Biochemistry of Thioredoxin Reductase

Crystal structure and catalysis of selenoprotein TrxR1

Crystal structure of selenoprotein TrxR1

TrxR1 electron map

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.

Crystal structure and catalysis of the selenoprotein thioredoxin reductase 1.
Cheng Q, Sandalova T, Lindqvist Y, Arnér E
J. Biol. Chem. 2009 Feb;284(6):3998-4008

Stereo view of the active site of reduced and oxidized TrxR1

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.

TrxR1 catalysis

The prerequisites for this model postulates the following: Cys-64 makes the FAD-charge-transfer complex; Cys-59 reduces the selenenylsulfide by an intermediary selenenylsulfide interchange with Sec-498 with His-472 and Glu-477 potentially acting as proton acceptors (in two successive steps, through His-472); Trx becomes reduced through an intermediate selenenylsulfide between Cys-32 of Trx and Sec-498 of TrxR, and Tyr-116 acting by (i) facilitating selenenylsulfide reduction in the reductive half-reaction through resonance effects and, possibly, (ii) facilitating release of the intermediate selenenylsulfide with Trx by its interaction with the selenenylsulfide between Cys-497 and Sec-498. A, details of the reductive half-reaction. This model is in agreement with data in the literature and with modeling based upon the crystal structure, postulating that an intermediate selenenylsulfide between Cys-59 and Sec-498 is formed. Note, however, that formation of an intermediate disulfide between Cys-59 and Cys-497 cannot be excluded as an alternative pathway for the reductive half-reaction. B, details of a model for Trx reduction. This model postulates that Trx makes a mixed selenenylsulfide between Cys-32 and Sec-498, whereupon a thiolate is formed at Cys-497 (proton transferred to Cys-35 of Trx), which attacks the mixed selenenylsulfide, thereby enabling formation of the selenenylsulfide at the C terminus of TrxR1; this motif is subsequently interacting with Tyr-116.

Redox Pathways in Schistosoma mansoni

Redox Pathways in Mammals and Schistosoma mansoni

In mammals (upper pathway), electrons from NADPH are transferred to an oxidoreductase flavoenzyme, either thioredoxin reductase (TrxR) or glutathione reductase (GR). Electrons are then transferred from the oxidoreductase flavoenzyme to the appropriate electron carrier, either oxidized thioredoxin (Trx-S2) or glutathione disulfide (GSSG) converting them to reduced thioredoxin (Trx-[SH]2) or glutathione (GSH), respectively. Trx-(SH)2 and GSH then supply reducing equivalents for a number of different reactions, including those that are glutaredoxin (Grx)-dependent. In S. mansoni (lower pathway), TrxR and GR are replaced with a unique oxidoreductase flavoenzyme, TGR, which provides reducing equivalents for Trx-, GSH- and Grx-dependent reactions.

Thioredoxin glutathione reductase from Schistosoma mansoni: an essential parasite enzyme and a key drug target.
Kuntz A, Davioud-Charvet E, Sayed A, Califf L, Dessolin J, Arnér E, et al
PLoS Med. 2007 Jun;4(6):e206

Mechanism of irreversible inactivation and concomitant induction of an NADPH oxidase activity upon derivatization of TrxR1 with DNCB

DNCB mechanism

Mechanism of irreversible inactivation and concomitant induction of an NADPH oxidase activity upon derivatization of TrxR1 with DNCB. 1-chloro-2,4-dinitrobenzene (DCNB, CDNB) was found to irreversibly inactivate reduced TrxR with a high affinity and specificity. This reaction gave a concomitant induction of an NADPH oxidase activity by the derivatized enzyme, as shown in

1-Chloro-2,4-dinitrobenzene is an irreversible inhibitor of human thioredoxin reductase. Loss of thioredoxin disulfide reductase activity is accompanied by a large increase in NADPH oxidase activity.
Arnér E, Björnstedt M, Holmgren A
J. Biol. Chem. 1995 Feb;270(8):3479-82.

Later knowing the selenoprotein nature and primary structure of TrxR1 we also showed that the selenocysteine-containing motif in fact was the primary target for dinitrophenyl-derivatization and that the NADPH oxidase activity produced superoxide. This was presented in

Mammalian thioredoxin reductase is irreversibly inhibited by dinitrohalobenzenes by alkylation of both the redox active selenocysteine and its neighboring cysteine residue.
Nordberg J, Zhong L, Holmgren A, Arnér E
J. Biol. Chem. 1998 May;273(18):10835-42

Taken together, the mechanistic model shown in the figure could be formulated. The model involves reduction of the C-terminal selenenylsulfide by NADPH (A - C), which then becomes a target for DNCB alkylation (E) explaining the irreversible inhibition prohibiting the normal cycle of reduction of substrates (D). The FAD of the dnp-derivatized enzyme can still be reduced by NADPH (F) but is proposed to participate in two consecutive one-electron transfers to the nitro groups of the dnp moieties, producing nitro anion radicals (G, I) which may react with molecular oxygen to form superoxide (H, J). The FAD may again be reduced by NADPH (F), thereby forming the NADPH oxidase activity observed experimentally. The model is further discussed in

Superoxide production by dinitrophenyl-derivatized thioredoxin reductase--a model for the mechanism and correlation to immunostimulation by dinitrohalobenzenes.
Arnér E
Biofactors 1999 ;10(2-3):219-26

We also investigated the cytotoxicity and caspase activation of both DNCB and other nitroaromatic compounds that we identified as new inhibitors of TrxR1. Specific inhibition of cellular TrxR1 over glutathione reductase correlated well with caspase activation and cytotoxicity in both lung and cervix carcinoma cell lines. This was a collaboration with Dr. Narimantas Cenas and coworkers, Vilnius, Lithuania, and the findings are described in

Interactions of nitroaromatic compounds with the mammalian selenoprotein thioredoxin reductase and the relation to induction of apoptosis in human cancer cells.
Cenas N, Prast S, Nivinskas H, Sarlauskas J, Arnér E
J. Biol. Chem. 2006 Mar;281(9):5593-603

Scheme of quinone metabolism by mammalian TrxR

Scheme of quinone metabolism by mammalian TrxR

Many quinone compounds may interact with TrxR, either as substrates in one- as well as two-electron transfer reactions or as inhibitors, either competitively or irreversibly. This figure schematically summarizes the three major redox states of mammalian TrxR (oxidized enzyme, two-electron reduced or four-electron reduced species), with the selenolthiol motif of species nr 6 reducing any of the many substrates of this enzyme. Shown is also the reduction of quinones directly by the reduced flavin moiety in species 2 or 5 of the unmodified enzyme, or species 8 of the enzyme irreversibly inhibited for its normal reactions by derivatisation of the selenolate residue in by an electrophilic compound (“X”), which may also be a quinone derivative.

This was a collaboration with Dr. Narimantas Cenas and coworkers, Vilnius, Lithuania, and the figure was taken from the following publication

Interactions of quinones with thioredoxin reductase: a challenge to the antioxidant role of the mammalian selenoprotein.
Cenas N, Nivinskas H, Anusevicius Z, Sarlauskas J, Lederer F, Arnér E
J. Biol. Chem. 2004 Jan;279(4):2583-92