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

Abstract

SUMMARY Protein 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.

Plant hemoglobins: a journey from unicellular green algae to vascular plants

“…On the other hand, green pigments derived from Lbs were observed in senescing soybean nodules (Jun et al , ,b) and almost 20 years later identified as nitri‐Lbs, unveiling the generation of nitrating reactive compounds in nodules (Navascués et al , ). By contrast, Lbs bearing nitro‐Tyr are more abundant in young nodules, suggesting that they originate as a result of active nodule metabolism (Sainz et al , ). Lbs could act as scavengers of nitrogen dioxide ( .…”

Protein Carbonylation and Glycation in Legume Nodules

“…In turn, Lb levels decline significantly during nodule senescence, although the mechanisms are still poorly known. Soybean (Glycine max) Lbs can be modified by reactive nitrogen species producing in vivo derivatives bearing nitrated hemes (Navascués et al, 2012) and nitro-Tyr residues (Sainz et al, 2015). Also, Lbs undergo cross-linking and dimerization after incubation with Figure 9.…”

Sinorhizobium meliloti Controls Nitric Oxide–Mediated Post-Translational Modification of a Medicago truncatula Nodule Protein

“…Indeed, leghemoglobin is metal-nitrosylated in nodules of soybean and this property was used to indirectly monitor NO levels (Sánchez et al 2010). On the other hand, recent studies revealed that leghemoglobin is also tyrosine nitrated (Sainz et al 2015). In addition, about 80 proteins have been shown recently to be S-nitrosylated in nodules of M. truncatula (Puppo et al 2013).…”

“…Interestingly, protein tyrosine nitration appears as a prevalent post-translational modification throughout pea root development and intensifies during root senescence (Begara-Morales et al 2013). Very recently, it has been shown that leghemoglobin, an abundant hemeprotein of legume nodules, is tyrosine-nitrated in bean and soybean (Sainz et al 2015). In M. truncatula, only one protein, the glutamine synthetase (GS), has been fully characterized as a target of tyrosine nitration in nodules (Melo et al 2011).…”