Thioredoxin/glutathione reductase (TGR) from the platyhelminthic parasitic worms has recently been identified as a drug target for the treatment of
schistosomiasis. Schistosomes lack
catalase, and so are heavily reliant on the regeneration of reduced
thioredoxin (Trx) and
glutathione (GSH) to reduce
peroxiredoxins that ameliorate oxidative damage from
hydrogen peroxide generated by the host immune response. This study focuses on the characterization of the catalytic mechanism of Schistosoma mansoni TGR (SmTGR). Variant forms of SmTGR were studied to assign the function of residues that participate in the electron distribution chain within the
enzyme. Using anaerobic transient state spectrophotometric methods, redox changes for the
FAD and
NADPH were observed and the function of specific residues was defined from observation of charge transfer absorption transitions that are indicative of specific complexations and redox states. The C159S variant prevented distribution of electrons beyond the
flavin and as such did not accumulate thiolate-
FAD charge transfer absorption. The lack of this absorption facilitated observation of a new charge transfer absorption consistent with proximity of
NADPH and
FAD. The C159S variant was used to confine electrons from
NADPH at the
flavin, and it was shown that
NADPH and
FAD exchange hydride in both directions and come to an equilibrium that yields only fractional
FAD reduction, suggesting that both have similar reduction potentials. Mutation of U597 to
serine resulted in sustained thiolate-
FAD charge transfer absorption and loss of the ability to reduce Trx, indicating that the C596-U597
disulfide functions in the catalytic sequence to receive electrons from the C154 C159 pair and distribute them to Trx. No kinetic evidence for a loss or change in function associated with the distal C28-C31
disulfide was observed when the C31S variant reductive half-reaction was observed. The Y296A variant was shown to slow the rate of but increase extent of reduction of the
flavin, and the dissociation of NADP+. The H571 residue was confirmed to be the residue responsible for the deprotonation of the C159
thiol, increasing its reactivity and generating the prominent thiolate-
FAD charge transfer absorption that accumulates with oxidation of the
flavin.