Characterization of the Heme Pocket Structure and Ligand Binding Kinetics of Non-symbiotic Hemoglobins from the Model Legume Lotus japonicus

Abstract

Plant hemoglobins (Hbs) are found in nodules of legumes and actinorhizal plants but also in non-symbiotic organs of monocots and dicots. Non-symbiotic Hbs (nsHbs) have been classified into two phylogenetic groups. Class 1 nsHbs show an extremely high O2 affinity and are induced by hypoxia and nitric oxide (NO), whereas class 2 nsHbs have moderate O2 affinity and are induced by cold and cytokinins. The functions of nsHbs are still unclear, but some of them rely on the capacity of hemes to bind diatomic ligands and catalyze the NO dioxygenase (NOD) reaction (oxyferrous Hb + NO → ferric Hb + nitrate). Moreover, NO may nitrosylate Cys residues of proteins. It is therefore important to determine the ligand binding properties of the hemes and the role of Cys residues. Here, we have addressed these issues with the two class 1 nsHbs (LjGlb1-1 and LjGlb1-2) and the single class 2 nsHb (LjGlb2) of Lotus japonicus, which is a model legume used to facilitate the transfer of genetic and biochemical information into crops. We have employed carbon monoxide (CO) as a model ligand and resonance Raman, laser flash photolysis, and stopped-flow spectroscopies to unveil major differences in the heme environments and ligand binding kinetics of the three proteins, which suggest non-redundant functions. In the deoxyferrous state, LjGlb1-1 is partially hexacoordinate, whereas LjGlb1-2 shows complete hexacoordination (behaving like class 2 nsHbs) and LjGlb2 is mostly pentacoordinate (unlike other class 2 nsHbs). LjGlb1-1 binds CO very strongly by stabilizing it through hydrogen bonding, but LjGlb1-2 and LjGlb2 show lower CO stabilization. The changes in CO stabilization would explain the different affinities of the three proteins for gaseous ligands. These affinities are determined by the dissociation rates and follow the order LjGlb1-1 > LjGlb1-2 > LjGlb2. Mutations LjGlb1-1 C78S and LjGlb1-2 C79S caused important alterations in protein dynamics and stability, indicating a structural role of those Cys residues, whereas mutation LjGlb1-1 C8S had a smaller effect. The three proteins and their mutant derivatives exhibited similarly high rates of NO consumption, which were due to NOD activity of the hemes and not to nitrosylation of Cys residues.

Redefining nitric oxide production in legume nodules through complementary insights from electron paramagnetic resonance spectroscopy and specific fluorescent probes

“…An alternative explanation, however, is that NO 3 − is present in the bacteroids after 3 d ( Arrese-Igor et al , 1998 ), but the generated NO is scavenged by metabolic reactions. Such scavenging may occur by nitrosylation of protein cysteine residues or, most probably, by the NO dioxygenase (NOD) activity of Lb 2+ O 2 that converts NO into NO 3 − in the cytosol of infected cells ( Calvo-Begueria et al , 2017 ). In this scenario, Lb 2+ NO would be detectable in nodules only when at least two mechanisms controlling NO concentration, NOD activity in the cytosol and NirK or Nor activities in the bacteroids, were overwhelmed ( Fig.…”

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

“…Regulation of ligand binding in support of function has been proposed by the detailed study of bimolecular and geminate ligand rebinding to Atha1 and Atha2 (Bruno et al , ; Spyrakis et al , ). Recently, extensive kinetic studies on the various Glbs of the model legume Lotus japonicus (Calvo‐Begueria et al , ) and Arabidopsis (Mot et al , ) strongly suggest non‐redundant functions.…”

Improving Air Quality by Nitric Oxide Consumption of Climate-Resilient Trees Suitable for Urban Greening

“…According to the biochemical activities of PGBs as NO dioxygenase [EC 1.14.12.17] ( Perazzolli et al., 2004 ), nitrite reductase [EC 1.7.2.1] ( Sturms et al., 2011a ; Tiso et al., 2012 ; Kumar et al., 2016 ), and hydroxylamine reductase [EC 1.7.1.10] ( Sturms et al., 2011b ), they seem to be of general importance for the nitrogen metabolism. Since different PGB isoforms differ in their kinetic properties regarding oxygen and NO binding as well as NO deoxygenase activity ( Smagghe et al., 2008 ; Calvo-Begueria et al., 2017 ; Eriksson et al., 2019 ), inducing the biosynthesis of PGBs and improving the biochemical features for NO-fixation might increase the uptake of atmospheric NO. We demonstrated that overexpression of Arabidopsis PGB 1 and 2 in grey poplar could indeed significantly enhance the NO uptake capacity.…”