Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

Thioredoxin Reductase

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_101928

Synonyms

Historical Background

Thioredoxin reductase was discovered in the course of studies on the biosynthesis of deoxyribonucleotides in Escherichia coli. Indeed in 1964, Laurent and coworkers discovered that thioredoxin is the reducing substrate of ribonucleotide reductase, the essential enzyme catalyzing de novo synthesis of 2′-deoxyribonucleotides. This discovery prompted the search of the enzyme responsible for the recycling of oxidized thioredoxin. Thioredoxin reductase from E. coli was isolated and purified shortly afterwards (Moore et al. 1964). Subsequent work by the same authors leads to the purification of thioredoxin reductase from mammalian neoplastic tissues (Moore 1967). The three-dimensional structure of thioredoxin reductase from E. coli was solved by X-ray crystallography in the early 1990s.

Molecular Structure of TrxRs

Thioredoxin reductase (TrxR; EC 1.8.1.9) is a ubiquitous flavoenzyme which is part of the cell armory against noxious oxidative agents (Gromer et al. 2004). It is a member of the thioredoxin network, comprising Thioredoxin, Trx; TrxR; and a host of enzymes that use Trx as a reductant (e.g., ribonucleotide reductase, peroxiredoxin, etc.). TrxR is member of a large family of pyridine nucleotide disulfide oxidoreductases, comprising TrxR, glutathione reductase (GR), thioredoxin glutathione reductase (TGR), mercuric ion reductase (MerR), lipoamide dehydrogenase (LipDH), trypanothione reductase (TryR), and alkyl hydroperoxide reductase (AhpF), all of them functioning as homodimers. The main physiological function of TrxR is to transfer electrons from NADPH to Trx, which in turn is responsible for the reduction of oxidants (Arnér and Holmgren 2000). Nevertheless TrxR is able to process and reduce substrates other than Trx, the difference in substrate preferences and mechanism of action reflecting the different type of TrxRs and their evolution. Two types of TrxRs have been recognized which share similar (but not identical) mechanism of action, similar topology but limited sequence identity. One type of TrxR is found in Archea, Bacteria, and Eukarya, including plants and fungi and some protozoan parasites; it has low molecular weight, approx. 35 kDa per monomer, and is designated L-TrxR. L-TrxRs are related to the bacterial alkyl hydroperoxide reductase (AhpF). The other type found in Metazoan Eukarya (including Homo sapiens, Drosophila melanogaster, and Caenorhabditis elegans) and in protozoan parasites (e.g., Plasmodia) has high molecular weight, i.e., 55 kDa per monomer, and is called H-TrxR. H-TrxRs are related to glutathione reductase (GR), thioredoxin-glutathione reductase (TGR), trypanothione reductase (TryR), mercuric-ion reductase (MerR), and lipoamide dehydrogenase (LipDH). Table 1 summarizes similarities and differences between the two classes of TrxRs and some of the other class of related enzymes.
Thioredoxin Reductase, Table 1

Conserved domains and features in some pyridine nucleotide disulfide oxidoreductases

 

FAD domain

NADPH

Interface domain

FAD Cys couple

NADPH Cys couple

C-term Cys couple

N-term Cys couple

Trx domain

Grx domain

GR

TGR

H-TrxR

L-TrxR

AhpF

The quaternary structure of the TrxR consists of two monomers arranged in a twofold symmetric dimer. Each monomer contains a FAD domain, a pyridine nucleotide-binding domain, where one molecule of NADPH binds, and at least one couple of catalytically active cysteine residues, located in close proximity of the isoalloxazine ring of FAD. Other redox-active Cys-Cys or SeC-Cys couples may be present (Table 1).

The pyridine nucleotide-binding motif, GXGXXA/G, is fully conserved among L- and H-TrxRs. The main differences between the two classes are detected in the specific interface domain of H-TrxR and in the number of the couples of active cysteines. The redox center of L-TrxRs, bearing the CXXC motif, is located in the NADPH domain while H-TrxRs have a redox center (bearing the CXXXXXC motif) in the FAD domain (Table 1). This is the basic arrangement which is shared among all members of each family, the prototype being glutathione reductase. H-TrxRs also possess a long C-terminal tail (not shared by GR) where one additional redox active couple formed by a cysteine and a seleno-cysteine (or, less commonly, two Cys) is located. The motif Gly-Cys-SeC-Gly-COOH is highly conserved but not invariant (see below). SeC is usually the second last residue of the polypeptide chain. The SeC-Cys couple on the C-terminal arm of each monomer of H-TrxRs is reduced by the FAD/Cys-Cys center of the partner dimer; hence the enzyme’s function requires both monomers. This is at variance with L-TxrRs that do not possess a redox active C-terminal arm (Kuriyan et al. 1991). Some variations from the basic pattern described above are known, e.g., the redox-active C-terminal couple is Cys-Cys in mercuric ion reductase, and Cys-XXXX-Cys in the TrxRs from Plasmodium parasites (Pai and Schulz 1983; Matthews and Williams 1976).

The redox activity of the Cys is enhanced by ionization of the sulfhydryl group to the thiolate ion. This is accomplished by a nearby acidic residue, whose function is to lower the pKa of Cys from >8 to 6: His in H-TrxR, GR, and LipD, and Asp in L-TrxR (Williams 1995). The presence of an aminoacid residue acting as a general acid/base catalytic group is necessary to shuttle protons from the nascent dithiol to the substrate disulfide (Mulrooney and Williams 1994; reviewed in Williams 1995).

Mammals possess multiple genes for TrxRs, usually at least one for the cytoplasmic variant of the enzyme (TrxR1), and another for the mitochondrial variant (TrxR2). They possess, in addition, a gene for a tissue-specific variant called Thioredoxin Glutathione Reductase (TrxR3 or TGR), which possesses an additional glutaredoxin domain at N-terminus together with an additional couple of Cys residues (Table 1).

From the viewpoint of protein evolution, it is accepted that L-TrxR and H-TrxR originated from the same common ancestor and evolved independently, thus explaining the marked structure differences (Hirt et al. 2002). Interestingly, so far, no genome is known to possess both L-TrxR and H-TrxR, suggesting that the presence of both the enzymes could be not be beneficial for the organism. Hirt and coworkers proposed that the common ancestor of all extant eukaryotes had both a GR and a L-TrxR to maintain redox homeostasis. The phylogenetic studies performed to date have shown that eukaryotic H-TrxRs cluster together with GR, LipDH, MerR, and TryR (Hirt et al. 2002) with GR more closely related to H-TrxR.

GR genes are detected both for metazoans and fungi but the latter lack of a genuine TrxR. Phylogenetic analysis revealed that H-TrxR from metazoans is more closely related to fungal GR than to other members of the reductase family. An interesting and puzzling case is that of parasitic nematodes (helminths) that, contrary to free-living, nonparasitic ones, entirely lack GR and only express TGR (TrxR3).

The presence of multiple copies of NADPH-dependent flavoreductases within the genome of many Metazoa complicates the analysis of the relationships within TrxR clade as well as the postulated horizontal gene transfer among organisms of different phyla and duplication event. This latter event seems to have given rise to the formation of TGR (Hirt et al. 2002).

Gene Architecture

The structural organization of TrxR genes within genomes of representative species from different phyla is complex, the more so, the more recent the appearance of the organism.

As one can see from Table 2 and Fig. 1, the number of coding sequences (exons) increases by going down along phyla as well as the number of noncoding ones (introns) and their lengths.
Thioredoxin Reductase, Table 2

Number of nucleotides within a single exon or intron in each variant of TrxR gene from various organisms. All but S. cerevisiae are members of Metazoan phylum. Data were retrieved from Ensembl genome browser for all species (http://www.ensembl.org/index.html)

 

S.cerevisiae

C.elegans

D.melanogaster

D.melanogaster

A.gambiae

S.mansoni

S.purpuratus

P.marinus

D.rerio

T.rupribes

L.chalumnae

X.tropicalis

H.sapiens

H.sapiens

 

TrxR1

TrxR2

TrxRl

TrxR2

TrxRl

TrxR2

TrxR

TGR

TrxRl

TrxR2

TrxR3

TrxR4

TrxRl

TrxRl

TrxR2

TrxRl

TrxR2

TrxRl

TrxR2

TrxR3

TrxRl

TrxR2

TrxR3

TrxRl

TrxR2

TrxR3

El

960

1029

85

315

204

1794

726

1788

28

13

192

69

223

312

279

117

59

137

96

37

105

95

163

115

292

408

I1

  

1066

48

3362

 

1778

34

719

60

6138

698

3115

2565

1264

365

182

5315

454

1226

1594

789

1177

35,633

10,602

7451

E2

  

663

137

346

 

2375

61

69

69

61

55

61

61

69

61

43

110

66

117

61

69

61

152

69

61

I2

  

53

46

70

  

33

1296

703

5796

641

1448

5226

162

719

873

13,280

1304

47

3415

3137

1260

6340

11,430

1015

E3

  

134

85

227

  

87

51

57

213

101

110

110

57

110

70

123

57

119

110

57

110

61

57

110

I3

  

63

349

71

  

36

678

418

11,930

2977

1369

1547

84

82

101

1690

781

1574

3045

1849

1615

30,851

538

1709

E4

  

279

537

1226

  

125

143

145

95

139

153

108

145

108

57

73

145

108

123

145

108

110

145

105

I4

  

147

50

   

2297

1779

459

389

963

4044

1828

1599

157

82

1460

305

145

876

1257

407

22,249

642

243

E5

  

456

343

   

142

49

75

77

113

73

73

75

73

145

120

75

73

73

75

73

123

75

73

I5

  

599

568

   

214

297

575

1208

989

287

637

1553

88

386

1335

430

1529

979

2331

425

1832

2299

1829

E6

  

207

542

   

54

1624

79

154

135

120

120

79

120

75

143

79

120

120

79

120

73

79

120

I6

  

47

    

2166

 

238

1128

543

428

504

96

77

727

3945

3119

39

1454

2477

288

2459

488

7980

E7

  

779

    

143

 

63

108

137

143

143

63

143

79

116

63

64

143

63

143

120

63

143

I7

       

840

 

412

691

1394

524

1190

1094

111

290

2086

1214

608

624

4019

457

3016

3766

2029

E8

       

116

 

71

96

153

116

116

71

116

63

385

71

33

116

71

116

143

71

116

I8

       

4232

 

440

639

 

3523

1812

8756

96

81

 

10,850

33

783

1230

1375

414

12,308

826

E9

       

226

 

20

135

 

226

226

20

232

71

 

20

43

226

20

226

116

20

226

I9

       

819

 

421

451

 

790

173

1268

2

3315

 

326

743

1126

2142

2687

1505

918

1240

E10

       

105

 

92

63

 

93

93

92

74

115

 

92

3

90

92

93

226

92

93

I10

       

1865

 

325

  

192

2971

1833

16

73

 

6209

2763

788

882

177

4020

2457

6838

Ell

       

74

 

172

  

77

77

175

97

175

 

175

116

77

175

77

93

175

77

I11

       

2,9

 

366

  

249

1249

9912

342

2995

 

1030

1429

216

11,935

986

917

11,945

565

E12

       

157

 

131

  

157

157

137

77

137

 

137

232

157

137

157

77

137

157

I12

       

1788

 

475

  

904

889

4039

731

1179

 

2155

899

2010

2211

1155

1091

2607

6279

E13

       

108

 

96

  

108

108

96

157

96

 

96

99

108

96

108

157

96

108

I13

       

2381

 

438

  

278

720

1177

140

283

 

3077

1273

1223

530

303

3862

350

4222

E14

       

93

 

93

  

96

96

93

108

93

 

93

77

96

93

96

108

93

96

I14

       

2491

 

415

  

355

297

642

224

818

 

5026

1609

6243

2183

2315

2595

1741

2403

E15

       

138

 

72

  

216

135

72

96

72

 

72

157

135

72

135

96

72

135

I15

       

170

 

823

   

1802

3188

108

138

 

527

1022

2481

1535

627

4806

178

525

E16

       

479

 

98

   

1041

98

135

98

 

98

108

63

98

1091

135

98

16

I16

         

306

    

195

268

186

 

1024

2749

 

2244

 

9079

855

15

E17

         

124

    

323

63

124

 

124

96

 

457

 

1931

195

 

I17

                   

1999

 

4192

  

1232

 

E18

                   

135

 

494

  

286

 

I18

                   

15

      

E19

                   

57

      

Total, N.

960

1029

4578

3020

5506

1794

4879

23,264,9

6733

8344

29,564

9107

19,478

26,386

38,806

5413

13,281

30,318

39,390

21,496

28,660

47,331

18,131

134,505

66,471

47,213

Exons, N.

960

1029

2603

1959

2003

1794

3101

3896

1964

1470

1194

902

1972

2976

1944

1887

1572

1207

1559

1794

1803

2388

2877

3836

2115

2044

Introns, N.

0

0

1975

1061

3503

0

1778

19,368,9

4769

6874

28,370

8205

17,506

23,410

36,862

3526

11,709

29,111

37,831

19,702

26,857

44,943

15,254

130,669

64,356

45,169

Exons/tot

100,0

100,0

56,9

64,9

36,4

100,0

63,6

16,7

29,2

17,6

4,0

9,9

10,1

11,3

5,0

34,9

11,8

4,0

4,0

8,3

6,3

5,0

15,9

2,9

3,2

4,3

Exons, n.

1

1

7

6

4

1

2

16

6

17

10

8

15

16

17

17

17

8

17

19

16

18

16

18

17

16

Introns, n.

0

0

6

5

3

 

1

15

5

16

9

7

14

15

16

16

16

7

16

18

15

17

15

17

16

15

Thioredoxin Reductase, Fig. 1

The plots summarize Table 1 information on TrxR gene architecture from various organisms. Panel A, total amount of exons relative to the sum exons + introns, in percentage; Panel B, total number of exons; Panel C, total number of nucleotides within the whole exons pool; Panel D, total number of nucleotides; Panel E, plot highlighting the relative amount of nucleotides within the exons and introns, in red and blue, respectively

However, the number of different isoforms (namely, splicing variants) does not parallel the evolutionary history: for example, among the two fish species reported (Danio rerio and Takifugu rupribes), the genome organization only shows two isoforms for TrxR, while the purple sea urchin (S. purpuratus) has four different isoforms. In addition, splicing variants number are higher in Arthropoda and Nematoda (here represented by Drosophila melanogaster, Anopheles gambiae, and Caenorhabditis elegans) than in Fishes. Clearly, to explain genetic variation and different occurrence of TrxR isoforms in different lineages, reasons other than organism complexity and evolutionary history should be considered. It is not straightforward to link gene organization, in terms of number of exons, and enzyme architecture, in terms of functional domains, even within a single species, because several exons are required to code for a single domain of the enzyme. Comparison among different species fails to show a clear pattern. E.g. comparison of TrxR2 genes from S. cerevisiae, D. melanogaster, and humans reveals that just one single exon codes for the whole protein in the former two species, whereas up to eighteen exons are required to code for the human ortholog. Moreover, TrxR from L. chalumnae (also known as coelacanth), the most ancient representative of extant tetrapods, shows the highest number of exons, nineteen. As a whole, we conclude that TrxR gene organization does not reflect the domain organization of the enzyme, and its relationships with the evolutionary history of the organism are at present unclear.

Mechanism of Action of H-TrxR

The basic mechanism of action of TrxR (both L- and H-) contemplates a series of reduction reactions, with the final goal to move electrons from NADPH to Trx: the electrons flow from NADPH to FAD, then to the first Cys couple located in the proximity of flavin moiety, and finally (in H-TrxRs) the C-terminal arm is reduced from the FAD cysteines and brings its redox center toward the cysteines located on Trx, reducing it (Fig. 2). Structural evidences confirm that the enzyme uses a flexible C-terminal arm acting as a fishline, first by uploading electrons from the FAD domain and then by moving them to Trx (Fig. 2; Fritz-Wolf et al. 2011).
Thioredoxin Reductase, Fig. 2

Tertiary and quaternary structural arrangement of TrxR in complex with Trx (PDB 3qfa). The quaternary structure is symmetric along the twofold axis within the dimerization interface. The picture shows the way the C-terminal tail of chain A (light blue) makes a disulphide bridge with Cys residue located on the Trx (magenta)

The differences in redox centers localization as well as structural organization reflect different catalytic mechanisms for L-TrxRs and H-TrxRs, in spite of the fact that they catalyze the same reaction, namely the NADPH-dependent reduction of Trx, by means of identical redox cofactors (Gromer et al. 2004). In L-TrxRs, both the disulphide (the Cys couple) and pyridine nucleotide are on the re face of the flavin (Fig. 3), whereas in H-TrxRs the Cys couple and the NADPH binding site are on opposite sides of the flavin.
Thioredoxin Reductase, Fig. 3

Structural superposition of FAD molecules from a L-TrxR (E. coli, PDB 1tde, orange) and H-TrxR (human, PDB 2zzc, gray); the picture highlights the main differences in active site arrangement and organization. For comparison, the structure from a GR (E. coli, PDB 1get, cyan) is shown as well. Displayed are the FAD moieties, the redox centers as Cys couples, NADPH molecules, the Tyr residues on the FAD domain of H-TtrR and GR, and the amino acids side chains serving as acid basic catalysts, His (in H-TrxR and GR) and Asp (in L-TrxR). Numbers refer to the position of residues in their respective structure. The two TrxR structures are oxidized, as shown by lacking the disulphide bridge between C59 and C64 (gray) and C135 and C138 (orange), whereas GR displays C42 and C47 reduced (green). Major differences are visible in the arrangement of NADPH domain, which can be assessed by comparing the position of the nicotinamide ring of NADPH in the L-TrxR and GR structures; the pyridinium ring of NADPH is not adjacent to isoalloxazine ring of FAD as seen in the other members of the family. Again the position of a Tyr residue in the NADPH domain is peculiar in that it keeps in place nicotinamide ring of NADPH when is present, by means of π-stacking interaction

When the Trx is bound to the surface of L-TrxR, NADPH is in position to reduce FAD and the dithiol is in position to reduce Trx. Extended movements are required for the L-TrxR in order to reduce a relatively large substrate like the Trx; one domain must rotate so that the N-terminal redox-active disulfide is carried to the hydrophilic surface where the oxidized substrate (Trx) is bound (Williams 1995). In oxidized L-TrxR, NADPH-binding domain is rotated by 66° relative to the orientation found in GR. In this conformation NADPH is >13 Å away from the FAD moiety; as a consequence, to achieve reduction of FAD, the entire domain must rearrange in order to bring NADPH in close proximity of flavin and this implies a very large movement. The confirmation of this peculiar mechanism comes from structural studies on E. coli TrxR (Lennon et al. 2000).

The catalytic mechanism of reduction of substrates by H-TrxR is quite different from that of L-TrxR, as demonstrated by several studies, and includes several intermediates, as depicted in Scheme 1 (Adapted from Cheng et al. 2007).
Scheme 1

Schematic representation of the catalytic cycle of H-TrxR (Adapted from Cheng et al. 2007). Legend: 1, Eox; 2, EH2(A); 3, EH2(B); 4, EH2(C); 5, EH2(D); 6, EH4(A); 7, EH4(B); 8, MDS; 9, TrxR-Trx complex; MC1, MC2, Michaelis complex. All steps are, in principle, reversible, as depicted by double arrow representing equilibrium between two thermodynamic states

In Scheme 1 the oxidized form of TrxR (Eox), the partially reduced form (EH2 and EH4), the Michaelis complexes (MCs) and the oxidized substrate (Trx, in this case) can be recognized. Many efforts were carried out to elucidate the mechanism as a whole together with the electron flow through the different steps. One major contribution comes from the study of D. melanogaster H-TrxR, where each reaction step has been elucidated (Bauer et al. 2003; Cheng et al. 2007), both kinetically and thermodynamically. In D. melanogaster, TrxR is of paramount importance because the fruit fly does not possess a genuine GR, so TrxR in addition to its normal role, must recycle GSSG. The results concerning this particular H-TrR proved to be general and their overall conclusions can be extended to all other the H-TrxR (see Scheme 1). The model for DmTrxR shows the path of electrons from NADPH to FAD, yielding FADH2; from reduced flavin, electrons flow toward the first Cys couple (Cys57-Cys62, in DmTrxR) in near proximity and hence the newly formed disulfide couple shuttles electrons toward C-terminal redox center (Cys489′-Cys490′) located in the neighbor subunit. The studies demonstrate that during catalysis the enzyme cycles between 2-electron and 4-electron reduced form, EH2 and EH4, respectively. As a general rule, one equivalent of NADPH is needed to reduce Eox to EH2 and hence a second NADPH molecule is required in order to achieve the fully reduced form EH4 (Bauer et al. 2003). The first equivalent yields a FADH − NADP+ charge transfer complex that reduces the first Cys couple to form a thiolate-flavin charge-transfer complex. A second equivalent is required to achieve the completely reduced form, EH4, which in turn readily reacts with Trx to produce EH2. The back formation of Eox from EH2 is slow compared to the reduction of Trx from EH4. An important finding is that site-directed mutants of H-TrxRs lacking C-terminal tail are not capable to reduce Trx and can only form EH2. This observation underlines the importance of the C-terminal couple to achieve reduction of Trx; in contrast, GRs and L-TrxRs possess a short C-terminus lacking any redox-active couple and the enzyme can only react with GSSG at the level of first Cys couple in the N-terminal domain.

The Role of Selenium in Catalytic Activity

The number of species whose genome encodes for at least one C-terminal SeC-containing H-TrxR isoform is large and has representatives within all phyla. Species with C-terminal Cys-containing H-TrxR isoforms are less common. In some cases, the same species has genes for isoforms of both types (e.g., C. elegans). The chemical properties of selenium may offer some specific advantages over sulfur, thus a brief comparison of the two may elucidate some catalytic properties of H-TrxRs. Compared to Cys, SeC is a more efficient nucleophile, because of its lower pKa, and its 15% longer bond length, that facilitates the formation of a selenenyl-sulfide bridge between the adjacent Cys-Sec residues (Arscott et al. 1997; Gromer et al. 1998a, b; Lee et al. 2000; Zhong and Holmgren 2000; Thorpe et al. 1976). Moreover, SeC is more resistant than Cys to overoxidation (Saccoccia et al. 2014b). Comparative studies between human’s and fruitfly’s TrxR reveals interesting results on the catalytic properties of Se-containing enzymes. While efficiency drops dramatically in the SeC -- > Cys mutant of mammalians enzyme (ultimately due to geometrical hindrance from juxtaposition of two Cys residues; Lee et al. 2000; Zhong and Holmgren 2000), the H-TrxR from D. melanogaster contains a Ser-Cys-Cys-Ser-COOH terminal arm and the kcat for oxidized Trx of the Drosophila enzyme is at least 50% that of the human enzyme (Kanzok et al. 2001). In this case the Ser residues flanking the dipeptide confer additional efficiency (Gromer et al. 2003). Thus, the local environment of the active site may confer to Cys a reactivity which closely resembles that of SeC (this may possibly, in other Cys TrxR as well). However, SeC (and selenium) confers to the protein: (i) intrinsic higher stability toward harsh conditions that mammalian cells should be able to face, like drop in pH (Bevensee et al. 1996) or high content in ROS whereby Se both is less prone to – and easier to restore from – overoxidized states (Saccoccia et al. 2014b) and, possibly, (ii) the ability to process a wider variety of substrates (Gromer et al. 2003, 2004).

Kinetic Studies

The mechanism of internal reduction of TrxR by NADPH and its reoxidation by Trx was extensively studied in vitro using TrxRs from many organisms. The seminal works from Massey et al. (1970), Williams (1995), Cheng et al. (2007), and Bauer et al. (2003) among others showed that, as a whole, in TrxR reductive half-reaction proceeds via three processes, corresponding to three separate events: the first event is the binding of NADPH and the formation of NADPH-FAD charge transfer complex, with a first-order catalytic constant (k1). This process is rapid, but can be monitored using stopped-flow or continuous-flow devices. The second event takes place when electrons equilibrate between FADH2 and disulphide in the second process, governed by the first order rate constant k2. The third process is complex and leads to the formation of EH4 from a second NADPH (Cheng et al. 2007). The spectral data for kinetic profiles obtained from rapid reaction studies of D. melanogaster TrxR were compatible with the following rate constants: k1 = 90–120 s−1 for flavin reduction by NADPH to give EH2, and k2 = 49 s−1 as FADH reduces the first Cys couple to give EH2B. In this case, two signals can be monitored to follow the reaction: one signal at 462 nm, corresponding to the reduction of FAD, and one at 540 nm, corresponding to the formation of a charge-transfer complex between thiolate and FAD (Bauer et al. 2003; Cheng et al. 2007). The third process was assigned k3 = 21 s−1 (Bauer et al. 2003) and was interpreted as the NADPH-driven reduction of EH2, in the specific arrangement EH2D, to yield EH4. The third process involves multiple steps, including a dithiol-disulphide exchange between the N-terminal couple and C-terminal redox center of the adjacent subunit and the reduction of EH2 to give EH4 (see Scheme 1). In the presence of oxidized Trx (Trx(S)2, see Scheme 1), a further fast process can be recorded with k = 140 s−1 corresponding to the reoxidation of EH4 by DmTrx (Cheng et al. 2007).

Redox Potentials of DmTrxR

The complete characterization of the catalytic cycle of TrxR involves the determination of the differences in the redox potentials of the cycle intermediates (ΔEm), that have been obtained using several methods. A convenient method to estimate small redox potential differences is from the chemical equilibrium. To adopt this method, one must determine the equilibrium constants of each redox step by redox titrations (Veine et al. 1998; Krauth-Siegel et al. 1998; Cenas et al. 2004; Rakauskiene et al. 1989; Cheng et al. 2007). The NADPH-dependent steps to which the method can be applied are:
$$ {\mathrm{E}}_{\mathrm{ox}}+\mathrm{NADPH}+{\mathrm{H}}^{+}<=>{\mathrm{E}\mathrm{H}}_2+{\mathrm{NADP}}^{+} $$
(1)
whose equilibrium constant results:
$$ {\mathrm{K}}_{\mathrm{eq},\mathrm{Eox}/\mathrm{EH}2,\mathrm{NADP}+/\mathrm{NADPH}}=\left[{\mathrm{NADP}}^{+}\right]\left[{\mathrm{E}\mathrm{H}}_2\right]/\left(\left[\mathrm{NADPH}\right]\left[{\mathrm{E}}_{\mathrm{ox}}\right]\right) $$
(2)
And
$$ {\mathrm{EH}}_2+\mathrm{NADPH}+{\mathrm{H}}^{+}\rightleftarrows {\mathrm{EH}}_4+{\mathrm{NADP}}^{+} $$
(3)
whose equilibrium constant results
$$ {\mathrm{K}}_{\mathrm{eq},\mathrm{EH}2/\mathrm{EH}4,\mathrm{NADP}+/\mathrm{NADPH}}=\left[{\mathrm{NADP}}^{+}\right]\left[{\mathrm{EH}}_4\right]/\left(\left[\mathrm{NADPH}\right]\left[{\mathrm{EH}}_2\right]\right) $$
(4)
The Nernst relationships for the two electron reduction of enzyme (Eox/EH2) by NADPH, at equilibrium, is given in (5)
$$ {\mathrm{E}}_{\mathrm{h}}={\mathrm{E}}_{\mathrm{m},\mathrm{NADP}+/\mathrm{NADPH}}+\mathrm{RT}/\mathrm{nF}\ \ln \left[{\mathrm{NADP}}^{+}\right]/\left[\mathrm{NADPH}\right]={\mathrm{E}}_{\mathrm{m},\mathrm{Eox}/\mathrm{EH}2}+\mathrm{RT}/\mathrm{nF}\ \ln \left[{\mathrm{E}}_{\mathrm{ox}}\right]/\left[{\mathrm{E}\mathrm{H}}_2\right] $$
(5)
Introducing (4) in (5), yields (6)
$$ {\displaystyle \begin{array}{l}{\mathrm{E}}_{\mathrm{m},\mathrm{Eox}/\mathrm{EH}2}={\mathrm{E}}_{\mathrm{m},\mathrm{NADP}+/\mathrm{NADPH}}+\\ {}\mathrm{RT}/\mathrm{nF}\ \ln\ {\mathrm{K}}_{\mathrm{eq},\mathrm{Eox}/\mathrm{EH}2,\mathrm{NADP}+/\mathrm{NADPH}}\end{array}} $$
(6)
The redox potential of the couple EH2/EH4 can be determined with the same procedure:
$$ {\displaystyle \begin{array}{l}{\mathrm{E}}_{\mathrm{m},\mathrm{EH}2/\mathrm{EH}4}={\mathrm{E}}_{\mathrm{m},\mathrm{NADP}+/\mathrm{NADPH}}+\\ {}\mathrm{RT}/\mathrm{nF}\ \ln\ {\mathrm{K}}_{\mathrm{eq},\mathrm{EH}2/\mathrm{EH}4,\mathrm{NADP}+/\mathrm{NADPH}}\end{array}} $$
(7)
A different method to measure the equilibrium constants is by steady-state kinetics (Cheng et al. 2007). This method has the advantage of using the apparent Michaelis and Menten parameters, easily obtained by standard enzymology measurements. According to the Haldane relation, the apparent equilibrium constants (Keq,app) can be inferred from the ratio of the specificity constants (kcat/Km) for the forward reaction to that of backward reaction. This is the approach used by Cheng et al. 2007 as well as Cenas et al. (2004) and Rakauskiene et al. (1989). The results obtained from these experiments were in full agreement with those obtained using spectral titration data. In these experiments Trx was the final electron acceptor, and the reaction rates were monitored by following the decrease in absorbance at 340 nm due to the oxidation of NADH. In these experiments, NADH was used instead of NADPH to avoid binding effects of NADP(H) on the measurements. For practical reasons, Cheng and coworkers (Cheng et al. 2007) used approximated equations to estimate the concentrations of each redox species at equilibrium:
$$ \left[\mathrm{NADH}\right]=\left({\mathrm{A}}_{360\mathrm{obs}}-{{\mathrm{A}}_{360}}_{\mathrm{Eox}}\right)/{\upvarepsilon}_{360},\mathrm{NADH} $$
(8)
$$ \left[{\mathrm{E}\mathrm{H}}_2\right]=\left({\mathrm{A}}_{540\mathrm{obs}}-{{\mathrm{A}}_{540}}_{\mathrm{E}\mathrm{ox}}\right)/{\Delta \upvarepsilon}_{540},{\mathrm{E}\mathrm{H}}_2/{\mathrm{E}}_{\mathrm{ox}} $$
(9)
for the 2-electron reduction reaction, and
$$ \left[\mathrm{NADH}\right]=\left({\mathrm{A}}_{360\mathrm{obs}}-{{\mathrm{A}}_{360}}_{\mathrm{EH}2}\right)/{\upvarepsilon}_{360},\mathrm{NADH} $$
(10)
$$ \left[{\mathrm{EH}}_4\right]=\left({\mathrm{A}}_{540\mathrm{obs}}-{\mathrm{A}}_{540,\mathrm{EH}2}\right)/{\Delta \upvarepsilon}_{540},{\mathrm{EH}}_4/{\mathrm{EH}}_2 $$
(11)
for the 4-electron reduction reaction.
A summary of the redox potentials measured using either or both of the above-described methods is reported in Table 3.
Thioredoxin Reductase, Table 3

Redox potentials of TrxRs from D. melanogaster, E. coli, and their substrates

Couple

mV

NADP+/NADP

−327a

Disulfide/dithiol couple

−243 (Ec)b

Eox/EH2

−272 (Dm)c/−254 (Ec)

EH2/EH4

−298 (Dm)c/−271 (Ec)

EH4/EH6

−400d

Trx(S)2/Trx(SH)2

−275 (Dm)c/284.2 (Ec)

Dithionite, SO2/HSO3

−660

Note: Redox potentials for the couples involved in catalysis. Uncertainties associated with values were not reported, since different methods were used to determine potentials in different original papers. The table aims at giving useful indications about the thermodynamics of internal reduction of TrxR and its reoxidation by Trx. The reader may refer to the original papers for additional information on the experimental methods. Dm, D. melanogaster; Ec, E. coli

The redox potential of the NADP+/NADPH couple is considerably more negative than that of the Eox/EH2 couple, so the formation of EH2 is greatly favored. The redox potential of the EH2/EH couple is closer to that of NADP+/NADPH and this explains why the first half-reduction from Eox to EH2 proceeds in much faster rate (90–120 s−1) than the reduction of NADPH with EH2 (21 s−1, Bauer et al. 2003). Assuming that the EH4/EH6 couple has a redox potential as low as −400 mV, the reduction of EH4 by NADPH is not allowed thermodynamically. It should be noticed that the only way to achieve full reduction of TrxR (namely, the EH6 state) is to use dithionite; this is a strong reductant, the calculated midpoint redox potential for the couple SO2/HSO3 at pH 7 and 25 °C being 660 mV (Mayhew 1978; Williams et al. 2000a).

aLennon et al. 2000

bFrom Williams 1995 (and references therein), the redox potential of the disulphide/dithiol couple is 11 mV lower than that of FAD/FADH2 couple and there is a 17 mV negative cooperativity, with the potential of the latter couple depending on the redox state of the former couple

cCheng et al. 2007

dFrom Bauer et al. 2003, reported in Cheng et al. 2007

Some caution is required to make use of the data reported in Table 3. Indeed, if the enzyme cycles between oxidized – Eox, partially reduced state – EH2, and fully reduced state – EH4, some of these states correspond to mixtures of more than a single chemical species. EH2 in particular should be considered an equilibrium mixture of at least two intermediates, in one of which the electrons are shared between the FAD and the nearest Cys couple, whereas in the other they are transferred to the C-terminal SeC-Cys couple. Kinetic evidences sustain this view with the conclusion that a full reduction of the first redox site (FAD and the Cys couple closest to it) does never occur (Williams et al. 2000b; Angelucci et al. 2010). Moreover, the intermediate state EH2 never accumulates in presence of the oxidized substrate, Trx. This is due again to the reversible internal electron transfer to first Cys couple near the FAD (Angelucci et al. 2010).

Inhibitors of TrxR

In view of the crucial physiological roles of Trx, TrxR is an attractive drug target, for pathological conditions such diverse as infections, chronic inflammatory diseases, and cancer (Holmgren and Lu 2010). An incomplete list of drugs targeting TrxR is presented in Table 4 (selected from Cai et al. 2012). The majority of these drugs act as irreversible inhibitors, by binding to the redox-active Cys residues. Some recovery of the activity of irreversibly inhibited TrxR may however be expected to occur in vivo because of transfer of the inhibitor to high or low molecular weight thiols present at high concentration in the cell (Schallreuter et al. 1990).
Thioredoxin Reductase, Table 4

Inhibitors of TrxR

Inhibitor

Clinical indications

Classification

IC50

Auranofin

Rheumatoid arthritis

(cancer, parasitoses)

Gold derivative

(gold is the actual inhibitor)

Irreversible

Aurothiomalate

Same as Auranofin

Same as Auranofin

Same as Auranofin

Aurothioglucose

Same as Auranofin

Same as Auranofin

Same as Auranofin

Cisplatin

Cancer

Platinum derivative

Irreversible

Emetic tartrate

Same as Auranofin

Antimony derivative

Irreversible?

Methylarsonous diiodide

None at present

Arsenic derivative

Irreversible

Curcumin

Same as Auranofin (?)

Alkylating agent

Irreversible

Nitrosoureas

 

Alkylating agent

Irreversible

Azelaic acid

None at present

 

1–10 mM (?)

13-cis-Retinoic acid

None at present

 

0.01 mM

Note: Several of the inhibitors listed in this table are declared in the literature as irreversible, yet have been assigned a non-zero IC50. These apparently self-contradictory cases have been exhaustively analyzed by Saccoccia et al. 2014a. For additional information, see Cai et al. 2012

Irreversible inhibitors of TrxR bind to the reduced enzyme only (EH2 or EH4) and may require long incubation times. Relatively high IC50 values have been repeatedly reported in the literature, presumably because of insufficient incubation of the enzyme with the chosen inhibitor (Saccoccia et al. 2014a).

An interesting property of typical TrxRs with respect to their reactivity with metal-based irreversible inhibitors is that the SeC residue in the C-terminal tail has greater reactivity that ordinary Cys greatly speeds up the binding of metal-based inhibitors. Once the metal-based inhibitor has been coordinated the C-terminal Cys-Sec couple, it can be transferred to the Cys-Cys couple in the FAD domain (Saccoccia et al. 2012, 2014a). Because of this mechanism, metal-based inhibitors present a strong kinetic (rather than thermodynamic) specificity for TrxR over GR, which lacks any SeC residue.

Reversible inhibitors of H-TrxR are comparatively rare, in keeping with the relatively large binding site of the macromolecular substrate (Trx) and the peculiar catalytic mechanism, which relies on the large movement of the flexible C-terminal arm for electron transfer to Trx. The well-defined binding site of NADPH is the main site to which reversible inhibitors may be designed or expected to bind. Unfortunately the large number of NADP-binding enzymes makes inhibitors that compete with NADPH unattractive as drugs, and relatively a few such compounds have been specifically studied as TrxR inhibitors.

Summary

Thioredoxin reductase is a key enzyme in the redox metabolism of a cell; in many parasitic organisms, a particular isoform of TrxR (namely TGR) is the central hub of the reducing machinery. In view of its involvement in many pathological issues as well as its central importance in parasites metabolism, TrxR/TGR inhibition and/or regulation should be regarded as an important clinical goal. A wide number of natural and synthetic compounds target the enzyme in a variety of different ways. The catalytic mechanism of internal reduction and electron transfer of the enzyme involves several intermediates, each characterized by subtle peculiarities in the arrangement of active site residues and cofactors, that may be the target of mechanism-specific inhibition. Such a complex scenario clarifies the reason why the development of a new class of specific inhibitors is an ambitious goal. However the paramount importance of TrxR is unquestionable and is reflected by the huge efforts spent to discover and develop inhibitors targeting species-specific TrxR and its isoforms.

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Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.National Research Council, Institute of Cell Biology and NeurobiologyCampus A. Buzzati-Traverso Monterotondo scaloRomeItaly
  2. 2.Istituto Pasteur – Fondazione Cenci-Bolognetti, Istituto di Biologia e patologia Molecolare del CNR, Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”Sapienza Università di RomaRomeItaly