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.

Fig. 1.

In vitro reconstitution and nitration of Lb. (A) ApoLbc was reconstituted with either protoheme (LbP) or mesoheme (LbM) and treated for 24 h at pH 6.5 with a 1,000-fold excess of NaNO₂. The products (LbP/N and LbM/N) were loaded on an analytical IEF gel and let to proceed until separation of Lbc₁ (Upper band) and Lbc₂+c₃ (Lower band). Green nitrated derivatives were formed from the Lb bearing heme with vinyls (LbP/N) and not from the Lb bearing heme with ethyl groups (LbM/N). (B) Soret and visible spectra of aliquot samples of the proteins loaded on the gel. Note that LbM and LbM/N have identical spectra, whereas LbP/N is being converted to green derivatives, with a Soret band of lower intensity and a hypsochromic shift of the 625-nm charge transfer absorption band. (C) Mass spectra of the hemes from Lba reconstituted with protoheme or mesoheme and then nitrated. Note the absence of nitration (m/z 620) in the mesoheme. Experiments shown in A and B were repeated three times and the experiment shown in C was repeated twice, each with a different apoLb preparation, producing identical results.

Fig. 2.

Nitration of Lba and separation of the nitrated products on preparative IEF gels. (Left) Mixture of Lba, Lbb, Lbc, and Lbc_m standards. The two Lbc protein bands correspond to Lbc₁ and Lbc₂+c₃. (Right) Lba (500 μM) purified from soybean nodules was nitrated with NaNO₂ (500 mM) for 48 h in citrate buffer (pH 5.5), yielding six derivatives (LbaN1 to LbaN6). (Center) A similar pattern of LbaN derivatives was obtained when nitration was performed in phosphate buffer (pH 7.0). These experiments were repeated at least twice with identical results.

Fig. 3.

Mechanisms that may be operative in the nitration of Tyr residues and heme groups of hemoproteins. The pathways have been exemplified for Lb but are also extensive to Mb and Hb. Some intermediates are indicated in square brackets only to mean that they are formed inside the heme pocket but, except for the nitrite and peroxynitrite complexes, are not necessarily bound to the heme. Experiments designed to test these pathways are described in the text. Additional abbreviations: Lb³⁺, ferric Lb; Lb⁴⁺=O, ferryl Lb; Lb³⁺(nitrovinyl), ferric Lb bearing a vinyl-bound NO₂ group in the heme.

Fig. 4.

Heme pocket of soybean Lba [PDB accession 1BIN (44)] showing relevant α-helices and amino acid residues. (A) Electrostatic potential surface of the whole protein, showing heme localization. (B) Detail of the heme pocket. Ribbon diagram showing side-chains B, E, and F, with stick representation of the relevant amino acid residues, including proximal His⁹² and distal His⁶¹. The vinyl groups of the heme are highlighted in yellow and the propionic groups in pink. Electrostatic potential surface is overlapped as transparency. Molecular structures were inspected, analyzed, and plotted with PyMol (56).