A tale of two controversies. Defining both the role of peroxidases in nitrotyrosine formation in vivo using eosinophil peroxidase and myeloperoxidase-deficient mice, and the nature of peroxidase-generated reactive nitrogen species

Nitrotyrosine is widely used as a marker of post-translational modification by the nitric oxide (^.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 (NO₂^-), 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 (^.NO₂), 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 ^.NO₂ formation using H₂O₂ and NO₂^- as substrates. However, formation of an additional oxidant was suggested since both enzymes promote NO₂^--dependent hydroxylation of targets under acidic conditions, a chemical reactivity shared with ONOO^- but not ^.NO₂. 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 NO₂^- is the one-electron oxidation product, ̇NO₂; 3) as a minor reaction, peroxidases may also catalyze the two-electron oxidation of NO₂^-, producing a ONOO^--like product. We speculate that the latter reaction generates a labile Fe-ONOO complex, which may be released following protonation under acidic conditions such as might exist at sites of inflammation.