The mechanism of high Mr thioredoxin reductase from Drosophila melanogaster

Drosophila melanogaster thioredoxin reductase-1 (DmTrxR-1) is a key flavoenzyme in dipteran insects, where it substitutes for glutathione reductase. DmTrxR-1 belongs to the family of dimeric, high Mr thioredoxin reductases, which catalyze reduction of thioredoxin by NADPH. Thioredoxin reductase has an N-terminal redox-active disulfide (Cys57-Cys62) adjacent to the flavin and a redox-active C-terminal cysteine pair (Cys489'-Cys490' in the other subunit) that transfer electrons from Cys57-Cys62 to the substrate thioredoxin. Cys489'-Cys490' functions similarly to Cys495-Sec496 (Sec = selenocysteine) and Cys535-XXXX-Cys540 in human and parasite Plasmodium falciparum enzymes, but a catalytic redox center formed by adjacent Cys residues, as observed in DmTrxR-1, is unprecedented. Our data show, for the first time in a high Mr TrxR, that DmTrxR-1 oscillates between the 2-electron reduced state, EH2, and the 4-electron state, EH4, in catalysis, after the initial priming reduction of the oxidized enzyme (Eox) to EH2. The reductive half-reaction consumes 2 eq of NADPH in two observable steps to produce EH4. The first equivalent yields a FADH--NADP+ charge-transfer complex that reduces the adjacent disulfide to form a thiolate-flavin charge-transfer complex. EH4 reacts with thioredoxin rapidly to produce EH2. In contrast, Eox formation is slow and incomplete; thus, EH2 of wild-type cannot reduce thioredoxin at catalytically competent rates. Mutants lacking the C-terminal redox center, C489S, C490S, and C489S/C490S, are incapable of reducing thioredoxin and can only be reduced to EH2 forms. Additional data suggest that Cys57 attacks Cys490' in the interchange reaction between the N-terminal dithiol and the C-terminal disulfide.     

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.     

Thioredoxin and related proteins in procaryotes

Thioredoxin is a small (Mr 12,000) ubiquitous redox protein with the conserved active site structure: -Trp-Cys-Gly-Pro-Cys-. The oxidized form (Trx-S2) contains a disulfide bridge which is reduced by NADPH and thioredoxin reductase; the reduced form [Trx(SH)2] is a powerful protein disulfide oxidoreductase. Thioredoxins have been characterized in a wide variety of prokaryotic cells, and generally show about 50% amino acid homology to Escherichia coli thioredoxin with a known three-dimensional structure. In vitro Trx-(SH)2 serves as a hydrogen donor for ribonucleotide reductase, an essential enzyme in DNA synthesis, and for enzymes reducing sulfate or methionine sulfoxide. E. coli Trx-(SH)2 is essential for phage T7 DNA replication as a subunit of T7 DNA polymerase and also for assembly of the filamentous phages f1 and M13 perhaps through its localization at the cellular plasma membrane. Some photosynthetic organisms reduce Trx-S2 by light and ferredoxin; Trx-(SH)2 is used as a disulfide reductase to regulate the activity of enzymes by thiol redox control. Thioredoxin-negative mutants (trxA) of E. coli are viable making the precise cellular physiological functions of thioredoxin unknown. Another small E. coli protein, glutaredoxin, enables GSH to be hydrogen donor for ribonucleotide reductase or PAPS reductase. Further experiments with molecular genetic techniques are required to define the relative roles of the thioredoxin and glutaredoxin systems in intracellular redox reactions.     

Mutation of conserved residues in Escherichia coli thioredoxin: effects on stability and function.

Mutations were made in three highly conserved residues in Escherichia coli thioredoxin. An internal charged residue, Asp-26, was changed to an alanine. The mutant protein was more stable than the wild type. It can function as a substrate for thioredoxin reductase with a 10-fold increase in the Km over the wild type. Although the redox potential was not substantially changed from that of the wild type, thioredoxin D26A was a poor reducing agent for ribonucleotide reductase. Asp-26 apparently serves to maintain an optimal charge distribution in the active site region for interaction with other proteins. Mutation of a surface Pro-34 in the active site disulfide ring to a serine had little effect on protein stability. A slight decrease in the redox potential (9 mV) made thioredoxin P34S a better reducing agent for ribonucleotide reductase. In contrast, mutation of the internal cis Pro-76 to an alanine destabilized the protein. The data indicate a change had also occurred in the charge distribution in the active site region. Thioredoxin P76A had a higher redox potential than the wild type protein and was not an effective reducing agent for ribonucleotide reductase. It was concluded that this residue is essential for maintaining the conformation of the active site and the redox potential of thioredoxin.     

Thioredoxin reductase from Plasmodium falciparum: evidence for interaction between the C-terminal cysteine residues and the active site disulfide-dithiol

Thioredoxin reductase (TrxR) catalyzes the reduction of thioredoxin by NADPH. TrxR from Plasmodium falciparum (PfTrxR) is a homodimer with a subunit Mr of 59 000. Each monomer contains one FAD and one redox active disulfide. Despite the high degress of similarity between PfTrxR and the human TrxR, their primary structures present a striking difference in the C-terminus. PfTrxR has two cysteine residues near the C-terminal Gly, while the human TrxR contains a Cys-SeCys dipeptide penultimate to the C-terminal Gly. It has been proposed that the C-terminal cysteines (as a cystine) of PfTrxR are involved in catalysis by an intramolecular dithiol-disulfide interchange with the nascent redox active dithiol. To investigate the proposed function of the C-terminal cysteines of PfTrxR, each has been changed to an alanine [Gilberger, T.-M., Bergmann, B., Walter, R. D., and Müller, S. (1998) FEBS Lett. 425, 407-410]. The single C-terminal cysteine remaining in each mutant was modified with 5,5'-dithiobis(2-nitrobenzoic acid) to form mixed disulfides consisting of the enzyme thiol and thionitrobenzoate (TNB). In reductive titrations of these mixed disulfide enzymes, 1 equiv of TNB anion was released upon reduction of the enzyme itself, while control experiments in which mutants without C-terminal cysteine were used showed little TNB anion release. This suggests that each of the C-terminal cysteines as a TNB mixed disulfide does mimic the proposed electron acceptor in the C-terminus. Analysis of the rapid reaction kinetics showed that the C-terminal mixed disulfide of the modified enzyme is reduced at a rate which is comparable with the turnover number of the wild type enzyme.     

Plasmoredoxin, a novel redox-active protein unique for malarial parasites.

Thioredoxins are a group of small redox-active proteins involved in cellular redox regulatory processes as well as antioxidant defense. Thioredoxin, glutaredoxin, and tryparedoxin are members of the thioredoxin superfamily and share structural and functional characteristics. In the malarial parasite, Plasmodium falciparum, a functional thioredoxin and glutathione system have been demonstrated and are considered to be attractive targets for antimalarial drug development. Here we describe the identification and characterization of a novel 22 kDa redox-active protein in P. falciparum. As demonstrated by in silico sequence analyses, the protein, named plasmoredoxin (Plrx), is highly conserved but found exclusively in malarial parasites. It is a member of the thioredoxin superfamily but clusters separately from other members in a phylogenetic tree. We amplified the gene from a gametocyte cDNA library and overexpressed it in E. coli. The purified gene product can be reduced by glutathione but much faster by dithiols like thioredoxin, glutaredoxin, trypanothione and tryparedoxin. Reduced Plrx is active in an insulin-reduction assay and reduces glutathione disulfide with a rate constant of 640 m-1.s-1 at pH 6.9 and 25 degrees C; glutathione-dependent reduction of H2O2 and hydroxyethyl disulfide by Plrx is negligible. Furthermore, plasmoredoxin provides electrons for ribonucleotide reductase, the enzyme catalyzing the first step of DNA synthesis. As demonstrated by Western blotting, the protein is present in blood-stage forms of malarial parasites. Based on these results, plasmoredoxin offers the opportunity to improve diagnostic tools based on PCR or immunological reactions. It may also represent a specific target for antimalarial drug development and is of phylogenetic interest.     

Protein disulfide-isomerase is a substrate for thioredoxin reductase and has thioredoxin-like activity

We have demonstrated that calf liver protein disulfide-isomerase (Mr 57,000) is a substrate for calf thymus thioredoxin reductase and catalyzes NADPH-dependent insulin disulfide reduction. This reaction can be used as a simple assay for protein disulfide-isomerase during purification in place of the classical method of reactivation of incorrectly oxidized ribonuclease A. Protein disulfide-isomerase contains two redox-active disulfides/molecule which were reduced by NADPH and calf thioredoxin reductase (Km approximately 35 microM). The isomerase was a poor substrate for NADPH and Escherichia coli thioredoxin reductase, but the addition of E. coli thioredoxin resulted in rapid reduction of two disulfides/molecule. Tryptophan fluorescence spectra were shown to monitor the redox state of protein disulfide-isomerase. Fluorescence measurements demonstrated that thioredoxin--(SH)2 reduced the disulfides of the isomerase and allowed the kinetics of the reaction to be followed; the reaction was also catalyzed by calf thioredoxin reductase. Equilibrium measurements showed that the apparent redox potential of the active site disulfide/dithiols of the thioredoxin domains of protein disulfide-isomerase was about 30 mV higher than the disulfide/dithiol of E. coli thioredoxin. Consistent with this, experiments using dithiothreitol or NADPH and thioredoxin reductase-dependent reduction and precipitation of insulin demonstrated differences between protein disulfide-isomerase and thioredoxin, thioredoxin being a better disulfide reductase but less efficient isomerase. Protein disulfide-isomerase is thus a high molecular weight member of the thioredoxin system, able to interact with both mammalian NADPH-thioredoxin reductase and reduced thioredoxin. This may be important for nascent protein disulfide formation and other thiol-dependent redox reactions in cells.     

Redox potential of human thioredoxin 1 and identification of a second dithiol/disulfide motif

Thioredoxin (Trx1) is a redox-active protein containing two active site cysteines (Cys-32 and Cys-35) that cycle between the dithiol and disulfide forms as Trx1 reduces target proteins. Examination of the redox characteristics of this active site dithiol/disulfide couple is complicated by the presence of three additional non-active site cysteines. Using the redox Western blot technique and matrix assisted laser desorption ionization time-of-flight mass spectrometry mass spectrometry, we determined the midpoint potential (E0) of the Trx1 active site (-230 mV) and identified a second redox-active dithiol/disulfide (Cys-62 and Cys-69) in an alpha helix proximal to the active site, which formed under oxidizing conditions. This non-active site disulfide was not a substrate for reduction by thioredoxin reductase and delayed the reduction of the active site disulfide by thioredoxin reductase. Within actively growing THP1 cells, most of the active site of Trx1 was in the dithiol form, whereas the non-active site was totally in the dithiol form. The addition of increasing concentrations of diamide to these cells resulted in oxidation of the active site at fairly low concentrations and oxidation of the non-active site at higher concentrations. Taken together these results suggest that the Cys-62-Cys-69 disulfide could provide a means to transiently inhibit Trx1 activity under conditions of redox signaling or oxidative stress, allowing more time for the sensing and transmission of oxidative signals.     

硫氧还蛋白的共价修饰

硫氧还蛋白是细胞中普遍存在的低分子量蛋白质,为生物体所必需。硫氧还蛋白、硫氧还蛋白还原酶和烟酰胺腺嘌呤二核苷磷酸组成硫氧还蛋白系统,调节细胞的氧化还原状态。硫氧还蛋白不仅维持细胞的氧化还原平衡,还具有抗凋亡及促进细胞增殖等功能。原核细胞的硫氧还蛋白仅含有两个半胱氨酸残基,真核细胞的硫氧还蛋白除了活性中心的两个半胱氨酸残基外,通常还有另外的半胱氨酸残基。这些半胱氨酸残基的共价修饰使硫氧还蛋白具有了更丰富的功能。硫氧还蛋白的共价修饰包括谷胱甘肽化、巯基氧化、亚硝基化和烷基化。     

Characterization of two active site mutations of thioredoxin reductase from Escherichia coli

Thioredoxin reductase (TRR), a member of the pyridine nucleotide-disulfide oxidoreductase family of flavoenzymes, undergoes two sequential thiol-disulfide interchange reactions with thioredoxin during catalysis. In order to assess the catalytic role of each nascent thiol of the active site disulfide of thioredoxin reductase, the 2 cysteines (Cys-136 and Cys-139) forming this disulfide have been individually changed to serines by site-directed mutageneses of the cloned trxB gene of Escherichia coli. Spectral analyses of TRR(Ser-136,Cys-139) as a function of pH and ionic strength have revealed two pKa values associated with the epsilon 456, one of which increases from 7.0 to 8.3 as the ionic strength is increased, and a second at 4.4 which is seen only at high ionic strength. epsilon 458 of wild type TRR(Cys-136,Cys-139) and epsilon 453 of TRR(Cys-136,Ser-139) are pH-independent. A charge transfer complex (epsilon 530 = 1300 M-1 cm-1), unique to TRR(Ser-136,Cys-139), has been observed under conditions of high ammonium cation concentration (apparent Kd = 54 microM) at pH 7.6. These results suggest the assignment of Cys-139 as the FAD-interacting thiol in the reduction of thioredoxin by NADPH via thioredoxin reductase. If, as with other members of this enzyme family, the two distinct catalytic functions are each carried out by a different nascent thiol, then Cys-136 would perform the initial thiol-disulfide interchange with thioredoxin. Steady state kinetic analyses of the proteins have revealed turnover numbers of 10 and 50% of the value of the wild type enzyme for TRR(Ser-136,Cys-139) and TRR(Cys-136,Ser-139), respectively, and no changes in the apparent Km values of TR(S2) or NADPH. The finding of activity in the mutants indicates that the remaining thiol can carry out interchange with the disulfide of thioredoxin, and the resulting mixed disulfide can be reduced by NADPH via the flavin.     

Thioredoxin and related proteins in procaryotes

Abstract Thioredoxin is a small ( M _r 12,000) ubiquitous redox protein with the conserved active site structure: -Trp-Cys-Gly-Pro-Cys-. The oxidized form (Trx-S₂) contains a disulfide bridge which is reduced by NADPH and thioredoxin reductase; the reduced form [Trx(SH)₂] is a powerful protein disulfide oxidoreductase. Thioredoxins have been characterized in a wide variety of prokaryotic cells, and generally show about 50% amino acid homology to Escherichia coli thioredoxin with a known three-dimensional structure. In vitro Trx-(SH)₂ serves as a hydrogen donor for ribonucleotide reductase, an essential enzyme in DNA synthesis, and for enzymes reducing sulfate or methionine sulfoxide. E. coli Trx-(SH)₂ is essential for phage T7 DNA replication as a subunit of T7 DNA polymerase and also for assembly of the filamentous phages f1 and M13 perhaps through its localization at the cellular plasma membrane. Some photosynthetic organisms reduce Trx-S₂ by light and ferrodoxin; Trx-(SH)₂ is used as a disulfide reductase to regulate the activity of enzymes by thiol redox control.
Thioredoxin-negative mutants ( trxA ) of E. coli are viable making the precise cellular physiological functions of thioredoxin unknown. Another small E. coli protein, glutaredoxin, enables GSH to be hydrogen donor for ribonucleotide reductase or PAPS reductase. Further experiments with molecular genetic techniques are required to define the relative roles of the thioredoxin and glutaredoxin systems in intracellular redox reactions.     

Characterization of mitochondrial thioredoxin reductase from C. elegans

Thioredoxin reductase catalyzes the NADPH-dependent reduction of the catalytic disulfide bond of thioredoxin. In mammals and other higher eukaryotes, thioredoxin reductases contain the rare amino acid selenocysteine at the active site. The mitochondrial enzyme from Caenorhabditis elegans, however, contains a cysteine residue in place of selenocysteine. The mitochondrial C. elegans thioredoxin reductase was cloned from an expressed sequence tag and then produced in Escherichia coli as an intein-fusion protein. The purified recombinant enzyme has a kcat of 610 min(-1) and a Km of 610 microM using E. coli thioredoxin as substrate. The reported kcat is 25% of the kcat of the mammalian enzyme and is 43-fold higher than a cysteine mutant of mammalian thioredoxin reductase. The enzyme would reduce selenocysteine, but not hydrogen peroxide or insulin. The flanking glycine residues of the GCCG motif were mutated to serine. The mutants improved substrate binding, but decreased the catalytic rate.     

Differential inhibition of high and low Mr thioredoxin reductases of parasites by organotelluriums supports the concept that low Mr thioredoxin reductases are good drug targets

Thioredoxin reductase (TrxR), a NADPH-dependent disulfide oxidoreductase, is vital in numerous cellular processes including defence against reactive oxygen species, cell proliferation and signal transduction. TrxRs occur in 2 forms, a high Mr enzyme characterized by those of mammals, the malaria parasite Plasmodium falciparum and some worms, and a low Mr form is present in bacteria, fungi, plants and some protozoan parasites. Our hypothesis is that the differences between the forms can be exploited in the development of selective inhibitors. In this study, cyclodextrin- and sulfonic acid-derived organotelluriums known to inhibit mammalian TrxR were investigated for their relative efficacy against P. falciparum TrxR (PfTrxR), a high Mr enzyme, and Trichomonas vaginalis TrxR (TvTrxR), a low Mr form of TrxR. The results suggest that selective inhibition of low Mr TrxRs is a feasible goal.     

硫氧还蛋白的结构及在生物抗氧化中的功能

硫氧还蛋白(thioredoxin,Trx)是广泛存在于原核与真核生物体内的氧化还原调节蛋白。Trx通过对目标蛋白质进行还原,从而调节机体的氧化还原平衡。Trx与硫氧还蛋白还原酶(thioredoxin reductase,TrxR)及NADPH共同组成硫氧还蛋白系统参与众多生理过程。细胞中的活性氧是导致生物氧化胁迫的一个主要方面。Trx可以通过对细胞内被氧化的二硫键的还原来修复机体的氧化损伤,并通过这种方式防止机体衰老。同时,Trx系统可以与其它氧化还原系统如谷胱甘肽(GSH)系统协调配合,并消除体内过多的活性氧。     

NADPH and oxidized thioredoxin mediate redox interconversion of calf-liver and Escherichia coli thioredoxin reductase

The activity of pure calf-liver and Escherichia coli thioredoxin reductases decreased drastically in the presence of NADPH or NADH, while NADP⁺, NAD⁺ and oxidized E. coli thioredoxin activated both enzymes significantly, particularly the bacterial one. The loss of activity under reducing conditions was time-dependent, thus suggesting an inactivation process: in the presence of 0.24 mM NADPH the half-lives for the E. coli and calf-liver enzymes were 13.5 and 2 min, respectively. Oxidized E. coli thioredoxin fully protected both enzymes from inactivation, and also promoted their complete reactivation after only 30 min incubation at 30° C. Lower but significant protection and reactivation was also observed with NADP⁺ and NAD⁺. EDTA protected thioredoxin reductase from NADPH inactivation to a great degree, thus indicating the participation of metals in the process; EGTA did not protect the enzyme from redox inactivation. Thioredoxin reductase was extensively inactivated by NADPH under aerobic and anaerobic conditions, thus excluding the participation of O₂ or oxygen active species in redox inactivation. The loss of thioredoxin reductase activity promoted by NADPH was much faster and complete in the presence of NAD⁺ glycohydrolase, thus suggesting that inactivation was related to full reduction of the redox-active disulfide. Those results indicate that thioredoxin reductase activity can be modulated in bacteria and mammals by the redox status of NADP(H) and thioredoxin pools, in a similar way to glutathione reductase. This would considerably expand the regulatory potential of the thioredoxin-thioredoxin reductase system with the enzyme being self-regulated by its own substrate, a regulatory protein.Abbreviations DTNB 5,5-dithiobis(2-nitrobenzoate) - EGTA Ethylenglycoltetraacetic Acid - TNB 5-thio-2-nitrobenzoate - Trx Thioredoxin - Trx(SH)2 Reduced Thioredoxin - Trx-S2 Oxidized Thioredoxin     

An f-type thioredoxin fromArabidopsis thaliana Leaves

Thioredoxin, a small redox protein with an active site disulfide/dithiol, is ubiquitous in bacteria, plants, and animals and functions as a reducing agent and modulator of enzyme activity. A thioredoxin has been purified to electrophoretic homogeneity from the leaves ofArabidopsis thaliana using procedures such as DE-52 ion exchange chromatography, Sephadex G-50 gel filtration, Q-Sepharose ion exchange chromatography, and DEAE-Sephadex A-25 chromatography. The purified thioredoxin was determined to be a single band on SDS-PAGE, and its molecular weight was estimated to be 21 KDa, which was much larger than those of most other known thioredoxins. It was proved to be an f-type thioredoxin, since it could activate fructose-l,6-bisphosphatase, but it could not activate NADP⁺-malate dehydrogenase. As a protein disulfide reductase, it could reduce the disulfide bonds contained in insulin. As a substrate, it showed a Km value of 20.2 μM onEscherichia coli thioredoxin reductase, and it had an optimal pH of 8.0. The molecular weight of the purified f-type thioredoxin is not consistent with those of the five divergent h-type thioredoxins already identified by cDNA cloning. The purified f-type thioredoxin is the first example isolated fromA. thaliana.     

Genomic organisation and alternative splicing of mouse and human thioredoxin reductase 1 genes

Background  

Thioredoxin reductase (TR) is a redox active protein involved in many cellular processes as part of the thioredoxin system. Presently there are three recognised forms of mammalian thioredoxin reductase designated as TR1, TR3 and TGR, that represent the cytosolic, mitochondrial and novel forms respectively. In this study we elucidated the genomic organisation of the mouse (Txnrd1) and human thioredoxin reductase 1 genes (TXNRD1) through library screening, restriction mapping and database mining.     

Characterization of Escherichia coli thioredoxins with altered active site residues

Escherichia coli thioredoxin is a small disulfide-containing redox protein with the active site sequence Cys-Gly-Pro-Cys-Lys. Mutations were made in this region of the thioredoxin gene and the mutant proteins expressed in E. coli strains lacking thioredoxin. Mutant proteins with a 17-membered or 11-membered disulfide ring were inactive in vivo. However, purified thioredoxin with the active site sequence Cys-Gly-Arg-Pro-Cys-Lys is still able to serve as a substrate for thioredoxin reductase and a reducing agent in the ribonucleotide reductase reaction, although with greatly reduced catalytic efficiency. A smaller disulfide ring, with the active site sequence Cys-Ala-Cys, does not turn over at a sufficient rate to be an effective reducing agent. Strain in the small ring favors the formation of intermolecular disulfide bonds. Alteration of the invariant proline to a serine has little effect on redox activity. The function of this residue may be in maintaining the stability of the active site region rather than participation in redox activity or protein-protein interactions. Mutation of the positively charged lysine in the active site to a glutamate residue raises the Km values with interacting enzymes. Although it has been proposed that the positive residue at position 36 is conserved to maintain the thiolate anion on Cys-32 (Kallis & Holmgren, 1985), the presence of the negative charge at this position does not alter the pH dependence of activity or fluorescence behavior. The lysine is most likely conserved to facilitate thioredoxin-protein interactions.     

Reaction mechanism and regulation of mammalian thioredoxin/glutathione reductase

Thioredoxin/glutathione reductase (TGR) is a recently discovered member of the selenoprotein thioredoxin reductase family in mammals. In contrast to two other mammalian thioredoxin reductases, it contains an N-terminal glutaredoxin domain and exhibits a wide spectrum of enzyme activities. To elucidate the reaction mechanism and regulation of TGR, we prepared a recombinant mouse TGR in the selenoprotein form as well as various mutants and individual domains of this enzyme. Using these proteins, we showed that the glutaredoxin and thioredoxin reductase domains of TGR could independently catalyze reactions normally associated with each domain. The glutaredoxin domain is a monothiol glutaredoxin containing a CxxS motif at the active site, which could receive electrons from either the thioredoxin reductase domain of TGR or thioredoxin reductase 1. We also found that the C-terminal penultimate selenocysteine was required for transfer of reducing equivalents from the thiol/disulfide active site of TGR to the glutaredoxin domain. Thus, the physiologically relevant NADPH-dependent activities of TGR were dependent on this residue. In addition, we examined the effects of selenium levels in the diet and perturbations in selenocysteine tRNA function on TGR biosynthesis and found that expression of this protein was regulated by both selenium and tRNA status in liver, but was more resistant to this regulation in testes.