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)-N-circle, 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)-N-circle), 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 helium-swept reaction mixtures provides direct evidence that leukocyte peroxidases catalytically generate (NO2)-N-circle 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)-N-circle. 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, circleNO(2); 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.