Charles H. Williams

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 M_r of 35 000, and a protein in higher eukaryotes having a M_r 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 M_r 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 M_r 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 M_r forms may be exploited for drug design.

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 138 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 Cys^l38 may be the thiol initiating dithiol‐disulfide interchange between thioredoxin reductase and thioredoxin.

Thioredoxin reductase (TrxR) from Escherichia coli consists of two globular domains connected by a two‐stranded β sheet: an FAD domain and a pyridine nucleotide binding domain. The latter domain contains the redox‐active disulfide composed of Cys 135 and Cys 138. TrxR is proposed to undergo a conformational change whereby the two domains rotate 66° relative to each other (Waksman G, Krishna TSR, Williams CH Jr, Kuriyan J, 1994, J Mol Biol 236:800‐816), placing either the redox active disulfide (FO conformation) or the NADPH binding site (FR conformation) adjacent to the flavin. This domain rotation model was investigated by using a Cys 138 Ser active‐site mutant. The flavin fluorescence of this mutant is only 7% that of wild‐type TrxR, presumably due to the proximity of Ser 138 to the flavin in the FO conformation. Reaction of the remaining active‐site thiol, Cys 135, with phenylmercuric acetate (PMA) causes a 9.5‐fold increase in fluorescence. Titration of the PMA‐treated mutant with the nonreducing NADP(H) analogue, 3‐aminopyridine adenine dinucleotide phosphate (AADP⁺), results in significant quenching of the flavin fluorescence, which demonstrates binding adjacent to the FAD, as predicted for the FR conformation. Wild‐type TrxR, with or without PMA treatment, shows similar quenching by AADP⁺, indicating that it exists mostly in the FR conformer. These findings, along with increased EndoGluC protease susceptibility of PMA‐treated enzymes, agree with the model that the FO and FR conformations are in equilibrium. PMA treatment, because of steric limitations of the phenylmercuric adduct in the FO form, forces the equilibrium to the FR conformer, where AADP⁺ binding can cause fluorescence quenching and conformational restriction favors proteolytic susceptibility.