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

Laura Calvo-Begueria,Cristiano Viappiani,Herald Berghmans,Manuel Becana,Stefano Bruno,Stefania Abbruzzetti,Javier Ramos,Bert Cuypers,Sabine Van Doorslaer,Sylvia Dewilde

doi:10.3389/fpls.2017.00407

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.

Highlights

The first plant hemoglobins (Hbs) were discovered in the root nodules of legumes and designated leghemoglobins (Appleby, 1984)
We found that the dimer of LjGlb1-1 precipitated if salt was omitted during purification and the spectroscopic studies of the wildtype LjGlb1-1 and its mutated forms were performed in buffer supplemented with 200 mM NaCl
Whereas the equilibrium constant for His binding (KH) of LjGlb1-1 is in line with those determined for other class 1 non-symbiotic Hbs (nsHbs) (Smagghe et al, 2009), LjGlb1-2 behaves more like class 2 nsHbs, showing complete bis-histidyl hexacoordination in the deoxyferrous state

Summary

Introduction

The first plant hemoglobins (Hbs) were discovered in the root nodules of legumes and designated leghemoglobins (Appleby, 1984). Class 1 nsHbs have an extremely high O2 affinity and are induced by hypoxia (Trevaskis et al, 1997; Smagghe et al, 2009) and by exposure to nitrate, nitrite, or nitric oxide (NO) (Sasakura et al, 2006) These proteins may play a role in plant survival by increasing the energy status of the cells under hypoxic conditions (Igamberdiev and Hill, 2004). The underlying molecular mechanism is thought to be the Hb/NO cycle, in which the NO dioxygenase (NOD) activity of Hb plays a critical role (Igamberdiev and Hill, 2004) In this reaction, the oxyferrous Hb dioxygenates NO to yield nitrate and ferric Hb. The NOD activities of a few class 1 nsHbs, including A. thaliana Hb1 (AtGlb1), have been measured in vitro (Perazzolli et al, 2004; Igamberdiev et al, 2006; Smagghe et al, 2008). Class 2 nsHbs have a moderate O2 affinity and are induced by low temperature and cytokinins but not by hypoxia (Hunt et al, 2001)

Methods

Results

Discussion

Conclusion