Thioredoxin reductase. Two modes of catalysis have evolved

Free Radical Biology and Medicine, 2012

Protein Science, 1998

Mutation of one of the cysteine residues in the redox active disulfide of thioredoxin reductase from Escherichia coli results in C135S with Cys138 remaining or C138S with Cys135 remaining. The expression system for the genes encoding thioredoxin reductase, wild-type enzyme, C135S, and C138S has been re-engineered to allow for greater yields of protein. Wild-type enzyme and C135S were found to be as previously reported, whereas discrepancies were detected in the characteristics of C138S. It was shown that the original C138S was a heterogeneous mixture containing C138S and wild-type enzyme and that enzyme obtained from the new expression system is the correct species. C138S obtained from the new expression system having 0.1% activity and 7% flavin fluorescence of wild-type enzyme was used in this study. Reductive titrations show that, as expected, only 1 mol of sodium dithionite/mol of FAD is required to reduce C138S. The remaining thiol in C135S and C138S has been reacted with 5,5′-dithiobis-(2-nitrobenzoic acid) to form mixed disulfides. The half time of the reaction was <5 s for Cys138 in C135S and approximately 300 s for Cys135 in C138S showing that Cys138 is much more reactive. The resulting mixed disulfides have been reacted with Cys32 in C35S mutant thioredoxin to form stable, covalent adducts C138S-C35S and C135S-C35S. The half times show that Cys138 is approximately fourfold more susceptible to attack by the nucleophile. These results suggest that Cysl38 may be the thiol initiating dithiol-disulfide interchange between thioredoxin reductase and thioredoxin.

FEBS Letters, 2005

The catalytic activity of selenocysteine-containing thioredoxin reductases can be mimicked by cysteine-variants if the local environment at the C-terminal redox center supports thiol activation. This concept of a linear catalytic site was challenged by structural data suggesting that the invariant residue His 106 functions as a base catalyst for the dithiol-disulphide exchange reaction between enzyme and substrate. As reported here, we changed His 106 to asparagine, glutamine, and phenylalanine in various C-terminal mutants of Drosophila melanogaster thioredoxin reductase. The catalytic activity dropped considerably, yet pH-profiles did not reveal differences, rendering a function for His 106 as a base catalyst unlikely. Interestingly, the phenylalanine-mutants, designed as negative controls were the most active mutants which suggests rather a structural role of His 106 .

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 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.

Journal of Biological Chemistry, 2003

Mammalian thioredoxin reductases (TrxR) are important selenium-dependent antioxidant enzymes. Quinones, a wide group of natural substances, human drugs, and environmental pollutants may act either as TrxR substrates or inhibitors. Here we systematically analyzed the interactions of TrxR with different classes of quinone compounds. We found that TrxR catalyzed mixed single-and two-electron reduction of quinones, involving both the selenium-containing motif and a second redox center, presumably FAD. Compared with other related pyridine nucleotide-disulfide oxidoreductases such as glutathione reductase or trypanothione reductase, the k cat /K m value for quinone reduction by TrxR was about 1 order of magnitude higher, and it was not directly related to the one-electron reduction potential of the quinones. A number of quinones were reduced about as efficiently as the natural substrate thioredoxin. We show that TrxR mainly cycles between the four-electron reduced (EH 4 ) and two-electron reduced (EH 2 ) states in quinone reduction. The redox potential of the EH 2 /EH 4 couple of TrxR calculated according to the Haldane relationship with NADPH/NADP ؉ was ؊0.294 V at pH 7.0. Antitumor aziridinylbenzoquinones and daunorubicin were poor substrates and almost inactive as reversible TrxR inhibitors. However, phenanthrene quinone was a potent inhibitor (approximate K i ‫؍‬ 6.3 ؎ 1 M). As with other flavoenzymes, quinones could confer superoxide-producing NADPH oxidase activity to mammalian TrxR. A unique feature of this enzyme was, however, the fact that upon selenocysteine-targeted covalent modification, which inactivates its normal activity, reduction of some quinones was not affected, whereas that of others was severely impaired. We conclude that interactions with TrxR may play a considerable role in the complex mechanisms underlying the diverse biological effects of quinones. Thioredoxin reductase (TrxR, 1 EC 1.8.1.9) catalyzes NADPHdependent reduction of the redox-active disulfide in thioredoxin (Trx), which serves a wide range of functions in cellular proliferation and redox control (1, 2). Thioredoxin reductases are homodimeric proteins that differ in properties between different classes of organisms. Low M r (34-kDa subunit) TrxRs of prokaryotes, plants, or yeast contain FAD and a redox-active disulfide/dithiol active site and display narrow substrate specificities. High M r (54 -58 kDa) TrxRs of animals have in contrast remarkably wide substrate specificities, explained by an additional easily accessible C-terminal redox center. This redox center is either a disulfide/dithiol as in TrxR of Plasmodium falciparum or Drosophila melanogaster or a selenocysteinecontaining selenenylsulfide/selenolthiol motif as found in TrxRs of mammals . In recent years, the catalytic mechanism of mammalian TrxR has been unraveled in significant detail. The three-dimensional crystal structure of rat TrxR is similar to that of glutathione reductase, including conserved FAD and NADP(H)-binding domains, but TrxR has a 16-residue C-terminal extension carrying the catalytic Cys-497/Sec-498 couple that in essence substitutes for glutathione as a substrate of the N-terminally located active site disulfide/dithiol motif (7). In the catalytic cycle of mammalian TrxR, NADPH first reduces FAD, which subsequently passes redox equivalents to the redox-active disulfide with formation of a dithiol, located within a conserved -CVNVGC-sequence. Finally, this dithiol reduces the selenenylsulfide formed by the Cys and Sec residues in a -GCUG sequence located at the C-terminal end of the other subunit in the dimeric enzyme . The so-formed selenolthiol is the proper active site of mammalian TrxR, reducing Trx or other substrates such as lipoic acid, ascorbic acid, or the synthetic model substrate, 5,5Ј-dithiobis-(2-nitrobenzoic acid) (DTNB) (9). Consecutive reduction of the three redox-active motifs of mammalian TrxR, i.e. the FAD, the N-terminal disulfide, and the C-terminal selenenylsulfide, gives two-, four-, and six-electron reduced states of the enzyme, with specific spectral properties that are well characterized (5, 8). It is believed that during normal catalysis, mammalian TrxR cycles between the two-and fourelectron reduced states with the two or four electrons shared mainly between the catalytic disulfide and the selenenylsulfide (5). The disulfide/dithiol motif also forms a charge transfer complex with the FAD (8). Recently, this mechanism was demonstrated also for D. melanogaster TrxR where, however, a

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, 1997

Glutaredoxins belong to the thioredoxin superfamily of structurally similar thiol-disulfide oxidoreductases catalyzing thiol-disulfide exchange reactions via reversible oxidation of two active-site cysteine residues separated by two amino acids (CX 1 X 2 C). Standard state redox potential (E°) values for glutaredoxins are presently unknown, and use of glutathione/glutathione disulfide (GSH/GSSG) redox buffers for determining E° resulted in variable levels of GSH-mixed disulfides. To overcome this complication, we have used reversephase high performance liquid chromatography to separate and quantify the oxidized and reduced forms present in the thiol-disulfide exchange reaction at equilibrium after mixing one oxidized and one reduced protein. This allowed for direct and quantitative pairwise comparisons of the reducing capacities of the proteins and mutant forms. Equilibrium constants from pair-wise reaction with thioredoxin or its P34H mutant, which have accurately determined E° values from their redox equilibrium with NADPH catalyzed by thioredoxin reductase, allowed for transformation into standard state values. Using this new procedure, the standard state redox potentials for the Escherichia coli glutaredoxins 1 and 3, which contain identical active site sequences CPYC, were found to be E° ‫؍‬ ؊233 and ؊198 mV, respectively. These values were confirmed independently by using the thermodynamic linkage between the stability of the disulfide bond and the stability of the protein to denaturation. Comparison of calculated E° values from a number of proteins ranging from ؊270 mV for E. coli Trx to ؊124 mV for DsbA obtained using this method with those determined using glutathione redox buffers provides independent confirmation of the standard state redox potential of glutathione as ؊240 mV. Determining redox potentials through direct proteinprotein equilibria is of general interest as it overcomes errors in determining redox potentials calculated from large equilibrium constants with the strongly reducing NADPH or by accumulating mixed disulfides with GSH.

PLoS ONE, 2013

Thioredoxin-like proteins contain a characteristic C-x-x-C active site motif and are involved in a large number of biological processes ranging from electron transfer, cellular redox level maintenance, and regulation of cellular processes. The mechanism for deprotonation of the buried C-terminal active site cysteine in thioredoxin, necessary for dissociation of the mixed-disulfide intermediate that occurs under thiol/disulfide mediated electron transfer, is not well understood for all thioredoxin superfamily members. Here we have characterized a 8.7 kD thioredoxin (BC3987) from Bacillus cereus that unlike the typical thioredoxin appears to use the conserved Thr8 side chain near the unusual C-P-P-C active site to increase enzymatic activity by forming a hydrogen bond to the buried cysteine. Our hypothesis is based on biochemical assays and thiolate pK a titrations where the wild type and T8A mutant are compared, phylogenetic analysis of related thioredoxins, and QM/MM calculations with the BC3987 crystal structure as a precursor for modeling of reduced active sites. We suggest that our model applies to other thioredoxin subclasses with similar active site arrangements.

The Journal of Physical Chemistry B, 2008

Thioredoxins (Trx) are enzymes with a characteristic CXYC active-site motif that catalyze the reduction of disulfide bonds in other proteins. We have theoretically explored this reaction mechanism, both in the gas phase and in water, using density functional theory. The mechanism of disulfide reduction involves two consecutive thiol-disulfide exchange reactions, that is, nucleophilic substitutions at sulfur (S N 2@S): first, by one Trx cysteine-thiolate group (Cys-32) at a sulfur atom of the disulfide substrate and, second, by the other Trx cysteine-thiolate group (the buried thiol of Cys-35) at the sulfur atom of the first Trx cysteine. We have investigated the intrinsic nature of such S N 2@S substitution using the simple CH 3 S -+ CH 3 SSCH 3 model and how it is affected by solvation in aqueous solution. Next, we have examined how the behavior of the elementary S N 2@S steps changes in the more realistic enzyme-substrate model CGPC + CH 3 SSCH 3 , which contains the active-site of Trx. In all model reactions, solvation turns the hypervalent trisulfide anion (i.e., the S N 2@S transition species) from a stable complex into a transition state. Importantly, our analyses suggest that the deprotonation of the buried thiol (which is required before the latter can enter into the second S N 2@S step) is done by the leaving group evolving from the first S N 2@S step. Finally, molecular dynamics (MD) simulations, in the gas phase and in water, of CGPC, CGGC, and the corresponding wild-type Trx and P34G Trx show that the activity of the thioredoxin active-site motif (CXYC) is determined not only by the structural rigidity associated with the particular variable residues (XY) but also by the number of amide N-H groups. The latter are involved in the stabilization of the Cys-32 thiolate and thus affect the acidity and nucleophilicity of this residue. * Corresponding authors.