Interactions of Quinones with Thioredoxin Reductase

European Journal of Biochemistry, 2000

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.

Free Radical Biology and Medicine, 2006

We have mutated the redox active C-terminal motif, Gly-Cys-Sec-Gly, of the mammalian selenoprotein thioredoxin reductase (TrxR) to mimic the C-terminal Ser-Cys-Cys-Ser motif of the non-selenoprotein orthologue of Drosophila melanogaster (DmTrxR). The activity of DmTrxR is almost equal to that of mammalian TrxR, which is surprising, because Cys mutants of selenoproteins are normally 1-2 orders of magnitude less active than their selenocysteine (Sec) containing counterparts. It was shown earlier that the flanking Ser residues were important for activating the Cys residues in DmTrxR (Gromer, et.al. (2003) PNAS 100, 12618-12623). However, the "Drosophila mimic" mutant of the mammalian enzyme studied herein had <0.5% activity compared to wild-type. Rapid kinetic studies revealed that all of the redox centers of the mutant were active, but that the C-terminal dithiols were not effective reductants of thioredoxin. The charge-transfer complex of the two-electron reduced enzyme slowly disappeared as the N-terminal dithiols reduced the C-terminal disulfide. In wild-type enzyme, the selenenylsulfide is more difficult to reduce and the charge-transfer complex is more stable. These findings suggest that features in addition to the flanking Ser residues are important for facilitating the high activity of the insect enzyme and that the corresponding features are absent in mammalian TrxR.

Journal of Molecular Biology, 2007

Human thioredoxin reductase (hTrxR) is a homodimeric flavoprotein crucially involved in the regulation of cellular redox reactions, growth and differentiation. The enzyme contains a selenocysteine residue at its C-terminal active site that is essential for catalysis. This redox center is located on a flexible arm, solvent-exposed and reactive towards electrophilic inhibitors, thus representing a target for antitumor drug development. During catalysis reducing equivalents are transferred from the cofactor NADPH to FAD, then to the N-terminal active site cysteine residues and from there to the flexible C-terminal part of the other subunit to be finally delivered to a variety of second substrates at the molecule's surface. Here we report the first crystal structure of hTrxR1 (Sec→Cys) in complex with FAD and NADP + at a resolution of 2.8 Å. From the crystals three different conformations of the carboxy-terminal arm could be deduced. The predicted movement of the arm is facilitated by the concerted action of the three sidechain residues of N418, N419 and W407, which act as a guiding bar for the C-terminal sliding process. As supported by previous kinetic data, the three visualized conformations might reflect different stages in enzymatic catalysis. Comparison with other disulfide reductases including human glutathione reductase revealed specific inhibitor binding sites in the intersubunit cavity of hTrxR that can be exploited for structure-based inhibitor development.

Journal of Molecular Biology, 2007

Some members of the glutathione peroxidase (GPx) family have been reported to accept thioredoxin as reducing substrate. However, the selenocysteine-containing ones oxidise thioredoxin (Trx), if at all, at extremely slow rates. In contrast, the Cys homolog of Drosophila melanogaster exhibits a clear preference for Trx, the net forward rate constant, k′ +2 , for reduction by Trx being 1.5 × 10 6 M − 1 s − 1 , but only 5.4 M − 1 s − 1 for glutathione. Like other CysGPxs with thioredoxin peroxidase activity, Drosophila melanogaster (Dm)GPx oxidized by H 2 O 2 contained an intra-molecular disulfide bridge between the active-site cysteine (C45; C P ) and C91. Site-directed mutagenesis of C91 in DmGPx abrogated Trx peroxidase activity, but increased the rate constant for glutathione by two orders of magnitude. In contrast, a replacement of C74 by Ser or Ala only marginally affected activity and specificity of DmGPx. Furthermore, LC-MS/MS analysis of oxidized DmGPx exposed to a reduced Trx C35S mutant yielded a dead-end intermediate containing a disulfide between Trx C32 and DmGPx C91. Thus, the catalytic mechanism of DmGPx, unlike that of selenocysteine (Sec)GPxs, involves formation of an internal disulfide that is pivotal to the interaction with Trx. Hereby C91, like the analogous second cysteine in 2-cysteine peroxiredoxins, adopts the role of a "resolving" cysteine (C R ). Molecular modeling and homology considerations based on 450 GPxs suggest peculiar features to determine Trx specificity: (i) a nonaligned second Cys within the fourth helix that acts as C R ; (ii) deletions of the subunit interfaces typical of tetrameric GPxs leading to flexibility of the C R -containing loop. Based of these characteristics, most of the nonmammalian CysGPxs, in functional terms, are thioredoxin peroxidases.

Journal of Biological Chemistry, 1992

Biochemistry, 2008

The flavoprotein quiescin-sulfhydryl oxidase (QSOX) rapidly inserts disulfide bonds into unfolded, reduced proteins with the concomitant reduction of oxygen to hydrogen peroxide. This study reports the first heterologous expression and enzymological characterization of a human QSOX1 isoform. Like QSOX isolated from avian egg white, recombinant HsQSOX1 is highly active toward reduced ribonuclease A (RNase) and dithiothreitol but shows a >100-fold lower k cat /K m for reduced glutathione. Previous studies on avian QSOX led to a model in which reducing equivalents were proposed to relay through the enzyme from the first thioredoxin domain (C70-C73) to a distal disulfide (C509-C512), then across the dimer interface to the FAD-proximal disulfide (C449-C452), and finally to the FAD. The present work shows that, unlike the native avian enzyme, HsQSOX1 is monomeric. The recombinant expression system enabled construction of the first cysteine mutants for mechanistic dissection of this enzyme family. Activity assays with mutant HsQSOX1 indicated that the conserved distal C509-C512 disulfide is dispensable for the oxidation of reduced RNase or dithiothreitol. The four other cysteine residues chosen for mutagenesis, C70, C73, C449, and C452, are all crucial for efficient oxidation of reduced RNase. C452, of the proximal disulfide, is shown to be the charge-transfer donor to the flavin ring of QSOX, and its partner, C449, is expected to be the interchange thiol, forming a mixed disulfide with C70 in the thioredoxin domain. These data demonstrate that all the internal redox steps occur within the same polypeptide chain of mammalian QSOX and commence with a direct interaction between the reduced thioredoxin domain and the proximal disulfide of the Erv/ALR domain.

Biochemistry, 2008

Thioredoxin reductase (TrxR) catalyzes the reduction of thioredoxin (Trx) by NADPH. Because dipteran insects such as Drosophila melanogaster lack glutathione reductase, their TrxRs are particularly important for antioxidant protection; reduced Trx reacts nonenzymatically with oxidized glutathione to maintain a high glutathione/glutathione disulfide ratio. Like other members of the pyridine nucleotidedisulfide oxidoreductase family, TrxR is a homodimer; in the enzyme from D. melanogaster (DmTrxR), each catalytically active unit consists of three redox centers: FAD and an N-terminal Cys-57-Cys-62 redox-active disulfide from one monomer and a Cys-489′-Cys-490′ C-terminal redox-active disulfide from the second monomer. A dyad of His-464′ and Glu-469′ in TrxR acts as the acid-base catalyst of the dithiol-disulfide interchange reactions required in catalysis ) Biochemistry 47, 1721-1731]. In this investigation, the role of Glu-469′ in catalysis by DmTrxR has been studied. The E469′A and E469′Q DmTrxR variants retain 28 and 35% of the wild-type activity, respectively, indicating that this glutamate residue is important but not critical to catalysis. The pH dependence of V max for both glutamate variants yields pK a values of 6.0 and 8.7, compared to those in the wild-type enzyme of 6.4 and 9.3, respectively, indicating that the basicity of His-464′ in TrxR in complex with its substrate, DmTrx-2, is significantly lower in the glutamate variants than in wild-type enzyme. The rates of some steps in the reductive half-reactions in both glutamate variants are much slower than those of the wild-type enzyme. On the basis of our observations, it is proposed that the function of Glu-469′ is to facilitate the positioning of His-464′ toward the interchange thiol, Cys-57, as suggested for the analogous residue in glutathione reductase.

Journal of Biological Chemistry, 2003

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 M r thioredoxin reductases, which catalyze reduction of thioredoxin by NADPH. Thioredoxin reductase has an N-terminal redox-active disulfide (Cys 57 -Cys 62 ) adjacent to the flavin and a redox-active C-terminal cysteine pair (Cys 489 -Cys 490 in the other subunit) that transfer electrons from Cys 57 -Cys 62 to the substrate thioredoxin. Cys 489 -Cys 490 functions similarly to Cys 495 -Sec 496 (Sec ‫؍‬ selenocysteine) and Cys 535 -XXXX-Cys 540 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 M r TrxR, that DmTrxR-1 oscillates between the 2-electron reduced state, EH 2 , and the 4-electron state, EH 4 , in catalysis, after the initial priming reduction of the oxidized enzyme (E ox ) to EH 2 . The reductive half-reaction consumes 2 eq of NADPH in two observable steps to produce EH 4 . The first equivalent yields a FADH ؊ -NADP ؉ chargetransfer complex that reduces the adjacent disulfide to form a thiolate-flavin charge-transfer complex. EH 4 reacts with thioredoxin rapidly to produce EH 2 . In contrast, E ox formation is slow and incomplete; thus, EH 2 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 EH 2 forms. Additional data suggest that Cys 57 attacks Cys 490 in the interchange reaction between the N-terminal dithiol and the C-terminal disulfide.

Journal of Biological Chemistry, 2006

High-M r thioredoxin reductase from the malaria parasite Plasmodium falciparum (PfTrxR) contains three redox active centers (FAD, Cys-88/Cys-93, and Cys-535/Cys-540) that are in redox communication. The catalytic mechanism of PfTrxR, which involves dithiol-disulfide interchanges requiring acidbase catalysis, was studied by steady-state kinetics, spectral analyses of anaerobic static titrations, and rapid kinetics analysis of wild-type enzyme and variants involving the His-509-Glu-514 dyad as the presumed acid-base catalyst. The dyad is conserved in all members of the enzyme family. Substitution of His-509 with glutamine and Glu-514 with alanine led to TrxR with only 0.5 and 7% of wild type activity, respectively, thus demonstrating the crucial roles of these residues for enzymatic activity. The H509Q variant had rate constants in both the reductive and oxidative half-reactions that were dramatically less than those of wild-type enzyme, and no thiolateflavin charge-transfer complex was observed. Glu-514 was shown to be involved in dithiol-disulfide interchange between the Cys-88/Cys-93 and Cys-535/Cys-540 pairs. In addition, Glu-514 appears to greatly enhance the role of His-509 in acid-base catalysis. It can be concluded that the His-509-Glu-514 dyad, in analogy to those in related oxidoreductases, acts as the acid-base catalyst in PfTrxR. Thioredoxin reductase (TrxR) 3 catalyzes the NADPHdependent reduction of the disulfide of thioredoxin (Trx), which in turn acts as an electron donor for proteins such as ribonucleotide reductase, methionine oxide reductase, 2-Cys peroxiredoxins, and a number of transcription factors (1-4). Thus, the function of the thioredoxin system is crucial for cell proliferation, protection against reactive oxygen species, and signal transduction. Levels and activities of the thioredoxin system were found to be increased by at least an order of magnitude in many tumor cell lines (1), indicating that TrxR is critical to their viability, possibly by enhancing both the antioxidant capacity of the cell and the production of nucleotides. The importance of TrxR to cancer cells suggests that it would be an attractive chemotherapeutic target (5-8). Tumor cells and malarial parasites have many common features, including high metabolic rates and rapid cell division. Approximately 500 million cases of malaria caused by infection with the protozoan parasite Plasmodium are reported annually, resulting in up to 3 million deaths, 75% of them being African children. Malaria not only has major health implications, but also causes substantial economic losses running into the billions of dollars in endemic areas. Because of increasing resistances of the parasites against antimalarial drugs, new and better chemotherapies are urgently required (9, 10). Plasmodium lacks both glutathione peroxidase and catalase, indicating that the thioredoxin system is particularly important for multiple roles, including reduction of glutathione, protection against oxidative stress, and biosynthesis of thymine. Indeed, TrxR has already been genetically validated in Plasmodium as a drug target (11). A first screen of a Pfizer chemical library identified nitrophenyl compounds as potential leads for development of clinical TrxR inhibitors (12). To date three compounds showing antimalarial activity as well as specific inhibition of PfTrxR over human TrxR are available (13, 14). To facilitate these investigations, a colorimetric microtiter assay has been developed that is suitable for high throughput inhibitor screening on PfTrxR (14). TrxR occurs in both high and low molecular weight forms in Nature. The low-M r TrxR class comprises enzymes found in Escherichia coli, fungi, plants, and the protozoan parasite, Trichomonas vaginalis (15-18). High-M r TrxR is present in mammals, insects, worms, and the malaria parasite, Plasmodium falciparum . The two classes are distinguished by molecular size, by the number of redox active centers, and by the mechanism by which they transfer reducing equivalents from the apolar part of the active center to the protein surface . High-M r TrxRs have many similarities to glutathione reductase and other disulfide oxidoreductases, but they are more complex ( ). These homodimeric proteins contain three

Journal of Biological Chemistry, 2009

Selenoproteins contain a highly reactive 21st amino acid selenocysteine (Sec) encoded by recoding of a predefined UGA codon. Because of a lack of selenoprotein supply, high chemical reactivity of Sec, and intricate translation machineries, selenoprotein crystal structures are difficult to obtain. Structural prerequisites for Sec involvement in enzyme catalysis are therefore sparsely known. Here we present the crystal structure of catalytically active rat thioredoxin reductase 1 (TrxR1), revealing surprises at the C-terminal Sec-containing active site in view of previous literature. The oxidized enzyme presents a selenenylsulfide motif in trans-configuration, with the selenium atom of Sec-498 positioned beneath the side chain of Tyr-116, thereby located far from the redox active moieties proposed to be involved in electron transport to the Sec-containing active site. Upon reduction to a selenolthiol motif, the Sec residue moved toward solvent exposure, consistent with its presumed role in reduction of TrxR1 substrates or as target of electrophilic agents inhibiting the enzyme. A Y116I mutation lowered catalytic efficiency in reduction of thioredoxin, but surprisingly increased turnover using 5-hydroxy-1,4-naphthoquinone (juglone) as substrate. The same mutation also decreased sensitivity to inhibition by cisplatin. The results suggest that Tyr-116 plays an important role for catalysis of TrxR1 by interacting with the selenenylsulfide of oxidized TrxR1, thereby facilitating its reduction in the reductive half-reaction of the enzyme. The interaction of a selenenylsulfide with the phenyl ring of a tyrosine, affecting turnover, switch of substrate specificity, and modulation of sensitivity to electrophilic agents, gives important clues into the mechanism of TrxR1, which is a selenoprotein that plays a major role for mammalian cell fate and function. The results also demonstrate that a recombinant selenoprotein TrxR can be produced in high amount and sufficient purity to enable crystal structure determination, which suggests that additional structural studies of these types of proteins are feasible.

Oxidative Medicine and Cellular Longevity, 2021

The mammalian cytosolic thioredoxin (Trx) system consists of Trx1 and its reductase, the NADPH-dependent seleno-enzyme TrxR1. These proteins function as electron donor for metabolic enzymes, for instance in DNA synthesis, and the redox regulation of numerous processes. In this work, we analysed the interactions between these two proteins. We proposed electrostatic complementarity as major force controlling the formation of encounter complexes between the proteins and thus the efficiency of the subsequent electron transfer reaction. If our hypothesis is valid, formation of the encounter complex should be independent of the redox reaction. In fact, we were able to confirm that also a redox inactive mutant of Trx1 lacking both active site cysteinyl residues (C32,35S) binds to TrxR1 in a similar manner and with similar kinetics as the wild-type protein. We have generated a number of mutants with alterations in electrostatic properties and characterised their interaction with TrxR1 in ki...

Journal of Biological …, 2003

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 nonactive 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 (E 0) of the Trx1 active site (؊230 mV) and identified a second redox-active dithiol/disulfide (Cys-62 and Cys-69) in an ␣ 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 nonactive 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.

Antioxidants, 2022

Ergothioneine (EGT) is a sulfur-containing amino acid analog that is biosynthesized in fungi and bacteria, accumulated in plants, and ingested by humans where it is concentrated in tissues under oxidative stress. While the physiological function of EGT is not yet fully understood, EGT is a potent antioxidant in vitro. Here we report that oxidized forms of EGT, EGT-disulfide (ESSE) and 5-oxo-EGT, can be reduced by the selenoenzyme mammalian thioredoxin reductase (Sec-TrxR). ESSE and 5-oxo-EGT are formed upon reaction with biologically relevant reactive oxygen species. We found that glutathione reductase (GR) can reduce ESSE, but only with the aid of glutathione (GSH). The reduction of ESSE by TrxR was found to be selenium dependent, with non-selenium-containing TrxR enzymes having little or no ability to reduce ESSE. In comparing the reduction of ESSE by Sec-TrxR in the presence of thioredoxin to that of GR/GSH, we find that the glutathione system is 10-fold more efficient, but Sec-T...

Journal of the American Chemical Society, 2006

Lipid peroxidation is a cellular process that takes place under physiological conditions and particularly after oxidative stress. 4-Hydroxy-2-nonenal (HNE), a major end product of lipid peroxidation, is known to exert a multitude of biological effects and has high reactivity to various cellular components, including DNA and protein. The thioredoxin system, composed of the selenoenzyme thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH, plays a key role in redox regulation and is involved in many signaling pathways. The selenocysteine (Sec) and cysteine (Cys) residues (Cys-496/Sec-497) in the active site of TrxR and a pair of Cys residues (Cys-32/Cys-35) in Trx are sensitive to various alkylating reagents. Herein, we report a mechanistic study on the inhibition of rat TrxR by HNE. The inhibition occurs with TrxR only in its reduced form and persists after removal of HNE. Inhibition of TrxR by HNE added to cultured HeLa cells is also observed. In addition, HNE inactivates reduced Escherichia coli Trx irreversibly. We proved that the redox residues (Cys-496/Sec-497 in TrxR and Cys-32/Cys-35 in Trx) were primary targets for HNE modification. The covalent adducts formed between HNE and Trx were also confirmed by mass spectrum. Because the thioredoxin system is one of the core regulation enzymes of cells&amp;amp;amp;amp;amp;amp;amp;#39; function, inhibition of both TrxR and Trx by HNE provides a possibly novel mechanism for explanation of its cytotoxic effect and signaling activity, as well as the further damage indirectly caused under oxidative stress conditions.

Journal of Biological Chemistry, 2008

Unlike other thioredoxins h characterized so far, a poplar thioredoxin of the h type, PtTrxh4, is reduced by glutathione and glutaredoxin (Grx) but not NADPH:thioredoxin reductase (NTR). PtTrxh4 contains three cysteines: one localized in an N-terminal extension (Cys 4 ) and two (Cys 58 and Cys 61 ) in the classical thioredoxin active site ( 57 WCGPC 61 ). The property of a mutant in which Cys 58 was replaced by serine demonstrates that it is responsible for the initial nucleophilic attack during the catalytic cycle. The observation that the C4S mutant is inactive in the presence of Grx but fully active when dithiothreitol is used as a reductant indicates that Cys 4 is required for the regeneration of PtTrxh4 by Grx. Biochemical and x-ray crystallographic studies indicate that two intramolecular disulfide bonds involving Cys 58 can be formed, linking it to either Cys 61 or Cys 4 . We propose thus a four-step disulfide cascade mechanism involving the transient glutathionylation of Cys 4 to convert this atypical thioredoxin h back to its active reduced form.