Thioredoxin reductase two modes of catalysis have evolved.

Thioredoxin reductase (EC 1.6.4.5) is a widely distributed flavoprotein that catalyzes the NADPH-dependent reduction of thioredoxin. Thioredoxin plays several key roles in maintaining the redox environment of the cell. Like all members of the enzyme family that includes lipoamide dehydrogenase, glutathione reductase and mercuric reductase, thioredoxin reductase contains a redox active disulfide adjacent to the flavin ring. Evolution has produced two forms of thioredoxin reductase, a protein in prokaryotes, archaea and lower eukaryotes having a Mr of 35 000, and a protein in higher eukaryotes having a Mr of 55 000. Reducing equivalents are transferred from the apolar flavin binding site to the protein substrate by distinct mechanisms in the two forms of thioredoxin reductase. In the low Mr enzyme, interconversion between two conformations occurs twice in each catalytic cycle. After reduction of the disulfide by the flavin, the pyridine nucleotide domain must rotate with respect to the flavin domain in order to expose the nascent dithiol for reaction with thioredoxin; this motion repositions the pyridine ring adjacent to the flavin ring. In the high Mr enzyme, a third redox active group shuttles the reducing equivalent from the apolar active site to the protein surface. This group is a second redox active disulfide in thioredoxin reductase from Plasmodium falciparum and a selenenylsulfide in the mammalian enzyme. P. falciparum is the major causative agent of malaria and it is hoped that the chemical difference between the two high Mr forms may be exploited for drug design.

Mixed disulfide with glutathione as an intermediate in the reaction catalyzed by glutathione reductase from yeast and as a major form of the enzyme in the cell.

Glutathione reductase catalyzes the reduction of glutathione disulfide by NADPH. The FAD of the reductase is reduced by NADPH, and reducing equivalents are passed to a redox-active disulfide to complete the first half-reaction. The nascent dithiol of two-electron reduced enzyme (EH(2)) interchanges with glutathione disulfide forming two molecules of glutathione in the second half-reaction. It has long been assumed that a mixed disulfide (MDS) between one of the nascent thiols and glutathione is an intermediate in this reaction. In addition to the nascent dithiol composed of Cys(45) and Cys(50), the enzyme contains an acid catalyst, His(456), having a pK(a) of 9.2 that protonates the first glutathione (residue numbers refer to the yeast enzyme sequence). Reduction of yeast glutathione reductase by glutathione and reoxidation of EH(2) by glutathione disulfide indicate that the mixed disulfide accumulates, in particular, at low pH. The reaction of glutathione disulfide with EH(2) is stoichiometric in the absence of an excess of glutathione. The equilibrium position among E(ox), MDS, and EH(2) is determined by the glutathione concentration and is not markedly influenced by pH between 6.2 and 8.5. The mixed disulfide is the principal product in the reaction of glutathione with oxidized enzyme (E(ox)) at pH 6. 2. Its spectrum can be distinguished from that of EH(2) by a slightly lower thiolate (Cys(50))-FAD charge-transfer absorbance at 540 nm. The high GSH/GSSG ratio in the cytoplasm dictates that the mixed disulfide will be the major enzyme species.

Redox potentials for yeast, Escherichia coli and human glutathione reductase relative to the NAD+/NADH redox couple: enzyme forms active in catalysis.

The flavoenzyme glutathione reductase catalyzes the NADPH-dependent reduction of glutathione disulfide, yielding two molecules of glutathione. The oxidation-reduction potentials, Eox/EH2 (two-electron reduced enzyme), for yeast, Escherichia coli, and human glutathione reductase have been determined between pH 6.0 and 9.8 relative to the nonphysiological substrate couple NAD+/NADH and were found to be -237, -243, and -227 mV (+/-5 mV) at pH 7.0 and 20 degreesC, respectively. The potential as a function of pH demonstrated slopes of -51, -45, and -42 mV/pH unit, respectively, at low pH and -37, -31, and -34 mV/pH unit, respectively, at high pH. The change in slope indicated pKa values of 7.4, 8.5, and 7.6, respectively. The slopes indicate that two protons are associated with the two-electron reduction of Eox at low pH and that only one proton is involved with the two-electron reduction of Eox at high pH, provided that the effects of nearby titratable residues are considered in the data analysis. The influence of four such groups, Cys50, Cys45, His456', and either Tyr107 or the flavin-(N3), has been included (residue numbering refers to the yeast sequence). The enzyme loses activity upon deprotonation of the acid-base catalyst at high pH. Since the pKa ascribed to the EH2-to-EH- ionization is lower than the pKa of the acid-base catalyst, both the EH2 and EH- forms of glutathione reductase must be catalytically active, in contrast to the closely related enzyme lipoamide dehydrogenase, for which only EH2 is active.

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.

Mutation of one of the cysteine residues in the redox active disulfide of thioredoxin reductase from Escherichia coli results in C135S with Cys138 remaining or C138S with Cys135 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 Cys138 in C135S and approximately 300 s for Cys135 in C138S showing that Cys138 is much more reactive. The resulting mixed disulfides have been reacted with Cys32 in C35S mutant thioredoxin to form stable, covalent adducts C138S-C35S and C135S-C35S. The half times show that Cys138 is approximately fourfold more susceptible to attack by the nucleophile. These results suggest that Cys138 may be the thiol initiating dithiol-disulfide interchange between thioredoxin reductase and thioredoxin.

Thioredoxin reductase from Escherichia coli: evidence of restriction to a single conformation upon formation of a crosslink between engineered cysteines.

Thioredoxin reductase is a flavoprotein which catalyzes the reduction of the small protein thioredoxin by NADPH. It contains a redox active disulfide and an FAD in each subunit of its dimeric structure. Each subunit is further divided into two domains, the FAD and the pyridine nucleotide binding domains. The orientation of the two domains determined from the crystal structure and the flow of electrons determined from mechanistic studies suggest that thioredoxin reductase requires a large conformational change to carry out catalysis (Williams CH Jr, 1995, FASEB J 9:1267-1276). The constituent amino acids of an ion pair, E48/R130, between the FAD and pyridine nucleotide binding domains, were mutagenized to cysteines to form E48C,R130C (CC mutant). Formation of a stable bridge between these cysteines was expected to restrict the enzyme largely in the conformation observed in the crystal structure. Crosslinking with the bifunctional reagent N,N,1,2 phenylenedimaleimide, spanning 4-9 A, resulted in a >95 % decrease in thioredoxin reductase and transhydrogenase activity. SDS-PAGE confirmed that the crosslink in the CC-mutant was intramolecular. Dithionite titration showed an uptake of electrons as in wild-type enzyme, but anaerobic reduction of the flavin with NADPH was found to be impaired. This indicates that the crosslinked enzyme is in the conformation where the flavin and the active site disulfide are in close proximity but the flavin and pyridinium rings are too far apart for effective electron transfer. The evidence is consistent with the hypothesis that thioredoxin reductase requires a conformational change to complete catalysis.

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 TrT 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 the 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 angstroms 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 Cys135 and Cys138, and the redox active disulfide of Tr is made up of Cys32 and Cys35. The complex C135S-C32S is prepared from forms of TrR and Tr altered by site-directed mutagenesis where Cys138 and Cys35 are remaining in TrR and Tr, respectively. The purified C135S-C32S presents a band on a nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis responding 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 Cys138 [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 Cys138 is participating in the mixed dissulfide. Reduction of C135S-C32S with dithiothreitol (DTT) results in a decrease of epsilon454 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 between Cys138 of TrR C135S and Cys35 of TrC32S that locks the structure in a conformation where FAD can be reduced by NADPH, but electrons cannot flow from FADH2 to the mixed disulfide bond.