A Tale of Two Controversies

Abstract

Nitrotyrosine is widely used as a marker of post-translational modification by the nitric oxide ( ⅐ NO, nitrogen monoxide)-derived oxidant peroxynitrite (ONOO ؊ ). However, since the discovery that myeloperoxidase (MPO) and eosinophil peroxidase (EPO) can generate nitrotyrosine via oxidation of nitrite (NO 2 ؊ ), several questions have arisen. First, the relative contribution of peroxidases to nitrotyrosine formation in vivo is unknown. Further, although evidence suggests that the one-electron oxidation product, nitrogen dioxide ( ⅐ NO 2 ), is the primary species formed, neither a direct demonstration that peroxidases form this gas nor studies designed to test for the possible concomitant formation of the two-electron oxidation product, ONOO ؊ , have been reported. Using multiple distinct models of acute inflammation with EPO-and MPO-knockout mice, we now demonstrate that leukocyte peroxidases participate in nitrotyrosine formation in vivo. In some models, MPO and EPO played a dominant role, accounting for the majority of nitrotyrosine formed. However, in other leukocyte-rich acute inflammatory models, no contribution for either MPO or EPO to nitrotyrosine formation could be demonstrated. Head-space gas analysis of heliumswept reaction mixtures provides direct evidence that leukocyte peroxidases catalytically generate ⅐ NO 2 formation using H 2 O 2 and NO 2 ؊ as substrates. However, formation of an additional oxidant was suggested since both enzymes promote NO 2 ؊ -dependent hydroxylation of targets under acidic conditions, a chemical reactivity shared with ONOO ؊ but not ⅐ NO 2 . Collectively, our results demonstrate that: 1) MPO and EPO contribute to tyrosine nitration in vivo; 2) the major reactive nitrogen species formed by leukocyte peroxidase-catalyzed oxidation of NO 2 ؊ is the one-electron oxidation product, ⅐ NO 2 ; 3) as a minor reaction, peroxidases may also catalyze the two-electron oxidation of NO 2 ؊

Inducible Peroxidases Mediate Nitration of Anopheles Midgut Cells Undergoing Apoptosis in Response to Plasmodium Invasion

“…In some models, MPO and EPO played a dominant role, accounting for the majority of nitrotyrosine formed. However, in other leukocyte-rich acute inflammatory models, neither MPO nor EPO contributed to nitrotyrosine formation, implying the existence of alternative nitration pathways (18).…”

“…NO formation resulted in nitrite accumulation, which was proposed to serve as a substrate for a myeloperoxidase (MPO)-mediated tyrosine nitration reaction (17). Experiments using multiple distinct models of acute inflammation with eosinophil peroxidase (EPO)-and MPO knock-out mice indicate that leukocyte peroxidases participate in nitrotyrosine formation in vivo (18). In some models, MPO and EPO played a dominant role, accounting for the majority of nitrotyrosine formed.…”

Rapid and Selective Oxygen-regulated Protein Tyrosine Denitration and Nitration in Mitochondria

“…Quantification of Total Nitrotyrosine and Tyrosine-The snap-frozen samples were thawed and analyzed by stable isotope dilution high pressure liquid chromatography-tandem mass spectrometry, as described previously in detail, using 300 g of protein per analysis (28).…”

Bioanalytical profile of the l-arginine/nitric oxide pathway and its evaluation by capillary electrophoresis

“…1 -5 NO-Tyr per 10 4 Tyr residues [175]. Values from 6-80 μmol/mol (Tyr/NO-Tyr) were reported in reference [176], and ~ 10 ng NO-Tyr/mg protein in plasma and tissue up to 170 ng/mg in actin-enreched fractions of tissue proteins have been reported in reference [177]. It has been demonstrated recently that N-terminal-blocked tryptophan moieties can effectively be nitrosated by N 2 O 3 [138,139], outlining the pivotal role of high nitrogen oxides in aromatic nitrosation.…”