Protein nitration is a physiological regulator of cardiac lactate dehydrogenase active site loop mobility and activity

Protein tyrosine nitration, detected as anti-nitrotyrosine immunoreactivity, is considered one of the most relevant disease biomarkers of oxidative stress. The mechanism of nitration, target protein and functional consequences remain often unclear. Here we first extend protein tyrosine nitration from pathology to physiology as additional mechanism of post-translational regulation. We focus on a prominent protein band surprisingly nitrotyrosine immunimmunoepositive under basal conditions in mouse, rat and pig heart and more so in diabetes and myocardial stress. Upon purification, we identify it as lactate dehydrogenase (LDH) and its basal nitration depending on NO synthase (NOS) and myeloperoxidase (MPO), respectively. Surprisingly, we locate LDH nitration by MALDI-TOF mass spectrometry not to a tyrosine but the C-terminal tryptophan, Trp-324. Molecular dynamics simulations suggested that Trp-324 nitration restricts the interaction of the active site loop with the C-terminal α-helix essential for activity, which was corroborated by an apparent lower Vmax. In summary, protein nitration is not merely a disease marker but also a physiological event involving both the eNOS/NO and the MPO/nitrite pathways. In the case of LDH, to our knowledge the first protein identified to be basally regulated by nitration, this limits its activity, which is aggravated under cardiac metabolic stress conditions.