Protein tyrosine nitration is a hallmark of oxidative stress related disease states, commonly detected as anti-nitrotyrosine immunoreactivity. The precise reactive oxygen sources, mechanisms of nitration as well as the modified target proteins and functional consequences, however, remain often unclear. Here we explore protein tyrosine nitration under basal conditions and find surprisingly physiologically nitrated proteins. Upon purifying a prominent physiologically nitrotyrosine immunopositive in hearts from mouse, rat and pig, we identify it as lactate dehydrogenase (LDH). Mechanistically, LDH's degree of basal nitration depended on two canonical sources, NO synthase (NOS) and myeloperoxidase (MPO), respectively. When validating the nitrated amino acid by MALDI-TOF mass spectrometry, we, surprisingly, located LDH nitration not to a tyrosine but the C-terminal tryptophan, Trp324. Molecular dynamics simulations suggested that Trp324 nitration restricts the interaction of the active site loop with the C-terminal a-helix essential for activity. This prediction was confirmed by enzyme kinetics revealing an apparent lower Vmax of nitrated LDH, although yet unidentified concurrent oxidative modifications may contribute. Protein nitration is, thus, not a by definition disease marker but reflects also physiological signaling by eNOS/NO, MPO/nitrite and possibly other pathways. The commonly used assay of anti-nitrotyrosine immunoreactivity is apparently cross-reactive to nitrotryptophan requiring a reevaluation of the protein nitration literature. In the case of LDH, nitration of Trp324 is aggravated under cardiac metabolic stress conditions and functionally limits maximal enzyme activity. Trp324-nitrated LDH may serve both as a previously not recognized disease biomarker and possibly mechanistic lead to understand the metabolic changes under these conditions.