Tryptophan nitration physiologically regulates cardiac lactate dehydrogenase active site loop mobility and activity

Protein tyrosine nitration is considered one of the most relevant disease biomarkers of oxidative stress. The mechanism of nitration, target protein and functional consequences remain often unclear. We focus on a prominent protein band surprisingly nitrotyrosine immunopositive 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 alfa-helix essential for activity, which was corroborated by an apparent lower Vmax. In summary, here we first extend protein nitration from pathology to physiology as an additional mechanism of post-translational regulation.