Protein tyrosine nitration is a well-established phenomenon under disease conditions, representing a shift from the physiological signaling functions of nitric oxide (NO) to oxidative and potentially pathogenic pathways. Tyrosine nitration is mediated by reactive nitrogen species such as peroxynitrite (ONOO⁻) and nitrogen dioxide (NO₂), formed from NO metabolism in the presence of oxidants like superoxide radicals (O₂⁻), hydrogen peroxide (H₂O₂), and transition metal centers. While peroxynitrite can mediate tyrosine nitration in vitro, its role in vivo remains debated, with alternative pathways such as the nitrite/H₂O₂/hemeperoxidase and transition metal-dependent mechanisms proposed. A balanced analysis of evidence indicates that multiple nitration pathways can contribute to tyrosine nitration in vivo, and most involve free radical biochemistry with carbonate radicals (CO₃⁻) and/or oxo-metal complexes oxidizing tyrosine to tyrosyl radical, followed by diffusion-controlled reaction with NO₂ to yield 3-nitrotyrosine. Although protein tyrosine nitration is a low-yield process in vivo, 3-nitrotyrosine serves as a relevant biomarker of NO-dependent oxidative stress. Site-specific nitration can modify protein function and promote biological effects. Understanding the tissue distribution, quantitation, and identification of nitrated proteins in disease states provides new insights into human pathologies. The reaction of NO with superoxide radicals leads to the formation of peroxynitrite, a transient species with a short biological half-life. Peroxynitrite can be involved in both oxidative damage and cytoprotection, and its role in nitration is complex. Transition metal-dependent pathways also contribute to the formation of strong oxidants and nitrogen dioxide. These pathways involve the oxidation of nitrite by hemeperoxidases and transition metal complexes, leading to the formation of NO₂ radicals. The interaction of peroxynitrite with transition metal centers can lead to the formation of oxo-metal complexes and NO₂ radicals, which can further contribute to nitration. Iron regulatory proteins (IRPs) play a role in regulating iron homeostasis and are involved in the regulation of iron-dependent nitration reactions. The free radical pathways to tyrosine nitration involve the formation of tyrosyl radicals and NO₂ radicals, which combine to form 3-nitrotyrosine. Electrophilic aromatic nitration can also occur, involving the formation of a nitronium cation from peroxynitrite and transition metal centers, which can attack tyrosine to form 3-nitrotyrosine. The efficiency of nitration reactions in biology is limited, with low yields of 3-nitrotyrosine under inflammatory conditions. The biological significance of protein tyrosine nitration includes alterations in protein function, conformation, andProtein tyrosine nitration is a well-established phenomenon under disease conditions, representing a shift from the physiological signaling functions of nitric oxide (NO) to oxidative and potentially pathogenic pathways. Tyrosine nitration is mediated by reactive nitrogen species such as peroxynitrite (ONOO⁻) and nitrogen dioxide (NO₂), formed from NO metabolism in the presence of oxidants like superoxide radicals (O₂⁻), hydrogen peroxide (H₂O₂), and transition metal centers. While peroxynitrite can mediate tyrosine nitration in vitro, its role in vivo remains debated, with alternative pathways such as the nitrite/H₂O₂/hemeperoxidase and transition metal-dependent mechanisms proposed. A balanced analysis of evidence indicates that multiple nitration pathways can contribute to tyrosine nitration in vivo, and most involve free radical biochemistry with carbonate radicals (CO₃⁻) and/or oxo-metal complexes oxidizing tyrosine to tyrosyl radical, followed by diffusion-controlled reaction with NO₂ to yield 3-nitrotyrosine. Although protein tyrosine nitration is a low-yield process in vivo, 3-nitrotyrosine serves as a relevant biomarker of NO-dependent oxidative stress. Site-specific nitration can modify protein function and promote biological effects. Understanding the tissue distribution, quantitation, and identification of nitrated proteins in disease states provides new insights into human pathologies. The reaction of NO with superoxide radicals leads to the formation of peroxynitrite, a transient species with a short biological half-life. Peroxynitrite can be involved in both oxidative damage and cytoprotection, and its role in nitration is complex. Transition metal-dependent pathways also contribute to the formation of strong oxidants and nitrogen dioxide. These pathways involve the oxidation of nitrite by hemeperoxidases and transition metal complexes, leading to the formation of NO₂ radicals. The interaction of peroxynitrite with transition metal centers can lead to the formation of oxo-metal complexes and NO₂ radicals, which can further contribute to nitration. Iron regulatory proteins (IRPs) play a role in regulating iron homeostasis and are involved in the regulation of iron-dependent nitration reactions. The free radical pathways to tyrosine nitration involve the formation of tyrosyl radicals and NO₂ radicals, which combine to form 3-nitrotyrosine. Electrophilic aromatic nitration can also occur, involving the formation of a nitronium cation from peroxynitrite and transition metal centers, which can attack tyrosine to form 3-nitrotyrosine. The efficiency of nitration reactions in biology is limited, with low yields of 3-nitrotyrosine under inflammatory conditions. The biological significance of protein tyrosine nitration includes alterations in protein function, conformation, and