A stable mixed disulfide between thioredoxin reductase and its substrate, thioredoxin: Preparation and characterization

The flavoenzyme thioredoxin reductase (TrR) catalyzes the reduction of the small redox protein thioredoxin (Tr) by NADPH. It has been proposed that a large conformational change is required in catalysis by TrR in order to visualize a complete pathway for reduction of equivalents. The proposal is based on the comparison of the crystal structures of TrR and glutathione reductase, the latter being a well-understood member of this enzyme family [Waksman, G., et al, (1994) J. Mol. Biol. 236, 800-816]. Bound NADPH is perfectly positioned for electron transfer to the FAD in glutathione reductase, but in TrR, these two components are 17 Angstrom apart. In order to provide evidence for the proposed conformational change, a complex between TrR and its substrate Tr involving a mixed disulfide between TrR and Tr was prepared. The redox active disulfide of TrR is composed of Cys(135) and Cys(138), and the redox active disulfide of Tr is made up of Cys(32) and Cys(35). The complex C135S-C32S is prepared from forms of TrR and Tr altered by site-directed mutagenesis where Cys(138) and Cys(35) remaining in TrR and Tr, respectively, The purified C135S-C32S presents a band on a nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular weight sum of one subunit of TrR and one of Tr. Several observations indicate that C135S-C32S can adopt only one conformation It was reported previously that TrR C135S can form a charge transfer complex in the presence of ammonium cation in which the donor is the remaining thiolate of Cys(138) [Prongay, A. J., et al., (1989) J. Biol. Chem. 264, 2656-2664], while titration of C135S-C32S with NH4Cl does not induce charge transfer, presumably because Cys(138) is participating in the mixed disulfide. Reduction of C135S-C32S with dithiothreitol (DTT) results in a decrease of epsilon(454) to a value similar to that of TrR C135S, and subsequent NH4Cl titration leads to charge transfer complex formation in the nascent TrR C135S. Reductive titrations show that approximately 1 equiv of sodium dithionite or NADPH is required to fully reduce C135S-C32S, and treatment with NH4Cl and DTT demonstrates that the mixed disulfide remains intact. These results indicate that C135S-C32S is a stable mixed disulfide between Cys(138) of TrR C135S and Cys(35) of Tr C32S that locks the structure in a conformation where FAD can be reduced by NADPH, but electrons cannot flow from FADH(2) to the mixed disulfide bond.