title: Leghemoglobin is nitrated in functional legume nodules in a tyrosine residue within the heme cavity by a nitrite/peroxide-dependent mechanism.
abstract: Protein tyrosine (Tyr) nitration is a post-translational modification yielding 3-nitrotyrosine (NO2 -Tyr). Formation of NO2 -Tyr is generally considered as a marker of nitro-oxidative stress and is involved in some human pathophysiological disorders, but has been poorly studied in plants. Leghemoglobin (Lb) is an abundant hemeprotein of legume nodules that plays an essential role as an O2 transporter. Liquid chromatography coupled to tandem mass spectrometry was used for a targeted search and quantification of NO2 -Tyr in Lb. 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 Lb revealed that Tyr nitration requires NO2- + H2 O2 and that peroxynitrite is not an efficient inducer of nitration, probably because Lb isomerizes 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 Lb is a consequence of active metabolism in functional nodules, where Lb may act as a sink of toxic peroxynitrite and may play a protective role in the symbiosis.
authors.affiliation: Departamento de Nutrición Vegetal, Estación Experimental de Aula Dei, Consejo Superior de Investigaciones Científicas (CSIC), 50080, Zaragoza, Spain.
authors.forename: Martha
authors.initials: M
authors.lastname: Sainz
authors.forename: Laura
authors.initials: L
authors.lastname: Calvo-Begueria
authors.forename: Carmen
authors.initials: C
authors.lastname: Pérez-Rontomé
authors.forename: Stefanie
authors.initials: S
authors.lastname: Wienkoop
authors.forename: Joaquín
authors.initials: J
authors.lastname: Abián
authors.forename: Christiana
authors.initials: C
authors.lastname: Staudinger
authors.forename: Silvina
authors.initials: S
authors.lastname: Bartesaghi
authors.forename: Rafael
authors.initials: R
authors.lastname: Radi
authors.forename: Manuel
authors.initials: M
authors.lastname: Becana
mesh_terms: D006418:Heme / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D007874:Leghemoglobin / Q000378:metabolism*; D009566:Nitrates / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism; D009585:Nitrogen Dioxide / Q000378:metabolism; D018384:Oxidative Stress / Q000235:genetics; D030421:Peroxynitrous Acid / Q000378:metabolism; D027805:Phaseolus / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013025:Glycine max / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism*
chemical_list: D007874:Leghemoglobin; D009566:Nitrates; D009573:Nitrites; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D006418:Heme; D006861:Hydrogen Peroxide; D009585:Nitrogen Dioxide
keywords: Glycine max; Phaseolus vulgaris; leghemoglobin; legume nodules; nitrogen dioxide; peroxynitrite; protein tyrosine nitration