Irreversible Inactivation of Glutathione Peroxidase 1 and Reversible Inactivation of Peroxiredoxin II by H₂O₂in Red Blood Cells

Abstract

Catalase, glutathione peroxidase1 (GPx1), and peroxiredoxin (Prx) II are the principal enzymes responsible for peroxide elimination in RBC. We have now evaluated the relative roles of these enzymes by studying inactivation of GPx1 and Prx II in human RBCs. Mass spectrometry revealed that treatment of GPx1 with H 2 O 2 converts the selenocysteine residue at its active site to dehydroalanine (DHA). We developed a blot method for detection of DHA-containing proteins, with which we observed that the amount of DHA-containing GPx1 increases with increasing RBC density, which is correlated with increasing RBC age. Given that the conversion of selenocysteine to DHA is irreversible, the content of DHA-GPx1 in each RBC likely reflects total oxidative stress experienced by the cell during its lifetime. Prx II is inactivated by occasional hyperoxidation of its catalytic cysteine to cysteine sulfinic acid during catalysis. We believe that the activity of sulfiredoxin in RBCs is sufficient to counteract the hyperoxidation of Prx II that occurs in the presence of the basal level of H 2 O 2 flux resulting from hemoglobin autoxidation. If the H 2 O 2 flux is increased above the basal level, however, the sulfinic Prx II begins to accumulate. In the presence of an increased H 2 O 2 flux, inhibition of catalase accelerated the accumulation of sulfinic Prx II, indicative of the protective role of catalase.

Selenium‐containing amino acids as direct and indirect antioxidants

“…Replacement of Cu 1 with a 2,2 0 -bipyridine-Cu 1 complex abolished the protection (34), suggesting that this was mediated, at least in part, by metal ion coordination, though whether this directly involves the selenium atom is less clear. Evidence for Fe 21 -Se complexes with both selenocystine and SeMet has also been provided in a Fe 21 /H 2 O 2 system (35), but in this case, no DNA protection was observed (35). It should be noted that in the Cu experiments the presence of the 2,2 0 -bipyridine ligand will affect the redox chemistry of the Cu 1 and this may explain, at least in part, these observations.…”

“…The products formed on two-electron oxidation of Sec include diselenides (RSe-SeR, analogous to disulfides) and mixed seleno-sulfur species (RSe-SR). Oxyacids analogous to sulfenic (RS-OH), sulfinic (RSO 2 H), and sulfonic (RSO 3 H) acids have been detected (21), and, in the case of HOSCN, an intermediate adduct RSe-SCN has been postulated (6). Evidence has also been presented for the conversion of Sec residues in proteins to dehydroalanine with loss of the selenium as inorganic selenite via b-elimination (21).…”

“…Oxyacids analogous to sulfenic (RS-OH), sulfinic (RSO 2 H), and sulfonic (RSO 3 H) acids have been detected (21), and, in the case of HOSCN, an intermediate adduct RSe-SCN has been postulated (6). Evidence has also been presented for the conversion of Sec residues in proteins to dehydroalanine with loss of the selenium as inorganic selenite via b-elimination (21). The dehydroalanine has been detected by a blotting method allowing this process to be examined in biological systems (22).…”

“…Severe deficiency (intake \ 20 lg day 21 ) has adverse biological effects with this initially associated with a fatal cardiomyopathy (Keshan disease), and more recently with other syndromes including some cancers, infections, neurodegenerative diseases (Alzheimer's, Parkinson's), decreased immune and thyroid function, and male infertility [reviewed (2)]. Adequate selenium intake (55-70 lg day 21 ) is, therefore, essential for good health. However, supplementation trials (e.g., NHANES, SELECT, ORDET; using up to 200 lg Se day 21 as SeMet) have failed to provide evidence of benefit against prostate cancer, and revealed that high plasma selenium levels ([140 lg Se L 21 ) is associated with increased risk of type 2 diabetes (3).…”

“…Adequate selenium intake (55-70 lg day 21 ) is, therefore, essential for good health. However, supplementation trials (e.g., NHANES, SELECT, ORDET; using up to 200 lg Se day 21 as SeMet) have failed to provide evidence of benefit against prostate cancer, and revealed that high plasma selenium levels ([140 lg Se L 21 ) is associated with increased risk of type 2 diabetes (3). It would therefore appear that adequate, but not excessive, selenium is required for human health; this mini-review focuses on the role of selenium-containing amino acids, both free and in proteins, in the defense against oxidants, as well as their potential pro-oxidant actions.…”