Selenium as an Electron Acceptor during the Catalytic Mechanism of Thioredoxin Reductase

Abstract

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.

Compensating for the Absence of Selenocysteine in High-Molecular Weight Thioredoxin Reductases: The Electrophilic Activation Hypothesis

“…Our experiments with hCys will also show that it is the penultimate Cys 2 residue that acts as both the acceptor of electrons from the N-terminal redox center, and the donor of electrons to the substrate. This is in complete agreement with both our recent results with the mammalian mitochondrial enzyme where we showed that the penultimate Sec residue of the Gly-Cys 1 -Sec 2 -Gly redox motif also acts an acceptor and donor of electrons in an analogous fashion (17), and as originally proposed by modeling studies of the oxidized Gly-Cys 1 -Sec 2 -Gly redox motif in the crystal structures of mouse mitochondrial enzyme (12) and the rat cytosolic enzyme (27). …”

“…In Tables 5 and 6, the mutants of mTR3 are abbreviated as mTR3-aa1aa2aa3aa4, using the same manner of abbreviation that is used for mutants of DmTR. The production of full-length, semi-synthetic WT enzyme and mutant enzymes by intein-mediated peptide ligation (IPL) have been previously described for mTR3 (enzymes 13–16 ) in (6–11, 17) and for DmTR (enzymes 1, 2 , and 11 ) in (8) as listed in Tables 2 and 3. For the production of enzymes 3–5, 10 , and 12 , a DmTRΔ3-intein fusion protein (missing the final three amino acids of DmTR) was cleaved in the presence of peptide and 50 mM N -methylmercaptoacetamide (NMA) in cleavage buffer (50 mM MOPS buffer at pH 8.0 with 150 mM NaCl).…”

“…Others have also examined this question in studies of the reaction mechanism of TR and other selenoenzymes (12–16). Selenium participates in the reaction mechanism of TR by two principal means as shown in Figure 1: First as the donor of electrons to the substrate – the disulfide bond of thioredoxin (Trx), and second, by acting as the acceptor of electrons in the thiol/disulfide exchange step that occurs between the N- and C-terminal redox centers (recently shown by us (17)). Most selenoenzymes characterized to date are oxidoreductases that make use of thiol/disulfide exchange reactions of the type shown in Figure 1 in which selenium has replaced sulfur.…”

“…We recently presented evidence that selenium is not the leaving group in the exchange reaction shown in Figure 1C, but is in fact the electrophile being attacked (17). Selenium is a superior electrophile in comparison to sulfur in thiol/disulfide exchange reactions of the type shown in Figure 1C (23–25).…”

A New Oxidative Stress Marker for Thiol-Disulphide Homeostasis in Seasonal Allergic Rhinitis

“…Chemical compounds that contain SH, such as selenium, alpha lipoic acid, and N-acetyl cysteine, may be used in new studies that concern SAR therapy. 29,30 …”