Thioredoxin reductase from Plasmodium falciparum: Evidence for interactionbetween the C-terminal cysteine residues and the active site disulfide-dithiol

Thioredoxin reductase (TrxR) catalyzes the reduction of thioredoxin by NADP H. TrxR from Plasmodium falciparum (PfTrxR) is a homodimer with a subunit M -r of 59 000. Each monomer contains one FAD and one redox active disulfide. Despite the high degress of similarity between PfTrxR and the human TrxR, their primary structures present a striking difference in the C-terminus. P fTrxR has two cysteine residues near the C-terminal Gly, while the human Tr xR contains a Cys-SeCys dipeptide penultimate to the C-terminal Gly. It has been proposed that the C-terminal cysteines las a cystine) of PfTrxR are i nvolved in catalysis by an intramolecular dithiol-disulfide interchange wit h the nascent redox active dithiol. To investigate the proposed function of the C-terminal cysteines of PfTrxR, each has been changed to an alanine [G ilberger, T.-M., Bergmann, B., Waiter, R. D., and Muller, S. (1998) FEES Le tt. 425, 407-410]. The single C-terminal cysteine remaining in each mutant was modified with 5,5'-dithiobis (2-nitrobenzoic acid) to form mixed disulf ides consisting of the enzyme thiol and thionitrobenzoate (TNB). In reducti ve titrations of these mixed disulfide enzymes, 1 equiv of TNB anion was re leased upon reduction of the enzyme itself, while control experiments in wh ich mutants without C-terminal cysteine were used showed little TNB anion r elease. This suggests that each of the C-terminal cysteines as a TNB mixed disulfide does mimic the proposed electron acceptor in the C-terminus. Anal ysis of the rapid reaction kinetics showed that the C-terminal mixed disulf ide of the modified enzyme is reduced at a rate which is comparable with th e turnover number of the wild type enzyme.