Mammalian thioredoxin reductase (TR) is a selenocysteine (Sec)-containing homodimeric pyridine nucleotide oxidoreductase which catalyzes the reduction of oxidized thioredoxin. We have previously demonstrated the full-length mitochondrial mammalian TR (mTR3) enzyme to be resistant to inactivation from exposure to 50 mM H2O2. Because a Sec residue oxidizes more rapidly than a cysteine (Cys) residue, it has been previously thought that Sec-containing enzymes are “sensitive to oxidation” compared to Cys-orthologs. Here we show for the first time a direct comparison of the abilities of Sec-containing mTR3 and the Cys-ortholog from D. melanogaster (DmTR) to resist inactivation by oxidation from a variety of oxidants including H2O2, hydroxyl radical, peroxynitrite, hypochlorous acid, hypobromous acid, and hypothiocyanous acid. The results show that the Sec-containing TR is far superior to the Cys-ortholog TR in resisting inactivation by oxidation. To further test our hypothesis that the use of Sec confers strong resistance to inactivation by oxidation, we constructed a chimeric enzyme in which we replaced the active site Cys nucleophile of DmTR with a Sec residue using semisynthesis. The chimeric Sec-containing enzyme has similar ability to resist inactivation by oxidation as the wild type Sec-containing TR from mouse mitochondria. The use of Sec in the chimeric enzyme “rescued” the enzyme from oxidant-induced inactivation for all of the oxidants tested in this study, in direct contrast to previous understanding. We discuss two possibilities for this rescue effect from inactivation under identical conditions of oxidative stress: (i) Sec resists over-oxidation and inactivation, whereas a Cys residue can be permanently over-oxidized to the sulfinic acid form, and (ii) Sec protects the body of the enzyme from harmful oxidation by allowing the enzyme to metabolize (turnover) various oxidants much better than a Cys-containing TR.

Mammalian thioredoxin reductase (TR) is a pyridine nucleotide disulfide oxidoreductase that uses the rare amino acid selenocysteine (Sec) in place of the more commonly used amino acid cysteine (Cys) in the redoxactive tetrapeptide Gly-Cys-Sec-Gly motif to catalyze thiol/disulfide exchange reactions. Sec can accelerate the rate of these exchange reactions by being: (i) a better nucleophile than Cys, (ii) a better electrophile than Cys, (iii) a better leaving group than Cys, or (iv) by using a combination of all three of these factors, being more chemically reactive than Cys. The role of the selenolate as a nucleophile in the reaction mechanism was recently demonstrated by creating a mutant of human thioredoxin reductase-1 in which the Cys497-Sec498 dyad of the C-terminal redox center was mutated to either a Ser497-Cys498 dyad or a Cys497-Ser498 dyad. Both mutant enzymes were incubated with human thioredoxin (Trx) in order to determine which mutant formed a mixed disulfide bond complex. Only the mutant containing the Ser497-Cys498 dyad formed a complex and this structure has been solved by X-ray crystallography [Fritz-Wolf, K., Kehr, S., Stumpf, M., Rahlfs, S., and Becker, K. (2011) Crystal structure of the human thioredoxin reductase-thioredoxin complex, Nat Commun 2, 383.] This experimental observation most likely means that the selenolate is the initial attacking nucleophile onto the disulfide bond of Trx because a complex only resulted when Cys was present in the second position of the dyad. As a nucleophile, the selenolate of Sec helps to accelerate the rate of this exchange reaction relative to Cys in the Sec → Cys mutant enzyme. Another thiol/disulfide exchange reaction that occurs in the enzymatic cycle of the enzyme is the transfer of electrons from the thiolate of the interchange Cys residue of the N-terminal redox center to the 8-membered selenosulfide ring of the C-terminal redox center. The selenium atom of the selenosulfide could accelerate this exchange reaction by being a good leaving group (attack at the sulfur atom), or by being a good electrophile (attack at the selenium atom). Here we provide strong evidence that the selenium atom is attacked in this exchange step. This was shown by creating a mutant enzyme containing a Gly-Gly-Seccoo- motif that had 0.5% of the activity of the wild type enzyme. This mutant lacks the adjacent, resolving Cys residue, which acts by attacking the mixed selenosulfide bond that occurs between enzyme and substrate. A similar result was obtained when Sec was replaced with homocysteine. These results highlight the role of selenium as an electron acceptor in the catalytic mechanism of thioredoxin reductase as well as its established role as an electron donor to the substrate.

Cytosolic thioredoxin reductase 1 is the best characterized of the class of high molecular weight (Mr) thioredoxin reductases (TRs). TR1 is highly dependent upon the rare amino acid selenocysteine (Sec) for the reduction of thioredoxin (Trx) and a host of small molecule substrates, as mutation of Sec to cysteine (Cys) results in large drop in catalytic activity for all substrate types. Previous work in our lab and others has shown that the mitochondrial TR (TR3) is much less dependent upon the use of Sec for the reduction of small molecules. Sec-dependent substrate utilization behavior of TR1 may be the exception and not the rule as we show that a variety of high Mr TRs from other organisms including D. melanogaster, C. elegans, and P. falciparum do not require Sec to reduce small molecule substrates including 5,5′dithio-bis(2-nitrobenzoic acid), lipoic acid, selenite, and selenocystine. The data shows that high Mr TRs can be divided into two groups based upon substrate utilization patterns: a TR1 group and a TR3-like group. We have constructed mutants of TR3-like enzymes from mouse, D. melanogaster, C. elegans, and P. falciparum and the kinetic data from these mutants show that these enzymes are less dependent upon the use of Sec for the reduction of substrates. We posit that the mechanistic differences between TR1 and the TR3-like enzymes in this study are due to the presence of a “guiding bar”, amino acids 407–422, found in TR1, but not TR3-like enzymes. The guiding bar, proposed by Becker and coworkers [Fritz-Wolf, K., Urig, S., and Becker, K. (2007) The structure of human thioredoxin reductase 1 provides insights into C-terminal rearrangements during catalysis, J Mol Biol 370, 116–127.], restricts the motion of the C-terminal tail containing the C-terminal Gly-Cys-Sec-Gly, redox-active tetrapeptide so that only this C-terminal redox center can be reduced by the N-terminal redox center, with the exclusion of most other substrates. This makes TR1 highly dependent upon the use of Sec because the selenium atom is responsible for both accepting electrons from the N-terminal redox center and donating them to the substrate in this model. Loss of both Se-electrophilicity and Se-nucleophilicity in the Sec → Cys mutant of TR1 greatly reduces catalytic activity. TR3-like enzymes in contrast are less dependent upon the use of Sec because the absence of the guiding bar in these enzymes allows for greater substrate access to the N-terminal redox center, and because they can make use of alternative mechanistic pathways that are not available to TR1.

No abstract

Thioredoxin reductase (TR) is an oxidoreductase responsible for maintaining thioredoxin in the reduced state, thereby contributing to proper cellular redox homeostasis. The C-terminal active site of mammalian TR contains the rare amino acid selenocysteine, which is essential to its activity. Alterations in thioredoxin-reductase activity due to changes in cellular redox homeostasis are found in clinical conditions such as cancer, viral infection, and various inflammatory processes, and quantification of thioredoxin-activity can therefore be a valuable indicator of clinical conditions. Here we describe a new, direct assay (termed the SC-TR assay) to determine the activity of TR based upon the reduction of selenocystine, a diselenide-bridged amino acid. Rather than being an end-point assay as is in older methods, the SC-TR assay directly monitors the continuous consumption of NADPH at 340 nm by TR as it reduces selenocystine. The SC-TR assay can be used in a cuvette using traditional spectrophotometry, or as a 96-well plate based format using a plate reader. In addition, the SC-TR assay is compatible with the use of non-ionic detergents, making it more versatile than other methods using cell lysates.