Characterization of the Active Center of Thioredoxin Reductase

Reduction of thioredoxin reductase with different reductants has been followed spectrally; by titration with TPNH in the presence of DPNase or with dithionite, it has been shown that this enzyme can accept 4 electrons per FAD. Thus, an additional acceptor group is postulated and is identified as a disulfide group which undergoes reversible reduction to a dithiol. The thioredoxin reductase activity is very sensitive to low concentrations of mercurial, whereas the transhydrogenase activity requires much higher levels for inhibition. Prereduction of the enzyme enhances the former, but not the latter process. Amperometric titrations show that the 4 cysteic acid residues per FAD, determined by amino acid analysis, arise from 2 free sulfhydryls and a disulfide bond which is reducible by TPNH. Mercurials are shown, in spectral experiments, to shift the electrons from the flavin the second redox group by trapping action on the nascent dithiol; after such a treatment, the enzyme is completely reduced by only 2 electrons per FAD and cannot be reoxidized by thioredoxin.