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

Xiaoming Fu,John W. Crabb,Thomas Koeck,Kulwant S. Aulak,Stanley L. Hazen,Dennis J. Stuehr

doi:10.1074/jbc.m401586200

Abstract

Growing evidence connects a cumulative formation of 3-nitrotyrosyl adducts in proteins as a marker for oxidative damage with the pathogenesis of various diseases and pathological conditions associated with oxidative stress. A physiological signaling role for protein nitration has also been suggested. Controlled "denitration" would be essential for such a contribution of protein nitration to cellular regulatory processes. Thus, we further characterized such a potentially controlled, reversible tyrosine nitration that occurs in respiring mitochondria during oxygen deprivation followed by reoxygenation, which we recently discovered. Mitochondria constitute cellular centers of protein nitration and are leading candidates for a "nitrative" regulation. Mitochondria are capable of completely eliminating 3-nitrotyrosyl adducts during 20 min of hypoxia-anoxia and undergoing a selective partial reduction after only 5 min. This denitration is independent of protein degradation but depends on the oxygen tension. Reoxygenation re-establishes protein tyrosine nitration patterns that are almost identical to the pattern that occurs before hypoxia-anoxia, with nitration levels that depend on the duration of hypoxia-anoxia. The identified mitochondrial targets of this process are critical for energy and antioxidant homeostasis and, therefore, cell and tissue viability. This cycle of protein nitration and denitration shows analogies to protein phosphorylation, and we demonstrate that the cycle meets most of the criteria for a cellular signaling mechanism. Taken together, our data reveal that protein tyrosine nitration in mitochondria can be controlled, is target-selective and rapid, and is dynamic enough to serve as a nitrative regulatory signaling process that likely affects cellular energy, redox homeostasis, and pathological conditions when these features become disturbed.

Highlights

The covalent addition of a nitro group to protein tyrosine in vivo, forming 3-nitro-L-tyrosine, is a selective post-translational process targeting a few tyrosine residues in a limited number of proteins [1]
Reoxygenation re-establishes protein tyrosine nitration patterns that are almost identical to the pattern that occurs before hypoxia-anoxia, with nitration levels that depend on the duration of hypoxia-anoxia
We recently discovered that isolated rat liver mitochondria are capable of nitrotyrosine clearance during a 20-min episode of hypoxia-anoxia and that nitrotyrosine immunoreactivity reappears during reoxygenation in an L-arginine-dependent way at protein spots that matched the spots before hypoxia-anoxia [18]

Summary

Introduction

The covalent addition of a nitro group to protein tyrosine in vivo, forming 3-nitro-L-tyrosine, is a selective post-translational process targeting a few tyrosine residues in a limited number of proteins [1]. Our data reveal that protein tyrosine nitration in mitochondria can be controlled, is target-selective and rapid, and is dynamic enough to serve as a nitrative regulatory signaling process that likely affects cellular energy, redox homeostasis, and pathological conditions when these features become disturbed.

Results

Conclusion