Soybean Leghemoglobin Research Articles

Soybean leghemoglobin gene family: normal, pseudo, and truncated genes.

Leghemoglobin (Lb) genes in soybean represent a small family of closely related genes. Three Lb sequences isolated from a genomic library were analyzed at the nucleotide sequence level. A Lb gene present on an 11.5-kilobase (kb) EcoRI genomic fragment spans approximately 1,200 nucleotides and is interrupted at amino acid positions 32 to 33, 68 to 69, and 103 to 104. The intervening sequences, as well as the 5' and 3' flanking regions of this gene, contain the consensus sequences found in other eukaryotic genes. The length of the 5'-untranslated region is 49 bases as determined by nuclease S1 mapping. R-loop analysis of the DNA from the recombinant phage containing the 11.5-kb EcoRI genomic fragment showed that another Lb gene is located 2.5 kb away. The nucleotide sequence of the second gene showed that this gene is incomplete, containing only exons 3 and 4. The deduced amino acid sequence of this gene, although showing 76% homology with the corresponding region of the other Lb gene, is not represented in any of the known Lb proteins. Both genes are oriented in the same direction with respect to the coding strand. Analysis of the sequence present on a second genomic clone containing a 4.2-kb EcoRI fragment revealed a truncated Lb gene showing homology with the last exon and the noncoding region at the 3' end of the two other Lb genes.

Differences in the heme environment of soybean leghemoglobin components shown by 1H-NMR spectroscopy

1H-NMR spectra have been recorded for the cyanide complexes of homogeneous soybean ferric leghemoglobin components a, c 1, c 3and a mixture of components c 2 + c 3, isolated by anion-exchange chromatography at pH 5.2. Small chemical shift differences are observed for the heme resonances in the spectra of the different components, indicating small differences in heme electronic structure which are attributed to substitutions amongst the heme contact residues. The heme environment is closely but not exactly conserved in the various soybean leghemoglobins and this might be reflected in their biological functions. The chemical shifts of the heme resonances of the component leghemoglobins in an unresolved mixture which was not subjected to low-pH chromatography are identical to those of the components a, c 1, c 2 and c 3 isolated at pH 5.2. The four major leghemoglobin components are therefore not artifacts of the separation procedure.

A Comment on Resonance Raman Spectroscopic Investigations of Axial Ligation of the Heme Iron Atom in Soybean Leghemoglobin a.

A Comment on Resonance Raman Spectroscopic Investigations of Axial Ligation of the Heme Iron Atom in Soybean Leghemoglobin a.

The nucleotide sequences of two leghemoglobin genes from soybean.

We present the complete nucleotide sequences of two leghemoglobin genes isolated from soybean DNA. Both genes contain three intervening sequences in identical positions. Comparison of the coding sequences with known amino-acid sequences of soybean leghemoglobins suggest that the two genes correspond to leghemoglobin C2 and leghemoglobin C3, respectively.

Protoheme conformations in low-spin ferrohemoproteins. Resonance raman spectroscopy

The low-frequency regions of resonance Raman spectra of various low-spin ferrous forms of normal human hemoglobin, soybean leghemoglobin a and of horse myoglobin are reported. Differences observed among the spectra of oxygenated and nitrosyl forms of these hemoproteins show that their globins impose various low-spin heme structures. A quantitative correlation between the variable frequency of resonance Raman band II (215–271 cm −1) and the iron atom-heme plane distance was observed for hemoproteins and heme models, either ferrous or ferric, high-spin or low-spin. From this correlation, the iron atom-heme plane distance should be 0.3 Å in nitrosyl and oxymyoglobin (band II at 256 cm −1) whereas the iron position should be near to or in the heme plane for nitrosyl and oxy forms of hemoglobin and leghemoglobin (band II between 266 and 273 cm −1). A new method is proposed for monitoring the photodissociation processes in ferrohemoproteins.

Resonance Raman spectra of deoxyhemoproteins. Heme structure in relation to dioxygen binding

The low-frequency regions of the resonance Raman spectra of deoxygenated ferrous forms of soybean leghemoglobin a, horse myoglobin, sperm whale myoglobin, Aplysia myoglobin, stripped normal human hemoglobin (T quaternary form) and of stripped human NESdesArg-hemoglobin (R quaternary form) are reported. Differences observed among these spectra show that the globins of these hemoproteins impose various heme structures. In particular, the variable frequencies of band II (210–224 cm −1) and of band I b (121–163 cm −1) show that an increase in dioxygen affinity corresponds to a decrease in Fe-N(pyrrole) bond length.

Resonance Raman studies of soybean leghemoglobin and myoglobin: Origin of the differences in O 2 dissociation rate constants

FEBS LettersVolume 133, Issue 2 p. 239-243 Full-length articleFree Access Resonance Raman studies of soybean leghemoglobin and myoglobin Origin of the differences in O2 dissociation rate constants M.J. Irwin, M.J. Irwin Department of Inorganic Chemistry, University of Sydney, Sydney, NSW 2006, AustraliaSearch for more papers by this authorR.S. Armstrong, R.S. Armstrong Department of Inorganic Chemistry, University of Sydney, Sydney, NSW 2006, AustraliaSearch for more papers by this authorP.E. Wright, P.E. Wright Department of Inorganic Chemistry, University of Sydney, Sydney, NSW 2006, AustraliaSearch for more papers by this author M.J. Irwin, M.J. Irwin Department of Inorganic Chemistry, University of Sydney, Sydney, NSW 2006, AustraliaSearch for more papers by this authorR.S. Armstrong, R.S. Armstrong Department of Inorganic Chemistry, University of Sydney, Sydney, NSW 2006, AustraliaSearch for more papers by this authorP.E. Wright, P.E. Wright Department of Inorganic Chemistry, University of Sydney, Sydney, NSW 2006, AustraliaSearch for more papers by this author First published: October 26, 1981 https://doi.org/10.1016/0014-5793(81)80514-5Citations: 18AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume133, Issue2October 26, 1981Pages 239-243 ReferencesRelatedInformation

Comparison of the heme electronic and molecular structure of soybean leghemoglobin and sperm whale myoglobin by proton NMR

The proton nuclear magnetic resonance spectra of soybean ferric leghemoglobin a in the low-spin cyanide and nicotinate complexes have been assigned by specific deuteration of heme methyl groups. The assignments differ from those obtained solely from nuclear Overhauser enhancement measurements and are indicative of a proximal histidyl imidazole-hemin interaction which is very similar to that found in sperm whale myoglobin. The absence of a hyperfine shifted exchangeable NH peak for the distal histidine in leghemoglobin suggests either a very different orientation for this distal ligand or a significantly faster exchange rate with bulk solvent than found in myoglobin.

Axial Ligands of the Heme Iron in Soybean Leghemoglobin a as Investigated Using Resonance Raman Spectroscopy.

Axial Ligands of the Heme Iron in Soybean Leghemoglobin a as Investigated Using Resonance Raman Spectroscopy.

13C nuclear magnetic resonance studies of the binding of alkyl isocyanides to soybean leghemoglobin; comparison with animal myoglobins

13C NMR of labelled alkyl isocyanide ligands has been used with a view to probe the protein environment around the heme site of Soybean leghemoglobin, and comparatively, those of sperm whale myoglobin and monomeric Glycera hemoglobin. The terminal carbon of the isocyanide, which is known to be highly sensitive to change in hybridization of the nitrogen, could be expected to reflect the movement of the alkyl group through steric interactions. Three alkyl isocyanides (alkyl = methyl, ethyl & n-butyl) have therefore been used and the 13C0 chemical shift values were measured for each ligand bound to the various proteins studied. In all cases, the 13C0 resonance of the bound ligand were shifted considerably down-field with respect to those of the free unbound species, but the pattern of these displacements revealed more pronounced steric hindrance in the case of some proteins compared to others. The modifications of the chemical shift values of binding delta delta = delta bound -- delta free) were least in the case of leghemoglobin; moreover, the delta delta values were insensitive to the length of the alkyl chain (methyl to n-butyl) when bound to leghemoglobin, in contrast to the other proteins examined. The results are interpreted as arising from a diminished steric hindrance to isocyanide binding with leghemoglobin, in conformity with the recently published X-ray structure which reports the existence of a large heme pocket on the distal side.

1H-NMR studies of ferric soybean leghemoglobin Assignment of hyperfine shifted resonances of complexes with cyanide, nicotinate, pyridine and azide

The 1H-NMR spectra of complexes of soybean ferric leghemoglobin a with cyanide, nicotinate, pyridine and azide have been recorded. Assignments of many of the hyperfine shifted resonances to specific groups on the periphery of the heme have been made on the basis of their intensities and chemical shifts, pH dependence, nuclear Overhauser effects, spin decoupling and the use of Gd 3+ as a relaxation probe. The resonances of the protons at positions 3 and 5 of pyridine and nicotinate ligands have also been assigned. The iron(III) atom in the cyanide, nicotinate and azide complexes is almost completely low spin. In the pyridine complex, which is predominantly low spin, a high-spin state is thermally populated at room temperature. Information on the conformation of the heme propionate and vinyl side-chains is obtained. The average rotational position of one of the heme vinyl groups appears to differ between the nicotinate and cyanide complexes. In both of these complexes conformational rearrangement of a heme propionic acid side-chain occurs upon deprotonation of its carboxylic acid group (p K approx. 5.0). A further change in the conformation of this group occurs in leghemoglobin nicotinate upon deprotonation of the distal histidine. The p K of the heme propionic acid side-chain in leghemoglobin pyridine is 5.6. Its conformation and environment appears to differ from that in the nicotinate and cyanide complexes. In leghemoglobin cyanide, evidence for an interaction between the protonated distal histidine and the cyanide ligand is obtained. In each of the complexes studied the unpaired electron spin distribution over the porphyrin ring is highly anisotropic. Considerable differences exist between the unpaired spin distribution in the cyanide complexes of leghemoglobin and myoglobin. The axial ligand field is stronger in leghemoglobin and this may be of significance in the reaction with oxygen.

Peroxidase content of soybean root nodules

A peroxidase has been isolated from soybean nodules and its main characteristics have been determined. Its molecular weight (48 000) and spectral properties are similar to those of usual plant peroxidases. Its activity is comparable to that of low-efficiency plant peroxidases. The rate constant of the reaction with H 2O 2 is 3 · 10 5 M −1 · s −1. In this reaction, nodule peroxidase yields an oxidized intermediate analogous to the compound I species of peroxidases already studied. A comparison is made with the pseudoperoxidatic activity of soybean leghemoglobin components. Leghemoglobins a and c react with H 2O 2 with rate constants of 5 · 10 3 and 2.5 · 10 3 M −1 · s −1, respectively, yielding the leghemoglobin (IV) species. During these reactions leghemoglobins are inactivated.

Resonance Raman evidence for constrained heme structure in soybean leghemoglobin and its derivatives

Resonance Raman spectra have been obtained for soybean leghemoglobin and many of its derivatives. The spectra reveal a stronger ligand field at the iron atom in leghemoglobin than in myoglobin or hemoglobin. Differences in the frequency of an anomalously polarized band near 1550 cm −1 indicate an expanded porphyrin core or greater ruffling or doming of the porphyrin ring in deoxyleghemoglobin than is present in deoxymyoglobin. These heme constraints are retained in the various low spin complexes. The significance of the unusual heme structure in leghemoglobin in relation to its rapid oxygen binding reaction is discussed.

Soybean Leghemoglobin with Covalently Bound Mesoheme.

Soybean Leghemoglobin with Covalently Bound Mesoheme.

Binding of alkylisocyanides with soybean leghemoglobin. Comparisons with sperm whale myoglobin.

The binding of various linear and branched chain alkylisocyanides to soybean leghemoglobin has been studied with respect to association and dissociation kinetics and the results compared with those obtained in parallel on sperm whale and horse heart myoglobins; the linear ligands used (methyl to n-heptyl) cover a greater distribution of chain lengths than hitherto used. The association rate constants are much higher for leghemoglobin than for myoglobin, while the dissociation rates are slower. For a given protein, the dissociation rate constants are not much different when different isocyanides are used (except for methyl), whereas the association rates show complex behavior in relation with the alkyl chain length; singular differences are observed between leghemoglobin and sperm whale myoglobin in this regard. For myoglobin, the binding rate constants decrease from methyl to n-propyl, but remain approximately the same when the ligand carries a still longer alkyl chain. In contrast, for leghemoglobin, although the rate constants decrease from methyl to n-propyl, they show a progressive and important rise with longer alkyl substituents: n-butyl and n-pentyl.

Separation and determination of the relative concentrations of the homogeneous components of soybean leghemoglobin by isoelectric focusing

The multiple components of soybean ferric leghemoglobin are readily separated by analytical and preparative flat bed isoelectric focusing in both the presence and also the absence of the ligand nicotinate. In the presence of nicotinate the separation by isoelectric focusing is more rapid and results in sharper bands of the very stable ferric leghemoglobin nicotinate complexes. The separation is sensitive enough to permit analytical experiments on leghemoglobin from single nodules. Leghemoglobins a and c 1 prepared by ion exchange chromatography are homogeneous by isoelectric focusing criteria. Leghemoglobin c 2 prepared by ion exchange chromatography is an approximately 1 : 2 mixture of leghemoglobins c 2 and c 3. Leghemoglobin d consists of three components. The ratio of leghemoglobin a to leghemoglobin c 3 content increases dramatically as very young nodules mature. The increase in relative leghemoglobin a content suggests that leghemoglobin a might be required for regulation of nodule O 2 concentration only when the nodule structure is complex. The ratio of leghemoglobin c 1 content to leghemoglobin c 3 content increases somewhat during the early period of nodule development, while the ratio of leghemoglobin c 2 content to leghemoglobin c 3 content increases slowly throughout nodule development. Ratios of leghemoglobin b content to leghemoglobin a content and of total leghemoglobin d content to total leghemoglobin c content were almost independent of nodule age. Leghemoglobins a and b might be related biosynthetically, as might leghemoglobins c and d.

Molecular basis for proton-dependent anion binding by soybean leghaemoglobin a

HAEM proteins are widespread in nature and exhibit diverse biological activities. The properties and function of a given haem protein depend critically on the environment of the haem group. The reactivity of the haem is often influenced by one or more haem-linked ionisable groups. Thus, oxygen affinities of mammalian haemoglobins, for example, exhibit considerable pH dependence (the Bohr effect1). Anion binding to some haem proteins is also a proton-dependent reaction (for example, peroxidase2, catalase2 and leghaemoglobin3,4) whereas for others (myoglobin, haemoglobin1) it is essentially independent of pH. Identification of the ionisable groups which influence the ligand-binding reactions of haem proteins is of fundamental importance in the elucidation of structure/function relationships. Nuclear magnetic resonance (NMR) spectroscopy is one of the most direct methods for observation of titrating groups in proteins. We report here on NMR studies of the nicotinate complex of soybean ferric leghaemoglobin a. On the basis of these studies we propose a role for a haem propionic acid group as a mediator of anion binding.

Determination of the blocked N-terminal of soybean leghemoglobin b

Soybean leghemoglobins ā and b̄were compared by microscale peptide mapping after heme removal with acid-acetone. Maps generated by trypsin or the combined action of trypsin and thermolysin indicated a large amount of homology between the proteins with the only variations detected being the N-terminal peptides. The N-terminal tryptic peptide of leghemoglobin b̄ was found to be both blocked and to lack the first amino acid of the corresponding leghemoglobin ā peptide. Nuclear magnetic resonance and gas chromatography/mass spectroscopy studies showed that the N-terminal of leghemoglobin b̄ was N-acetyl-alanine. It is possible that leghemoglobin b̄ arises from leghemoglobin ā by a two-stage modification involving cleavage of the N-terminal valyl residue and subsequent acetylation of the exposed alanyl residue.

Systems utilizing oxygenated leghemoglobin and myoglobin as sources of free dissolved O 2 at low concentrations for experiments with bacteria

Oxygenated soybean leghemoglobin and sperm whale myoglobin have been used as sources of O 2 for respiring bacteria in experiments with no gas phase. These O 2-carrying hemoproteins provide dispersed free O 2 at concentrations defined by the kinetic constants of their oxygenation and deoxygenation, and their optical absorption spectra indicate the average concentration of free O 2. They can thus be used to study relationships between concentrations of free O 2, rates of respiration, and other metabolic processes in bacteria suspended in solutions of them. Three types of apparatus are described and examples of studies of respiration and nitrogen fixation by Rhizobium spp. are given.

Cloning of soybean leghemoglobin structural gene sequences synthesized in vitro.

Double-stranded soybean leghemoglobin DNA was synthesized from leghemoglobin mRNA isolated from soybean nodules. The dsDNA was inserted into the Bam H1 site of plasmid pBR322 using the poly-dAT-joiner method. A cloned DNA fragment of one recombinant plasmid was isolated and characterized by restriction endonuclease digestion. The restriction cleavage map and the DNA sequence of a selected part of the inserted DNA are in complete accordance with the amino-acid sequence of soybean leghemoglobin.