S and approximately 300 s for C Y S '~~ in (2138s showing that CysI3' is much more reactive. The resulting mixed disulfides have been reacted with Cys32 in C35S mutant thioredoxin to form stable, covalent adducts C138S-C35S and C135S-C35S. The half times show that Cys"' is approximately fourfold more susceptible to attack by the nucleophile. These results suggest that Cys 13' may be the thiol initiating dithiol-disulfide interchange between thioredoxin reductase and thioredoxin.Keywords: dithiol-disulfide interchange; flavoprotein; thioredoxin reductase Thioredoxin reductase from Escherichia coli is a pyridine nucleotidedisulfide oxidoreductase containing FAD and a disulfide in each active site. The redox active disulfide is composed of C Y S "~ and Cys'". Reducing equivalents move from NADPH to FAD, from Abbreviations: DTNB, 5,5'-dithiobis-(2-nitrobenzoic acid); TNB anion, 5-thio-2-nitrobenzoate anion: DTT, 1,4-dithiothreitol; DPDS, 4,4'-dithio-C138S and C135S, thioredoxin reductase in which C Y S '~~ or C Y S '~~ has dipyridine: PDS, 4-thiopyridone; IPTG, isopropyl P-D-thiogalactoside; been changed to Ser, respectively; C32S and C35S, thioredoxin in which Cys32 or has been changed to Ser, respectively: C138S-C35S, a covalent adduct where the remaining active site thiol of C138S is linked via a disulfide to the remaining active site thiol of C35S, analogous designations are used for the three other possible combinations of mutated thioredoxin reductases and thioredoxins. reduced flavin to the active site disulfide; dithiol-disulfide interchange effects reduction of the substrate thioredoxin. The redox active disulfide of thioredoxin is composed of Cys3' and Cys3'. Modification by site-directed mutagenesis of the active site in the enzyme has produced the single thiol mutants C138S (with CysI3' remaining from the active site disulfide) and C135S (with CysI3' remaining) (Prongay et al., 1989). The spectral characteristics...

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