Leghemoglobin green derivatives with nitrated hemes evidence production of highly reactive nitrogen species during aging of legume nodules

Abstract

Globins constitute a superfamily of proteins widespread in all kingdoms of life, where they fulfill multiple functions, such as efficient O 2 transport and modulation of nitric oxide bioactivity. In plants, the most abundant Hbs are the symbiotic leghemoglobins (Lbs) that scavenge O 2 and facilitate its diffusion to the N 2 -fixing bacteroids in nodules. The biosynthesis of Lbs during nodule formation has been studied in detail, whereas little is known about the green derivatives of Lbs generated during nodule senescence. Here we characterize modified forms of Lbs, termed Lba m , Lbc m , and Lbd m , of soybean nodules. These green Lbs have identical globins to the parent red Lbs but their hemes are nitrated. By combining UV-visible, MS, NMR, and resonance Raman spectroscopies with reconstitution experiments of the apoprotein with protoheme or mesoheme, we show that the nitro group is on the 4-vinyl. In vitro nitration of Lba with excess nitrite produced several isomers of nitrated heme, one of which is identical to those found in vivo. The use of antioxidants, metal chelators, and heme ligands reveals that nitration is contingent upon the binding of nitrite to heme Fe, and that the reactive nitrogen species involved derives from nitrous acid and is most probably the nitronium cation. The identification of these green Lbs provides conclusive evidence that highly oxidizing and nitrating species are produced in nodules leading to nitrosative stress. These findings are consistent with a previous report showing that the modified Lbs are more abundant in senescing nodules and have aberrant O 2 binding.

Nitric oxide from a “green” perspective

“…In functional N2-fixing nodules, NO appears to inhibit nitrogenase activity, suggesting that modulation of NO levels in rhizobia might be necessary for efficient symbiosis [47], with the formation of nitric oxide-leghemoglobin complexes playing an essential role in this process [48]. In senescing nodules, an increase in ROS and RNS causing nitro-oxidative stress has been reported, leading to a reduction in the ability of symbiotic leghemoglobins to scavenge oxygen due to modifications mediated by these ROS/RNS [49,50].…”

Reactive Oxygen/Nitrogen Species and Antioxidant Defenses in Lotus japonicus

“…Other RNS with highly oxidizing and nitrating potential can be formed by interaction of ROS (Navascués et al 2012). …”

Control of NO level in rhizobium-legume root nodules

“…Hence it might be important to scavenge NO before it can nitrate hemes of leghemoblobins which then would be deficient in O 2 binding and therefore impair nitrogen fixation. 15 Alternately, we could hypothesize that Hmp is more efficient in degrading NO than plant hemoglobins. Also, bacterial proteins could be more sensitive to NO than plant proteins and have to be protected by a bacterial cytosolic globin.…”

“…In plant systems, among the molecular mechanisms underlying NO signaling role, are the post-translational modifications of target proteins. In that framework, at least two proteins from legumes are post-translationally affected by NO: leghemoglobin 15 which helps to provide oxygen to the nitrogenfixing bacteroids, and glutamine synthetase, 16 a crucial plant enzyme responsible for the assimilation of NH 3 or ammonium by the plant. These may explain some of the deleterious effects of NO.…”