Protein Tyrosine Nitration: Biochemical Mechanisms and Structural Basis of Functional Effects

In proteins, the nitration of tyrosine residues to 3-nitro-tyrosine represents an oxidative post-translational modification that disrupts nitric oxide (•NO) signaling and skews metabolism towards pro-oxidant processes. Indeed, excess levels of reactive oxygen species in the presence of •NO or •NO-derived metabolites lead to the formation of nitrating species such as peroxynitrite. Thus, protein 3-nitrotyrosine has been established as a biomarker of cell, tissue, and systemic "nitroxidative stress". Moreover, tyrosine nitration modifies key properties of the amino acid: phenol group pKa, redox potential, hydrophobicity, and volume. Thus, the incorporation of a nitro group (-NO2) into protein tyrosines can lead to profound structural and functional changes, some of which contribute to altered cell and tissue homeostasis.In this Account, I describe our current efforts to define (1) biologically-relevant mechanisms of protein tyrosine nitration and (2) how this modification can cause changes in protein structure and function at the molecular level. First, I underscore the relevance of protein tyrosine nitration via free-radical-mediated reactions (in both peroxynitrite-dependent and -independent pathways) involving a tyrosyl radical intermediate (Tyr•). This feature of the nitration process is critical because Tyr• can follow various fates, including the formation of 3-nitrotyrosine. Fast kinetic techniques, electron paramagnetic resonance (EPR) studies, bioanalytical methods, and kinetic simulations have all assisted in characterizing and fingerprinting the reactions of tyrosine with peroxynitrite and one-electron oxidants and its further evolution to 3-nitrotyrosine. Recent findings show that nitration of tyrosines in proteins associated with biomembranes is linked to the lipid peroxidation process via a connecting reaction that involves the one-electron oxidation of tyrosine by lipid peroxyl radicals (LOO•).Second, immunochemical and proteomic-based studies indicate that protein tyrosine nitration is a selective process in vitro and in vivo, preferentially directed to a subset of proteins, and within those proteins, typically one or two tyrosine residues are site-specifically modified. The nature and site(s) of formation of the proximal oxidizing or nitrating species, the physicochemical characteristics of the local microenvironment, and the structural features of the protein account for part of this selectivity. How this relatively subtle chemical modification in one tyrosine residue can sometimes cause dramatic changes in protein activity has remained elusive. Herein, I analyze recent structural biology data of two pure and homogenously nitrated mitochondrial proteins (i.e., cytochrome c and manganese superoxide dismutase, MnSOD) to illustrate regioselectivity and structural effects of tyrosine nitration and subsequent impact in protein loss- or even gain-of-function.