Direct real-time evaluation of nitration with green fluorescent protein in solution and within human cells reveals the impact of nitrogen dioxide vs. peroxynitrite mechanisms

3-Nitrotyrosyl adducts in proteins have been detected in a wide range of diseases. The mechanisms by which reactive nitrogen oxide species may impede protein function through nitration were examined by using a unique model system, which exploits a critical tyrosyl residue in the fluorophoric pocket of recombinant green fluorescent protein (GFP). Exposure of purified GFP suspended in phosphate buffer to synthetic peroxynitrite in either 0.5 or 5 μM steps resulted in progressively increased 3-nitrotyrosyl immunoreactivity concomitant with disappearance of intrinsic fluorescence (IC₅₀ ≈ 20 μM). Fluorescence from an equivalent amount of GFP expressed within intact MCF-7 tumor cells was largely resistant to this bolus treatment (IC₅₀ > 250 μM). The more physiologically relevant conditions of either peroxynitrite infusion (1 μM/min) or de novo formation by simultaneous, equimolar generation of nitric oxide (NO) and superoxide (e.g., 3-morpholinosydnonimine; NONOates plus xanthine oxidase/hypoxanthine, menadione, or mitomycin C) were examined. Despite robust oxidation of dihydrorhodamine under each of these conditions, fluorescence decrease of both purified and intracellular GFP was not evident regardless of carbon dioxide presence, suggesting that oxidation and nitration are not necessarily coupled. Alternatively, both extra- and intracellular GFP fluorescence was exquisitely sensitive to nitration produced by heme-peroxidase/hydrogen peroxidecatalyzed oxidation of nitrite. Formation of nitrogen dioxide (NO₂) during the reaction between NO and the nitroxide 2-phenyl-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide indicated that NO₂ can enter cells and alter peptide function through tyrosyl nitration. Taken together, these findings exemplified that heme-peroxidasecatalyzed formation of NO₂ may play a pivotal role in inflammatory and chronic disease settings while calling into question the significance of nitration by peroxynitrite.