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 (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 ((*)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 helium-swept reaction mixtures provides direct evidence that leukocyte peroxidases catalytically generate (*)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 (*)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, (*)NO(2); 3) as a minor reaction, peroxidases may also catalyze the two-electron oxidation of NO(2)(-), 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.
Brennan
Marie-Luise
ML
Department of Cell Biology, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Wu
Weijia
W
Fu
Xiaoming
X
Shen
Zhongzhu
Z
Song
Wei
W
Frost
Heather
H
Vadseth
Caryn
C
Narine
Laura
L
Lenkiewicz
Elizabeth
E
Borchers
Michael T
MT
Lusis
Aldons J
AJ
Lee
James J
JJ
Lee
Nancy A
NA
Abu-Soud
Husam M
HM
Ischiropoulos
Harry
H
Hazen
Stanley L
SL
D000818:Animals; D002177:Candidiasis / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D007962:Leukocytes / Q000201:enzymology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009573:Nitrites / Q000378:metabolism; D009585:Nitrogen Dioxide / Q000378:metabolism; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000502:physiology*; D026361:Reactive Nitrogen Species / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000096:biosynthesis*
D009573:Nitrites; D026361:Reactive Nitrogen Species; C002744:3-nitrotyrosine; D014443:Tyrosine; D006861:Hydrogen Peroxide; D009195:Peroxidase; D009585:Nitrogen Dioxide