Mammalian Thioredoxin Reductase Is Irreversibly Inhibited by Dinitrohalobenzenes by Alkylation of Both the Redox Active Selenocysteine and Its Neighboring Cysteine Residue*

The immunostimulatory dinitrohalobenzene compound 1-chloro-2,4-dinitrobenzene (DNCB) irreversibly inhibits mammalian thioredoxin reductase (TrxR) in the presence of NADPH, inducing an NADPH oxidase activity in the modified enzyme (Arnér, E. S. J., Björnstedt, M., and Holmgren, A. (1995) J. Biol. Chem. 270, 3479–3482). Here we have further analyzed the reactivity with the enzyme of DNCB and analogues with varying immunomodulatory properties. We have also identified the reactive residues in bovine thioredoxin reductase, recently discovered to be a selenoprotein. We found that 4-vinylpyridine competed with DNCB for inactivation of TrxR, with DNCB being about 10 times more efficient, and only alkylation with DNCB but not with 4-vinylpyridine induced an NADPH oxidase activity. A number of nonsensitizing DNCB analogues neither inactivated the enzyme nor induced any NADPH oxidase activity. The NADPH oxidase activity of TrxR induced by dinitrohalobenzenes generated superoxide, as detected by reaction with epinephrine (the adrenochrome method). Addition of superoxide dismutase quenched this reaction and also stimulated the NADPH oxidase activity. By peptide analysis using mass spectrometry and Edman degradation, both the cysteine and the selenocysteine in the conserved carboxyl-terminal sequence Gly-Cys-Sec-Gly (where Sec indicates selenocysteine) were determined to be dinitrophenyl-alkylated upon incubation of native TrxR with NADPH and DNCB. A model for the interaction between TrxR and dinitrohalobenzenes is proposed, involving a functional FAD in the alkylated TrxR generating an anion nitroradical in a dinitrophenyl group, which in turn reacts with oxygen to generate superoxide. Production of reactive oxygen species and inhibited reduction of thioredoxin by the modified thioredoxin reductase after reaction with dinitrohalobenzenes may play a major role in the inflammatory reactions provoked by these compounds.

[1]  A. Holmgren,et al.  Thioredoxin and glutaredoxin systems. , 2019, The Journal of biological chemistry.

[2]  Elias S. J. Arnér,et al.  Rat and Calf Thioredoxin Reductase Are Homologous to Glutathione Reductase with a Carboxyl-terminal Elongation Containing a Conserved Catalytically Active Penultimate Selenocysteine Residue* , 1998, The Journal of Biological Chemistry.

[3]  L. Montagnier,et al.  Oxidative Stress in Cancer, AIDS, and Neurodegenerative Diseases , 1997 .

[4]  R. Schirmer,et al.  The mechanism of thioredoxin reductase from human placenta is similar to the mechanisms of lipoamide dehydrogenase and glutathione reductase and is distinct from the mechanism of thioredoxin reductase from Escherichia coli. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[5]  S. Ramaswamy,et al.  Crystal structure of Arabidopsis thaliana NADPH dependent thioredoxin reductase at 2.5 A resolution. , 1996, Journal of molecular biology.

[6]  Elias S. J. Arnér,et al.  Efficient reduction of lipoamide and lipoic acid by mammalian thioredoxin reductase. , 1996, Biochemical and biophysical research communications.

[7]  T C Stadtman,et al.  Selenocysteine, identified as the penultimate C-terminal residue in human T-cell thioredoxin reductase, corresponds to TGA in the human placental gene. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[8]  T. Stadtman,et al.  A new selenoprotein from human lung adenocarcinoma cells: purification, properties, and thioredoxin reductase activity. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[9]  G. Powis,et al.  Cloning and sequencing of a human thioredoxin reductase , 1995, FEBS letters.

[10]  C. Williams,et al.  Characterization of lipoamide dehydrogenase from Escherichia coli lacking the redox active disulfide: C44S and C49S. , 1995, Biochemistry.

[11]  M. Hamberg,et al.  Human Thioredoxin Reductase Directly Reduces Lipid Hydroperoxides by NADPH and Selenocystine Strongly Stimulates the Reaction via Catalytically Generated Selenols(*) , 1995, The Journal of Biological Chemistry.

[12]  M. Radman,et al.  Mismatch Binding Protein-Based Mutation Detection Systems , 1995 .

[13]  A. Holmgren,et al.  Thioredoxin structure and mechanism: conformational changes on oxidation of the active-site sulfhydryls to a disulfide. , 1995, Structure.

[14]  A. Holmgren,et al.  1-Chloro-2,4-dinitrobenzene Is an Irreversible Inhibitor of Human Thioredoxin Reductase , 1995, The Journal of Biological Chemistry.

[15]  V. Massey.  Activation of molecular oxygen by flavins and flavoproteins. , 1994, The Journal of biological chemistry.

[16]  L. Liu,et al.  Reversible modification of rat liver glutathione S-transferase 3-3 with 1-chloro-2,4-dinitrobenzene: specific labelling of Tyr-115. , 1993, The Biochemical journal.

[17]  G. Powis,et al.  Purification of human thioredoxin reductase: properties and characterization by absorption and circular dichroism spectroscopy. , 1993, Biochemistry.

[18]  A. Holmgren,et al.  Selenite is a substrate for calf thymus thioredoxin reductase and thioredoxin and elicits a large non-stoichiometric oxidation of NADPH in the presence of oxygen. , 1992, European journal of biochemistry.

[19]  A. Holmgren,et al.  Selenodiglutathione is a highly efficient oxidant of reduced thioredoxin and a substrate for mammalian thioredoxin reductase. , 1992, The Journal of biological chemistry.

[20]  G. Powis,et al.  Mechanism-based inhibition of thioredoxin reductase by antitumor quinoid compounds. , 1992, Biochemical pharmacology.

[21]  G. Powis,et al.  Inhibition of cellular thioredoxin reductase by diaziquone and doxorubicin. Relationship to the inhibition of cell proliferation and decreased ribonucleotide reductase activity. , 1992, Biochemical pharmacology.

[22]  J. Kuriyan,et al.  Convergent evolution of similar function in two structurally divergent enzymes , 1991, Nature.

[23]  J. Yodoi,et al.  Adult T-cell leukemia-derived factor/thioredoxin, produced by both human T-lymphotropic virus type I- and Epstein-Barr virus-transformed lymphocytes, acts as an autocrine growth factor and synergizes with interleukin 1 and interleukin 2. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[24]  A. Stoppani,et al.  Catalysis of nitrofuran redox-cycling and superoxide anion production by heart lipoamide dehydrogenase. , 1990, Biochemical pharmacology.

[25]  J. Wood,et al.  The mechanism of action of the nitrosourea anti-tumor drugs on thioredoxin reductase, glutathione reductase and ribonucleotide reductase. , 1990, Biochimica et biophysica acta.

[26]  J. Wood,et al.  The stereospecific suicide inhibition of human melanoma thioredoxin reductase by 13-cis-retinoic acid. , 1989, Biochemical and biophysical research communications.

[27]  B. Mannervik,et al.  Reduction of 2,4,6-trinitrobenzenesulfonate by glutathione reductase and the effect of NADP+ on the electron transfer. , 1986, The Journal of biological chemistry.

[28]  A. Ahmed,et al.  Delayed-Type Hypersensitivity Skin Testing: A Review , 1983 .

[29]  A. Holmgren,et al.  Rat liver thioredoxin and thioredoxin reductase: purification and characterization. , 1982, Biochemistry.

[30]  A. Holmgren,et al.  Enzymatic reduction of alloxan by thioredoxin and NADPH-thioredoxin reductase. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[31]  A. Holmgren.  Bovine thioredoxin system. Purification of thioredoxin reductase from calf liver and thymus and studies of its function in disulfide reduction. , 1977, The Journal of biological chemistry.

[32]  R. Mason,et al.  The role of catalytic superoxide formation in the O2 inhibition of nitroreductase. , 1975, Biochemical and biophysical research communications.

[33]  W B Jakoby,et al.  Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. , 1974, The Journal of biological chemistry.

[34]  I. Fridovich,et al.  The univalent reduction of oxygen by reduced flavins and quinones. , 1972, The Journal of biological chemistry.

[35]  G. Powis,et al.  Redox signalling and the control of cell growth and death. , 1995, Pharmacology & therapeutics.

[36]  A. Holmgren,et al.  [21] Thioredoxin and thioredoxin reductase , 1995 .

[37]  K. Albermann,et al.  Nuclear Factor Kb: An Oxidative Stress-Responsive Transcription Factor of Eukaryotic Cells (A Review) , 1992 .

[38]  J. Yodoi,et al.  ADF, a growth-promoting factor derived from adult T cell leukemia and homologous to thioredoxin: involvement in lymphocyte immortalization by HTLV-I and EBV. , 1991, Advances in cancer research.