A Tale of Two Controversies

doi:10.1074/jbc.M112400200

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*

From the ^‡Department of Cell Biology and the ^§Department of Cardiovascular Medicine and Center for Cardiovascular Diagnostics and Prevention, Cleveland Clinic Foundation, Cleveland, Ohio 44195, the ^¶Stokes Research Institute, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, Pennsylvania 19104, the ^‖Departments of Biochemistry and Molecular Biology, Mayo Clinic Scottsdale, Scottsdale, Arizona 85259, and the ^**Departments of Medicine, Human Genetics, Microbiology, Immunology, and Molecular Genetics, UCLA, Los Angeles, California 90095

Abstract

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 $\text{[math]}$ ), 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 helium-swept reaction mixtures provides direct evidence that leukocyte peroxidases catalytically generate ^⋅NO₂formation using H₂O₂ and NO $\text{[math]}$ as substrates. However, formation of an additional oxidant was suggested since both enzymes promote NO $\text{[math]}$ -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 NO $\text{[math]}$ is the one-electron oxidation product, ^⋅NO₂; 3) as a minor reaction, peroxidases may also catalyze the two-electron oxidation of NO $\text{[math]}$ , 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.

Footnotes

↵* This work was supported by National Institutes of Health Grants HL61878 and HL62526 (to S. L. H.), HL30568 (to A. J. L.), HL60793 (to N. A. L.), HL65228 (to J. J. L.), and HL54926 (to H. I.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ To whom correspondence should be addressed: Dept. of Cell Biology, Cleveland Clinic Foundation, 9500 Euclid Ave., NC-10, Cleveland, OH 44195. Tel.: 216-445-9763. Fax: 216-444-9404; E-mail: hazens@ccf.org.
Published, JBC Papers in Press, February 27, 2002, DOI 10.1074/jbc.M112400200
Abbreviations:

MPO

myeloperoxidase

BHT

butylated hydroxytoluene

DHB

dihydroxybenzoic acid

DOPA

3,4-dihydroxyphenylalanine

DTPA

diethylenetriaminepentaacetic acid

EPO

eosinophil peroxidase

ESI

electrospray ionization

GC

gas chromatography

HPA

3-(4-hydroxyphenyl)propanoic acid

HPLC

high performance liquid chromatography

KO

knock-out

LC

liquid chromatography

MS

mass spectrometry

m/z

mass-to-charge-ratio

PBS

phosphate buffered saline

SA

salicylate

WT

wild-type
- Received December 27, 2001.
- Revision received February 22, 2002.
The American Society for Biochemistry and Molecular Biology, Inc.