NIN-like protein transcription factors regulate leghemoglobin genes in legume nodules.

Leghemoglobins enable the endosymbiotic fixation of molecular nitrogen (N2) in legume nodules by channeling O2 for bacterial respiration while maintaining a micro-oxic environment to protect O2-sensitive nitrogenase. We found that the NIN-like protein (NLP) transcription factors NLP2 and NIN directly activate the expression of leghemoglobins through a promoter motif, resembling a "double" version of the nitrate-responsive elements (NREs) targeted by other NLPs, that has conserved orientation and position across legumes. CRISPR knockout of the NRE-like element resulted in strongly decreased expression of the associated leghemoglobin. Our findings indicate that the origins of the NLP-leghemoglobin module for O2 buffering in nodules can be traced to an ancient pairing of NLPs with nonsymbiotic hemoglobins that function in hypoxia.

Assessment of the potential allergenicity and toxicity of Pichia proteins in a novel leghemoglobin preparation.

The production of soy leghemoglobin C2 (LegH) by Pichia pastoris (syn. K. phaffii) was developed by Impossible Foods to serve as a sustainable source of flavor and aroma in plant-based meats. The potential allergenicity and toxicity of a LegH from a new production process was analyzed using bioinformatics, proteomics and a pepsin digestion assay on leghemoglobin, and residual host proteins. LegH in the new preparation had the same proteoform as in the previous preparations as well as in soy root nodule extracts. Results of seven Pichia proteins, each representing ≥1% of the total protein content, showed no significant sequence matches to any known allergens with the exception of one, which matched the highly conserved wheat GAPDH, whose protein homolog is found in fungi and humans. Based on the data, it is unlikely that there is any risk of cross reactivity between LegH Prep and GAPDH. Pichia protein sequences showed very good alignment to homologous proteins from many common yeasts including Saccharomyces sp. In addition, LegH and Pichia proteins were all rapidly digested in a pepsin digest assay. In conclusion, LegH Prep from this P. pastoris production process is unlikely to pose a risk of food allergenicity.

Comparative analysis of the heme iron electronic structure and stereochemistry in tetrameric rabbit hemoglobin and monomeric soybean leghemoglobin a using Mössbauer spectroscopy with a high velocity resolution.

A comparative study of tetrameric rabbit hemoglobin and monomeric soybean leghemoglobin a in the oxy- and deoxy-forms was carried out using 57Fe Mössbauer spectroscopy with a high velocity resolution in order to analyze the heme iron electronic structure and stereochemistry in relation to the Mössbauer hyperfine parameters. The Mössbauer spectra of tetrameric rabbit hemoglobin in both forms were fitted using two quadrupole doublets related to the 57Fe in ɑ- and ß-subunits. In contrast, the Mössbauer spectra of monomeric soybean leghemoglobin a were fitted using: (i) two quadrupole doublets for the oxy-form related to two conformational states of the distal His E7 imidazole ring and different hydrogen bonding of oxygen molecule in the oxy-form and (ii) using three quadrupole doublets for deoxy-form related to three conformational states of the proximal His F8 imidazole ring. Small variations of Mössbauer hyperfine parameters related to small differences in the heme iron electronic structure and stereochemistry in tetrameric rabbit hemoglobin and monomeric soybean leghemoglobin a are discussed.

Molecular dynamics simulations indicate that tyrosineB10 limits motions of distal histidine to regulate CO binding in soybean leghemoglobin.

Myoglobin (Mb) uses strong electrostatic interaction in its distal heme pocket to regulate ligand binding. The mechanism of regulation of ligand binding in soybean leghemoglobin a (Lba) has been enigmatic and more so due to the absence of gaseous ligand bound atomic resolution three-dimensional structure of the plant globin. While the 20-fold higher oxygen affinity of Lba compared with Mb is required for its dual physiological function, the mechanism by which this high affinity is achieved is only emerging. Extensive mutational analysis combined with kinetic and CO-FT-IR spectroscopic investigation led to the hypothesis that Lba depended on weakened electrostatic interaction between distal HisE7 and bound ligand achieved by invoking B10Tyr, which itself hydrogen bonds with HisE7 thus restricting it in a single conformation detrimental to Mb-like strong electrostatic interaction. Such theory has been re-assessed here using CO-Lba in silico model and molecular dynamics simulation. The investigation supports the presence of at least two major conformations of HisE7 in Lba brought about by imidazole ring flip, one of which makes hydrogen bonds effectively with B10Tyr affecting the former's ability to stabilize bound ligand, while the other does not. However, HisE7 in Lba has limited conformational freedom unlike high frequency of imidazole ring flips observed in Mb and in TyrB10Leu mutant of Lba. Thus, it appears that TyrB10 limits the conformational freedom of distal His in Lba, tuning down ligand dissociation rate constant by reducing the strength of hydrogen bonding to bound ligand, which the freedom of distal His of Mb allows.

Denaturation properties and folding transition states of leghemoglobin and other heme proteins.

This work reports unfolding transitions of monomeric heme proteins leghemoglobin (Lb), myoglobin (Mb), and cytochrome c (Cyt c) utilizing UV-Vis spectral and steady-state and time-resolved fluorescence methods. Conformational stabilities of the native "folded" state of the proteins and their "unfolded" states were investigated in the light of a two-state transition model. Two-state transition values for ΔGD (298K) were obtained by denaturation with the chaotropic agents urea and guanidium hydrochloride (GdnHCl). The free energy value of Lb is the lowest compared to Cyt c and Mb along the denaturation pathway. The m value is also the lowest for Lb compared to Cyt c and Mb. The m value (a measure of dependence of ΔGD on denaturant concentration) for Cyt c and Mb is lower when it is denatured with urea compared to GdnHCl. The UV-Vis absorbance maximum and steady state fluorescence emission maximum were drastically red shifted in the presence of a certain denaturant concentration both in cases of Mb and Lb, but the scenario is different for Cyt c. The results are analyzed using a two-state transition model. The lifetime data clearly indicate the presence of an intermediate state during denaturation. The unfolding transition can modulate the conformation, stability, and surface exposure of these biologically important proteins.

Phosphorylation of Leghemoglobin at S45 is Most Effective to Disrupt the Molecular Environment of Its Oxygen Binding Pocket.

In leguminous plants, nitrogenase that catalyzes anaerobic symbiotic nitrogen fixation is protected by the sequestration of O2 by Leghemoglobin (LegH). The modulation of the oxygen binding capacity of Hemoglobin (Hb) by different post-translational modifications is well studied; whereas similar studies on LegH's O2 binding are not yet benchmarked. Our results show that in vitro serine phosphorylation of recombinant LegH from Lotus japonicus, a model legume, by a homologous kinase caused a reduction in its oxygen consumption as determined by Clark type electrode. Although mass spectrometry revealed a few phosphorylated serine residues in the LegH, molecular modeling study showed that particularly S45 is the most critical one, along with S55, however the latter with lesser impact on its molecular environment responsible for oxygen consumption. Separate S45D and S55D mutants of recombinant LegH also corroborated the results obtained from molecular modeling study. Thus, this work lays groundwork for further investigation of structural and functional role of serine phosphorylation as one of the mechanisms by which oxygen consumption by LegH may possibly be regulated during nodulation.

pH-Induced conformational isomerization of leghemoglobin from Arachis hypogea.

The pH dependence of proteins is related to the thermodynamic stability and electrostatic interactions in the native state of a protein. Here we report the pH-induced conformational transition of the heme protein leghemoglobin (Lb) isolated from root nodules of the leguminous plant Arachis hypogea. Unlike the other heme proteins myoglobin, hemoglobin, and cytochrome c, the structural characteristics and interactions of Lb is almost unknown, though its functional importance is already established since it binds oxygen to maintain the environment for N2 fixation. We investigated pH-induced unfolding of this protein and identified a number of conformational isomers using multiple fluorescence observables as a function of pH titration. We have characterized the acid- and base-induced conformational transitions among the structural states over the pH range 2-11. Depending on the solution conditions, Lb can exist in one of three phases: pH 2, 3, 4; pH 5, 6, 7; pH 8, 9, 10. The secondary structure as revealed by CD spectroscopy indicated the maximum percentage of α-helix to be present at pH 7, where the structure of Lb is also most rigid according to fluorescence anisotropy experiments. The fluorescence lifetime of tryptophan was observed to be maximum at pH 10 and minimum at pH 6, suggesting unfolding transitions of Lb. Thus, alteration of the microenvironment of the globin moiety during pH transition ultimately leads to the conformational change of this monomeric protein Lb.

Similar structures to the E-to-H helix unit in the globin-like fold are found in other helical folds.

A protein in the globin-like fold contains six alpha-helices, A, B, E, F, G and H. Among them, the E-to-H helix unit (E, F, G and H helices) forms a compact structure. In this study, we searched similar structures to the E-to-H helix of leghomoglobin in the whole protein structure space using the Dali program. Several similar structures were found in other helical folds, such as KaiA/RbsU domain and Type III secretion system domain. These observations suggest that the E-to-H helix unit may be a common subunit in the whole protein 3D structure space. In addition, the common conserved hydrophobic residues were found among the similar structures to the E-to-H helix unit. Hydrophobic interactions between the conserved residues may stabilize the 3D structures of the unit. We also predicted the possible compact regions of the units using the average distance method.

Phenolphthalein false-positive reactions from legume root nodules.

Presumptive tests for blood play a critical role in the examination of physical evidence and in the determination of subsequent analysis. The catalytic power of hemoglobin allows colorimetric reactions employing phenolphthalein (Kastle-Meyer test) to indicate "whether" blood is present. Consequently, DNA profiles extracted from phenolphthalein-positive stains are presumed to be from blood on the evidentiary item and can lead to the identification of "whose" blood is present. Crushed nodules from a variety of legumes yielded phenolphthalein false-positive reactions that were indistinguishable from true bloodstains both in color quality and in developmental time frame. Clothing and other materials stained by nodules also yielded phenolphthalein false-positive reactivity for several years after nodule exposure. Nodules from leguminous plants contain a protein (leghemoglobin) which is structurally and functionally similar to hemoglobin. Testing of purified leghemoglobin confirmed this protein as a source of phenolphthalein reactivity. A scenario is presented showing how the presence of leghemoglobin from nodule staining can mislead investigators.

Characterization of haemoglobin from actinorhizal plants--an in silico approach.

Plant haemoglobins (Hbs), found in both symbiotic and non-symbiotic plants, are heme proteins and members of the globin superfamily. Hb genes of actinorhizal Fagales mostly belong to the non-symbiotic type of haemoglobin; however, along with the non-symbiotic Hb, Casuarina sp. posses a symbiotic one (symCgHb), which is expressed specifically in infected cells of nodules. A thorough sequence analysis of 26 plant Hb proteins, currently available in public domain, revealed a consensus motif of 29 amino acids. This motif is present in all the members of symbiotic class II Hbs including symCgHb and non-symbiotic Class II Hbs, but is totally absent in Class I symbiotic and non-symbiotic Hbs. Further, we constructed 3D structures of Hb proteins from Alnus and Casuarina through homology modelling and peeped into their structural properties. Structure-based studies revealed that the Casuarina symbiotic haemoglobin protein shows distinct stereochemical properties from that of the other Casuarina and Alnus Hb proteins. It also showed considerable structural similarities with leghemoglobin structure from yellow lupin (pdb id 1GDI). Therefore, sequence and structure analyses point to the fact that symCgHb protein shows significant resemblance to symbiotic haemoglobin found in legumes and may thus eventually play a similar role in shielding the nitrogenase from oxygen as seen in the case of leghemoglobin.

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

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.

[Formation of glycated recombinant leghemoglobin in Escherichia coli cells].

A nonenzymatic glycation of the recombinant leghemoglobin expressed in Escherichia coli cells was demonstrated for the first time. This process involved the heme pocket and gave low-spin leghemoglobin species. A correlation between the degree of E. coli protein glycation and synthesis of poly-beta-hydroxybutyric acid was found, suggesting that the accumulation of reserve carbon sources and nonenzymatic glycation could be alternative processes.

Iterative non-sequential protein structural alignment.

Structural similarity between proteins gives us insights into their evolutionary relationships when there is low sequence similarity. In this paper, we present a novel approach called SNAP for non-sequential pair-wise structural alignment. Starting from an initial alignment, our approach iterates over a two-step process consisting of a superposition step and an alignment step, until convergence. We propose a novel greedy algorithm to construct both sequential and non-sequential alignments. The quality of SNAP alignments were assessed by comparing against the manually curated reference alignments in the challenging SISY and RIPC datasets. Moreover, when applied to a dataset of 4410 protein pairs selected from the CATH database, SNAP produced longer alignments with lower rmsd than several state-of-the-art alignment methods. Classification of folds using SNAP alignments was both highly sensitive and highly selective. The SNAP software along with the datasets are available online at http://www.cs.rpi.edu/~zaki/software/SNAP.

Review: correlations between oxygen affinity and sequence classifications of plant hemoglobins.

Plants express three phylogenetic classes of hemoglobins (Hb) based on sequence analyses. Class 1 and 2 Hbs are full-length globins with the classical eight helix Mb-like fold, whereas Class 3 plant Hbs resemble the truncated globins found in bacteria. With the exception of the specialized leghemoglobins, the physiological functions of these plant hemoglobins remain unknown. We have reviewed and, in some cases, measured new oxygen binding properties of a large number of Class 1 and 2 plant nonsymbiotic Hbs and leghemoglobins. We found that sequence classification correlates with distinct extents of hexacoordination with the distal histidine and markedly different overall oxygen affinities and association and dissociation rate constants. These results suggest strong selective pressure for the evolution of distinct physiological functions. The leghemoglobins evolved from the Class 2 globins and show no hexacoordination, very high rates of O(2) binding ( approximately 250 muM(-1) s(-1)), moderately high rates of O(2) dissociation ( approximately 5-15 s(-1)), and high oxygen affinity (K(d) or P(50) approximately 50 nM). These properties both facilitate O(2) diffusion to respiring N(2) fixing bacteria and reduce O(2) tension in the root nodules of legumes. The Class 1 plant Hbs show weak hexacoordination (K(HisE7) approximately 2), moderate rates of O(2) binding ( approximately 25 muM(-1) s(-1)), very small rates of O(2) dissociation ( approximately 0.16 s(-1)), and remarkably high O(2) affinities (P(50) approximately 2 nM), suggesting a function involving O(2) and nitric oxide (NO) scavenging. The Class 2 Hbs exhibit strong hexacoordination (K(HisE7) approximately 100), low rates of O(2) binding ( approximately 1 muM(-1) s(-1)), moderately low O(2) dissociation rate constants ( approximately 1 s(-1)), and moderate, Mb-like O(2) affinities (P(50) approximately 340 nM), perhaps suggesting a sensing role for sustained low, micromolar levels of oxygen.

The kinetic and equilibrium molten globule intermediates of apoleghemoglobin differ in structure.

An important question in protein folding is whether molten globule states formed under equilibrium conditions are good structural models for kinetic folding intermediates. The structures of the kinetic and equilibrium intermediates in the folding of the plant globin apoleghemoglobin have been compared at high resolution by quench-flow pH-pulse labeling and interrupted hydrogen/deuterium exchange analyzed in dimethyl sulfoxide. Unlike its well studied homolog apomyoglobin, where the equilibrium and kinetic intermediates are quite similar, there are striking structural differences between the intermediates formed by apoleghemoglobin. In the kinetic intermediate, formed during the burst phase of the quench-flow experiment, protected amides and helical structure are found mainly in the regions corresponding to the G and H helices of the folded protein, and in parts of the E helix and CE loop regions, whereas in the equilibrium intermediate, amide protection and helical structure are seen in parts of the A and B helix regions, as well as in the G and H regions, and the E helix remains largely unfolded. These results suggest that the structure of the molten globule intermediate of apoleghemoglobin is more plastic than that of apomyoglobin, so that it is readily transformed depending on the solution conditions, particularly pH. Thus, in the case of apoleghemoglobin at least, the equilibrium molten globule formed under destabilizing conditions at acid pH is not a good model for the compact intermediate formed during kinetic refolding experiments. Our high-precision kinetic analysis also reveals an additional slow phase during the folding of apoleghemoglobin, which is not observed for apomyoglobin. Hydrogen exchange pulse-labeling experiments show that the slow-folding phase is associated with residues in the CE loop, which probably forms non-native structure in the intermediate that must be resolved before folding can proceed to completion.

Cloning and characterization of a caesalpinoid (Chamaecrista fasciculata) hemoglobin: the structural transition from a nonsymbiotic hemoglobin to a leghemoglobin.

Nonsymbiotic hemoglobins (nsHbs) and leghemoglobins (Lbs) are plant proteins that can reversibly bind O(2) and other ligands. The nsHbs are hexacoordinate and appear to modulate cellular concentrations of NO and maintain energy levels under hypoxic conditions. The Lbs are pentacoordinate and facilitate the diffusion of O(2) to symbiotic bacteroids within legume root nodules. Multiple lines of evidence suggest that all plant Hbs evolved from a common ancestor and that Lbs originated from nsHbs. However, little is known about the structural intermediates that occurred during the evolution of pentacoordinate Lbs from hexacoordinate nsHbs. We have cloned and characterized a Hb (ppHb) from the root nodules of the ancient caesalpinoid legume Chamaecrista fasciculata. Protein sequence, modeling data, and spectral analysis indicated that the properties of ppHb are intermediate between that of nsHb and Lb, suggesting that ppHb resembles a putative ancestral Lb. Predicted structural changes that appear to have occurred during the nsHb to Lb transition were a compaction of the CD-loop and decreased mobility of the distal His inhibiting its ability to coordinate directly with the heme-Fe, leading to a pentacoordinate protein. Other predicted changes include shortening of the N- and C-termini, compaction of the protein into a globular structure, disappearance of positive charges outside the heme pocket and appearance of negative charges in an area located between the N- and C-termini. A major consequence for some of these changes appears to be the decrease in O(2)-affinity of ancestral nsHb, which resulted in the origin of the symbiotic function of Lbs.

Plant hemoglobins: a molecular fossil record for the evolution of oxygen transport.

The evolution of oxygen transport hemoglobins occurred on at least two independent occasions. The earliest event led to myoglobin and red blood cell hemoglobin in animals. In plants, oxygen transport "leghemoglobins" evolved much more recently. In both events, pentacoordinate heme sites capable of inert oxygen transfer evolved from hexacoordinate hemoglobins that have unrelated functions. High sequence homology between hexacoordinate and pentacoordinate hemoglobins in plants has poised them for potential structural analysis leading to a molecular understanding of this important evolutionary event. However, the lack of a plant hexacoordinate hemoglobin structure in the exogenously ligand-bound form has prevented such comparison. Here we report the crystal structure of the cyanide-bound hexacoordinate hemoglobin from barley. This presents the first opportunity to examine conformational changes in plant hexacoordinate hemoglobins upon exogenous ligand binding, and reveals structural mechanisms for stabilizing the high-energy pentacoordinate heme conformation critical to the evolution of reversible oxygen binding hemoglobins.

Plant hemoglobins: what we know six decades after their discovery.

This review describes contributions to the study of plant hemoglobins (Hbs) from a historical perspective with emphasis on non-symbiotic Hbs (nsHbs). Plant Hbs were first identified in soybean root nodules, are known as leghemoglobins (Lbs) and have been characterized in detail. It is widely accepted that a function of Lbs in nodules is to facilitate the diffusion of O(2) to bacteroids. For many years Hbs could not be identified in plants other than N(2)-fixing legumes, however in the 1980s a Hb was isolated from the nodules of the non-legume dicot plant Parasponia, a hb gene was cloned from the non-nodulating Trema, and Hbs were detected in nodules of actinorhizal plants. Gene expression analysis showed that Trema Hb transcripts exist in non-symbiotic roots. In the 1990s nsHb sequences were also identified in monocot and primitive (bryophyte) plants. In addition to Lbs and nsHbs, Hb sequences that are similar to microbial truncated (2/2) Hbs were also detected in plants. Plant nsHbs have been characterized in detail. These proteins have very high O(2)-affinities because of an extremely low O(2)-dissociation constant. Analysis of rice Hb1 showed that distal His coordinates heme Fe and stabilizes bound O(2); this means that O(2) is not released easily from oxygenated nsHbs. Non-symbiotic hb genes are expressed in specific plant tissues, and overexpress in organs of stressed plants. These observations suggest that nsHbs have functions additional to O(2)-transport, such as to modulate levels of ATP and NO.

On optima: the case of myoglobin-facilitated oxygen diffusion.

The process of myoglobin/leghemoglobin-facilitated oxygen diffusion is adapted to function in different environments in diverse organisms. We enquire how the functional parameters of the process are optimized in particular organisms. The ligand-binding properties of the proteins, myoglobin and plant symbiotic hemoglobins, we discover, suggest that they have been adapted under genetic selection pressure for optimal performance. Since carrier-mediated oxygen transport has probably evolved independantly many times, adaptation of diverse proteins for a common functionality exemplifies the process of convergent evolution. The progenitor proteins may be built on the myoglobin scaffold or may be very different.

Oxygen affinity controlled by dynamical distal conformations: the soybean leghemoglobin and the Paramecium caudatum hemoglobin cases.

The binding of diatomic ligands, such as O(2), NO, and CO, to heme proteins is a process intimately related with their function. In this work, we analyzed by means of a combination of classical Molecular Dynamics (MD) and Hybrid Quantum-Classical (QM/MM) techniques the existence of multiple conformations in the distal site of heme proteins and their influence on oxygen affinity regulation. We considered two representative examples: soybean leghemoglobin (Lba) and Paramecium caudatum truncated hemoglobin (PcHb). The results presented in this work provide a molecular interpretation for the kinetic, structural, and mutational data that cannot be obtained by assuming a single distal conformation.