Selenocysteine Confers Resistance to Inactivation by Oxidation in Thioredoxin Reductase: Comparison of Selenium and Sulfur Enzymes

Abstract

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.

RETRACTED: Glutathione peroxidase׳s reaction intermediate selenenic acid is stabilized by the protein microenvironment

“…Overall, in the presence of EDTA, GPx1’s Sec is stable and not prone to hyperoxidation or selenium elimination. This is in agreement with several reports about selenoproteins resistance to inactivation by oxidants [12, 34, 35]. …”

Selenoproteins: Molecular Pathways and Physiological Roles

“…Second, the higher reactivity of Sec compared with Cys in thiol-disulfide-like exchange reactions could be attributed to inherent high nucleophilicity of Sec and its higher reactivity with electrophiles (11). At the same time, electrophilicity of the Se atom could also allow the enzyme to resist irreversible oxidative inactivation, in contrast to Cys whose overoxidized forms are not easily reduced (91,320,321). In addition, evidence was presented that thiol-disulfide exchange reactions could be accelerated by the presence of Se instead of sulfur as Se could serve as a better leaving group as well as the fact that it could stabilize a favorable enzyme conformation due to longer sulfur-selenium bond length compared with the sulfur-sulfur bond (200).…”

Genetic code flexibility in microorganisms: novel mechanisms and impact on physiology

“…For example, mammalian Met- R -sulfoxide reductases containing Sec in the active site are 100-fold more active than the Cys-containing variants 58 . Sec is also found to be more resistant to irreversible oxidation than Cys 59 and has utility as a probe for protein structure and function 60 .…”