Leghemoglobin is nitrated in functional legume nodules in a tyrosine residue within the heme cavity by a nitrite/ peroxide-dependent mechanism

Abstract

Trabajo presentado en la 12th International Conference on Reactive Oxygen and Nitrogen Species in Plants: from model systems to field, celebrada en Verona, Italia, del 24 al 26 de junio de 2015Protein Tyr nitration is a post-translational modification yielding 3-nitrotyrosine (NO2-Tyr). Formation of NO2-Tyr is generally considered as a marker of nitroxidative stress and is involved in some human pathophysiological disorders, but it has been poorly studied in plants. Leghemoglobin (Lb) is an abundant hemeprotein of legume nodules that plays an essential role as O2 transporter. Liquid chromatography coupled to tandem mass spectrometry was used for a targeted search and quantification of NO2-Tyr in Lbs. For all Lbs examined, Tyr30, located in the distal heme pocket, is the major target of nitration. Lower amounts were found for NO2-Tyr25 and NO2-Tyr133. Nitrated Lb and other as yet unidentified nitrated proteins were also detected in nodules of plants not receiving NO3- and were found to decrease during senescence. This demonstrates formation of nitric oxide (NO) and NO2- by alternative means to nitrate reductase, probably via a NO synthase-like enzyme, and strongly suggests that nitrated proteins perform biological functions and are not merely metabolic byproducts. In vitro assays with purified Lbs revealed that Tyr nitration requires NO2- + H2O2 and that peroxynitrite is not an efficient inducer of nitration, possibly by isomerizing it to NO3-. Nitrated Lb is formed via oxoferryl Lb, which generates nitrogen dioxide and tyrosyl radicals. This mechanism is distinctly different from that involved in heme nitration. Formation of NO2-Tyr in Lbs is a consequence of active metabolism in functional nodules, where Lbs may act as a sink of toxic peroxynitrite and may play a protective role in the symbiosis.Peer Reviewe