Protein Science, Vol 7, Issue 6 1441-1450, Copyright © 1998 by Cold Spring Harbor Laboratory Press

ARTICLE

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

D. M. VEINE, S. B. MULROONEY, P. F. WANG and C. H. WILLIAMS-JR.
Department of Veterans Affairs Medical Center, Ann Arbor, Michigan 48105 and Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109

Mutation of one of the cysteine residues in the redox active disulfide of thioredoxin reductase from Escherichia coli results in C135S with Cys(138) remaining or C138S with Cys(135) 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 Cys(138) in C135S and approximately 300 s for Cys(135) in C138S showing that Cys(138) is much more reactive. The resulting mixed disulfides have been reacted with Cys(32) in C35S mutant thioredoxin to form stable, covalent adducts C138S-C35S and C135S-C35S. The half times show that Cys(138) is approximately fourfold more susceptible to attack by the nucleophile. These results suggest that Cys(138) may be the thiol initiating dithiol-disulfide interchange between thioredoxin reductase and thioredoxin.
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