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PubMedDate not supplied

[Structure of leghemoglobin].

The structure of the high spin complex of lupin leghemoglobin with acetate is refined at 2.0 A resolution using Diamond and Jack-Levitt technique (R=19%). The 0.05 A displacement of the iron atom from porphyrin plane to the distal side and the weakening of iron-nitrogen bond to the proximal histidine (2.37 A) give an explanation of the high affinity of the leghemoglobin for molecular oxygen. The ruffling of the porphyrin core may also favour it. Binding of nicotinate is accompanied by small conformational movements. Swinging out of the distal histidine from the heme pocket is the most prominent feature of this complex. The known amino acid sequences of leghemoglobin are analysed in view of the spatial structure of lupin hemoglobin.

Amino Acid Sequence↗
PubMed1973

Nicotinic Acid as a ligand affecting leghemoglobin structure and oxygen reactivity.

A small molecule, hitherto called X, which is present in legume root nodules and ligates reversibly to the monomeric protein, leghemoglobin, with formation of a hemochrome structure, is identified as nicotinic acid. The binding constants at pH 5.3 are K = 7.3 x 10(5) M(-1) and K = 3.0 x 10(4) M(-1) for combination of nicotinic acid with ferric and ferrous leghemoglobin, respectively. This high affinity binding of ligand requires an unsubstituted pyridine ring nitrogen atom and an ionized carboxyl group in the 3-position of the ring. Binding of nicotinic acid is favored at acid pH and is reflected by diminished apparent affinity of leghemoglobin for oxygen.

Journal Article↗
PubMed1988

Regulation of o(2) concentration in soybean nodules observed by in situ spectroscopic measurement of leghemoglobin oxygenation.

A fiber optic spectrophotometric system was used to monitor the in vivo oxygenation of leghemoglobin in intact, attached soybean root nodules (Glycine max L. Merr. x USDA 16 Bradyrhizobium japonicum) which were flattened during development by growth in narrow, glass-walled cuvettes. When equilibrated at an external pO(2) of 20 kilopascals, leghemoglobin was 36.6 +/- 5.4% oxygenated, a value estimated to represent an infected cell O(2) concentration of 21.5 nanomolar. Increasing the external pO(2) from 20 to 25 kilopascals caused a rapid increase in leghemoglobin oxygenation, followed by a recovery to the initial level, all within 7.5 minutes. At 25 kilopascals O(2), the rates of H(2) and CO(2) evolution were similar to those at 20 kilopascals. Since respiration had not increased, the results support the proposal that nodules adapt to increased external pO(2) by regulating their resistance to O(2) diffusion.

Journal Article↗
PubMed1989

Measurement of the fractional oxygenation of leghemoglobin in intact detached pea nodules by reflectance spectroscopy.

A method is presented for the rapid measurement of the spectral properties of detached nodules of pea (Pisum sativum L. cv "Sparkle") by diffuse reflectance spectroscopy. After correcting the spectra for surface light scattering, the spectrum of leghemoglobin is obtained. From this, the fractional oxygenation of leghemoglobin and the internal O(2) concentration can be calculated. With this method, we determined internal O(2) while measuring nitrogenase activity (C(2)H(2)) in detached pea nodules over a range of external O(2) concentrations. Nitrogenase activity was maximum when leghemoglobin was 25% oxygenated, corresponding to a calculated free O(2) concentration of 45 nanomolar in infected cells. Advantages of this method over previous methods which employed transmitted light are: (a) many nodules can be assayed simultaneously, (b) nitrogenase activity (C(2)H(2)) can be determined at the same time as spectra are recorded, and (c) spectra can be obtained from nodules submerged in buffer containing metabolic effectors.

Journal Article↗
PubMed1991

Purification and characterization of soybean root nodule ferric leghemoglobin reductase.

A ferric leghemoglobin reductase from the cytosol of soybean (Glycine max) root nodules was purified to homogeneity and partially characterized. The enzyme is a flavoprotein with flavin adenine dinucleotide as the prosthetic group and consists of two identical subunits, each having a molecular mass of 54 kilodaltons. The pure enzyme shows a high activity for ferric leghemoglobin reduction with NADH as the reductant in the absence of any exogenous mediators. The enzyme also exhibits NADH-dependent 2,6-dichloroindophenol reductase activity. A sequence of the first 50 N-terminal amino acids of the purified protein was obtained. Comparisons with known protein sequences have shown that the sequence of the ferric leghemoglobin reductase is highly related to those of the flavin-nucleotide disulfide oxido-reductases, especially dihydrolipoamide dehydrogenase of the pyruvate dehydrogenase complex.

Journal Article↗
PubMed1993

Comparative sequence analysis of cis elements present in Glycine max L. leghemoglobin lba and lbc3 genes.

The soybean leghemoglobin lba gene promoter sequence was determined and aligned with the promoter sequence of the soybean lbc3 gene from the same gene family. Five highly conserved regions were found. There are two large conserved regions, one of which overlaps the basic promoter while the other defines a minimal enhancer in the upstream positive elements. Within the minimal enhancer, an inverted repeat with similarity to the binding site of a yeast transcription factor, GCN4, was found. This particular repeat is conserved in the promoters of all functional soybean lb genes as well as in lb gene promoters from other legumes. This suggests that the inverted repeat is important for leghemoglobin gene expression.

Base Sequence↗
PubMed1987

Molecular cloning of lupin leghemoglobin cDNA.

Poly(A)+ RNA isolated from root nodules of yellow lupin (Lupinus luteus, var. Ventus) has been used as a template for the construction of a cDNA library. The ds cDNA was synthesized and inserted into the Hind III site of plasmid pBR 322 using synthetic Hind III linkers. Clones containing sequences specific for nodules were selected by differential colony hybridization using 32P-labeled cDNA synthesized either from nodule poly(A)+ RNA or from poly(A)+ RNA of uninfected root as probes. Among the recombinant plasmids, the cDNA gene for leghemoglobin was identified. The protein structure derived from its nucleotide sequence was consistent with known amino acid sequence of lupin Lb II. The cloned lupin Lb cDNA hybridized to poly(A)+ RNA from nodules only, which is in accordance with the general concept, that leghemoglobin is expressed exclusively in nodules.

Amino Acid Sequence↗
PubMed1993

How is leghemoglobin involved in peribacteroid membrane degradation during nodule senescence?

An increase in the rate of succinate and glutamate uptake by isolated symbiosomes from French bean nodules was observed in the presence of iron plus H2O2. The lipid bilayer, and not proteins involved in transport, seems to be the major target of radical attack. Leghemoglobin in the presence of a 6-fold excess of H2O2 (where heme breakdown and iron release occurred) provoked also an increase in peribacteroid membrane permeability. In contrast, this hemoprotein in the presence of a 2-fold excess of H2O2 (where a protein radical was generated) was without effect. We suggest that in vivo the release of heme iron may constitute the major process concerning the involvement of leghemoglobin in the degradation of the peribacteroid membrane during nodule senescence.

Cell Membrane↗
PubMedDate not supplied

1H NMR studies of heme pocket conformation in zinc-substituted leghemoglobin, a diamagnetic analog of deoxyleghemoglobin.

Reconstitution of apoleghemoglobin with zinc protoporphyrin IX is reported. NMR spectra show that the reconstitution is orientation specific and that there is no detectable heme isomerism or conformational heterogeneity. Resonances of heme substituents and distal and proximal amino acid protons have been assigned. Only minor differences in porphyrin-protein packing occur between zinc leghemoglobin and the CO complex of ferrous leghemoglobin. The zinc is five-coordinate and is ligated by the proximal histidine. Comparisons with diamagnetic six-coordinate complexes show that the distal His-61 and Leu-65 side chains move away from the binding site upon coordination of exogenous ligands. Conformational changes are minimal when the ligand is O2.

Heme↗
PubMed1991

Kinetics and mechanisms of reduction of Cu(II) and Fe(III) complexes by soybean leghemoglobin alpha.

The reduction of low-molecular-weight Cu(II) and Fe(III) complexes by soybean leghemoglobin alpha was characterized using both kinetic analysis and 1H-NMR experiments. Whereas Fe(III) (CN)6(3-) was reduced through an outer sphere transfer over the exposed heme edge, all other Cu(II) and Fe(III) complexes investigated were reduced via a site-specific binding of the metal to the protein. Reduction of all metal complexes was enhanced by decreasing pH while only Fe(III)NTA reduction kinetics were altered by changes in ionic strength. Rates of reduction for both Cu(II) and Fe(III) were also affected inversely by the effective binding constant of the metal chelate used. NMR data confirmed that both Cu(II)NTA and Fe(III)NTA were bound to specific sites on the protein. Cu(II) bound preferentially to distal His-61 and Fe(III) exerted its greatest effect on two surface lysine residues with epsilon proton resonances at 3.04 and 3.12 ppm. The Fe(III)NTA complex also had a mild but noticeable line broadening effect on the distal His-61 singlet resonance near 5.3 ppm. Like hemoglobin and myoglobin, leghemoglobin might function not only as an oxygen carrier, but also as a biological reductant for low-molecular-weight Cu(II) and Fe(III) complexes.

Binding Sites↗
PubMed1995

Reaction of ferric leghemoglobin with H2O2: formation of heme-protein cross-links and dimeric species.

Ferric leghemoglobin in the presence of H2O2 is known to give rise to protein radicals, at least one of which is centred on a tyrosine residue. These radicals are quenched by at least two processes. The first one involves an intramolecular heme-protein cross-link probably involving the tyrosine radical; this leads to the formation of a green compound with spectral characteristics differing markedly from those of ferryl and ferric leghemoglobin. This green compound cannot be reduced by dithionite or ascorbate, precluding any role for this species as an oxygen carrier. It exhibits modified EPR and pyridine haemochromogen spectra, indicating that alterations occur at the porphyrin macrocycle level. The additional compound previously described [Puppo, A., Monny, C. and Davies, M.J. (1993) Biochem. J. 289, 435-438] appears to be a mixture of ferry Lb and this green compound. The second quenching route results in the formation of intermolecular cross-links and hence dimeric forms of the protein. Ascorbate and glutathione inhibit both this intermolecular dimer formation and the formation of the intramolecular haem-protein cross-links and are likely to play a protective role in vivo.

Electron Spin Resonance Spectroscopy↗
PubMed1998

Direct detection of radicals in intact soybean nodules: presence of nitric oxide-leghemoglobin complexes.

Electron paramagnetic resonance spectroscopy has been employed to examine the nature of the metal ions and radicals present in intact root nodules of soybean plants grown in the absence of nitrate. The spectra obtained from nodules of different ages using this non-invasive technique show dramatic differences, suggesting that there are both qualitative and quantitative changes in the metal ion and radical species present. A major component of the spectra obtained from young nodules is assigned to a complex (Lb-NO) of nitric oxide (NO.) with the heme protein leghemoglobin (Lb). This Lb-NO species, which has not been previously detected in intact root nodules of plants grown in the absence of nitrate, is thought to be formed by reaction of nitric oxide with iron(II) leghemoglobin. The nitric oxide may be generated from arginine via a nitric oxide synthase-like activity present in the nodules of the soybean plants, in a manner analogous to that recently described for Lupinus albus. This Lb-NO complex is present at lower concentrations in older nodules, and is almost completely absent from senescent nodules. Exposure of young and mature nodules to oxidant stress, in the form of hydrogen peroxide, results in changes in the EPR spectra, with the loss of the signals from the Lb-NO complex and appearance of absorptions similar to those from untreated senescent nodules. These results suggest that there are characteristic changes in both the metal ion complexes and radicals present in intact root nodules of different ages, and support the theory that nitric oxide and other radicals play a significant role in determining the nitrogen fixing activity of root nodules; the modulatory activity of NO. may involve regulation of gene activity.

Electron Spin Resonance Spectroscopy↗
PubMed1993

Role of hydrogen bonding to bound dioxygen in soybean leghemoglobin.

Electron spin echo envelope modulation (ESEEM) spectroscopy was applied to oxy cobaltous soybean leghemoglobin (oxyCoLb) in D2O at various pH values to investigate electron nuclear superhyperfine coupling to N epsilon of the proximal histidyl imidazole and to exchangeable deuterons. Two spectroscopically distinct forms of oxyCoLb, acid and neutral, were identified. In the acid form, a 0.82-MHz hyperfine coupling to 2H was found, indicating the presence of a hydrogen bond to bound O2. No hyperfine-coupled 2H was found in the neutral form. Nuclear hyperfine and nuclear quadrupole couplings to the proximal histidyl N epsilon in the acid form are smaller than those in the neutral form: Aiso = 2.22 MHz and e2qQ = 1.98 MHz for the acid form; Aiso = 2.90 MHz and e2qQ = 2.22 MHz for the neutral form. The differences are believed to result from the presence of a hydrogen bond to bound O2 in the acid form. A discussion of the contribution of this hydrogen bond to the pH-dependent O2 affinity of leghemoglobin is presented.

Cobalt↗
PubMed1975

Characterization and improved separation of soybean leghemoglobins.

An improved separation procedure is described for isolating five leghemoglobin components from the nodules of soybean plants. After a preliminary oxidation with ferricyanide, and separation from endogenous nicotinate at pH 9.2, the ferrileghemoglobins are separated by DEAE-cellulose chromatography using gradient elution with acetate buffer (pH 5.2). The components have been characterized by their acetate and nicotinate binding affinities, gel electrophoretic, visible, and circular dichroic spectra in the ultraviolet, Soret and visible regions. Two formerly unresolved components of leghemoglobin c have indistinguishable circular dichroic, electrophoretic, and ligand binding properties, but differ in their spin states as judged by their visible spectra, their amino acid analyses, and their tryptic maps.

Amino Acids↗
PubMed1993

Site-directed mutagenesis of the organ-specific element in the soybean leghemoglobin lbc3 gene promoter.

The expression of a soybean leghemoglobin 5'lbc3-GUS-3'nos chimeric gene was analyzed in Lotus corniculatus after site-specific mutagenesis of the nodulin consensus sequences, AAAGAT and CTCTT, present in the organ-specific element (OSE) (-139 to -102). Full-length promoters (-1956, +46) carrying clustered point mutations in the CTCTT sequence or in both the AAAGAT and the CTCTT sequences were inactive. Point mutations in the AAAGAT sequence had only minor effects on the expression level. Substitution of the ATTG sequence between the AAAGAT and the CTCTT sequences in the OSE reduced the activity in nodules to 10%. This, together with the conservation of the ATTGT sequence in the same position of leghemoglobin genes from other legumes, indicates that these sequences, in addition to the nodulin consensus sequences of the OSE, are important for high-level nodule-specific expression. Substitution of the CTCTT sequences outside the OSE (-44, -40 and -79, -75) results in promoter activities of approximately 50%.

Base Sequence↗
PubMed2000

CPP1, a DNA-binding protein involved in the expression of a soybean leghemoglobin c3 gene.

Nodulin genes are specifically expressed in the nitrogen-fixing root nodules. We have identified a novel type of DNA-binding protein (CPP1) interacting with the promoter of the soybean leghemoglobin gene Gmlbc3. The DNA-binding domain of CPP1 contains two similar Cys-rich domains with 9 and 10 Cys, respectively. Genes encoding similar domains have been identified in Arabidopsis thaliana, Caenorhabditis elegans, the mouse, and human. The domains also have some homology to a Cys-rich region present in some polycomb proteins. The cpp1 gene is induced late in nodule development and the expression is confined to the distal part of the central infected tissue of the nodule. A constitutively expressed cpp1 gene reduces the expression of a Gmlbc3 promoter-gusA reporter construct in Vicia hirsuta roots. These data therefore suggest that CPP1 might be involved in the regulation of the leghemoglobin genes in the symbiotic root nodule.

Amino Acid Sequence↗
PubMed1983

Chromosomal arrangement of leghemoglobin genes in soybean.

A cluster of four different leghemoglobin (Lb) genes was isolated from AluI-HaeIII and EcoRI genomic libraries of soybean in a set of overlapping clones which together include 45 kilobases (kb) of contiguous DNA. These four genes, including a pseudogene, are present in the same orientation and are arranged in the order: 5'-Lba-Lbc1-Lb psi-Lbc3-3'. The intergenic regions average 2.5 kb. In addition to this main Lb locus, there are other Lb genes which do not appear to be contiguous to this locus. A sequence probably common to the 3' region of Lb loci was found flanking the Lbc3 gene. The 3' flanking region of the main Lb locus also contains a sequence that appears to be expressed more abundantly in root tissue. Another sequence which is primarily expressed in root and leaf is found 5' to two Lb loci. Overall, the main leghemoglobin locus is similar in structure to the mammalian globin gene loci.

Base Sequence↗
PubMed1985

Primary structure of the soybean nodulin-23 gene and potential regulatory elements in the 5'-flanking regions of nodulin and leghemoglobin genes.

The nodulin-23 gene of soybean is one of the most abundantly transcribed genes induced during symbiosis with Rhizobium. Using a plasmid (pNod25) from a nodule cDNA library, we have isolated the nodulin-23 gene from a soybean genomic library. Nucleotide sequence analysis of the cDNA and of the genomic clone indicated that the coding region of this gene is 669 bp long and is interrupted by a single intron of about 530 bp. The deduced protein sequence suggests that nodulin-23 may have a signal sequence. The 5'-flanking sequence of two other nodulin genes, nodulin-24 encoding for a membrane polypeptide and one of the leghemoglobin genes (LbC3), were obtained. Comparison of these sequences revealed three conserved regions, one of which, an octanucleotide (GTTTCCCT), has 100% homology. The conserved sequences are arranged in a unique fashion and have a spatial organization with respect to order and position, which may suggest a potential regulatory role in controlling the expression of nodulin and leghemoglobin genes during symbiosis.

Amino Acid Sequence↗