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.
Mutation of one of the cysteine residues in the redox active disulfide of thioredoxin reductase from Escherichia coli results in C135S with Cys138 remaining or C138S with Cys135 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 Cys138 in C135S and approximately 300 s for Cys135 in C138S showing that Cys138 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 Cys138 is approximately fourfold more susceptible to attack by the nucleophile. These results suggest that Cys138 may be the thiol initiating dithiol-disulfide interchange between thioredoxin reductase and thioredoxin.
Veine
D M
DM
Department of Veterans Affairs Medical Center, Ann Arbor, Michigan 48105, USA.
Mulrooney
S B
SB
Wang
P F
PF
Williams
C H
CH
D003545:Cysteine / Q000235:genetics; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D004227:Dithionite / Q000737:chemistry; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000737:chemistry; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D011487:Protein Conformation; D015202:Protein Engineering; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013879:Thioredoxins / Q000737:chemistry / Q000378:metabolism*
D004220:Disulfides; D013438:Sulfhydryl Compounds; D005182:Flavin-Adenine Dinucleotide; D004227:Dithionite; D013879:Thioredoxins; D009249:NADP; D004228:Dithionitrobenzoic Acid; D013880:Thioredoxin-Disulfide Reductase; D003545:Cysteine; D004229:Dithiothreitol