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PubMed2003

Distal heme pocket regulation of ligand binding and stability in soybean leghemoglobin.

Leghemoglobins facilitate diffusion of oxygen through root tissue to a bacterial terminal oxidase in much the same way that myoglobin transports oxygen from blood to muscle cell mitochondria. Leghemoglobin serves an additional role as an oxygen scavenger to prevent inhibition of nitrogen fixation. For this purpose, the oxygen affinity of soybean leghemoglobin is 20-fold greater than myoglobin, resulting from an 8-fold faster association rate constant combined with a 3-fold slower dissociation rate constant. Although the biochemical mechanism used by myoglobin to bind oxygen has been described in elegant detail, an explanation for the difference in affinity between these two structurally similar proteins is not obvious. The present work demonstrates that, despite their similar structures, leghemoglobin uses methods different from myoglobin to regulate ligand affinity. Oxygen and carbon monoxide binding to a comprehensive set of leghemoglobin distal heme pocket mutant proteins in comparison to their myoglobin counterparts has revealed some of these mechanisms. The "distal histidine" provides a crucial hydrogen bond to stabilize oxygen in myoglobin but has little effect on bound oxygen in leghemoglobin and is retained mainly for reasons of protein stability and prevention of heme loss. Furthermore, soybean leghemoglobin uses an unusual combination of HisE7 and TyrB10 to sustain a weak stabilizing interaction with bound oxygen. Thus, the leghemoglobin distal heme pocket provides a much lower barrier to oxygen association than occurs in myoglobin and oxygen dissociation is regulated from the proximal heme pocket.

Amino Acids↗
PubMed1991

Nicotinate, nicotinamide, and the reactivity of leghemoglobin in soybean root nodules.

Nicotinate has been postulated to interfere with the binding of O(2) to ferrous leghemoglobin in soybean (Glycine max) root nodules. For such a function, the levels of nicotinate in nodules must be sufficiently high to bind a significant amount of leghemoglobin. We have measured levels of nicotinate, nicotinamide, and leghemoglobin in soybean nodules from plants 34 to 73 days after planting in a glasshouse. On a per gram nodule fresh weight basis, levels between 10.4 and 21 nanomoles for nicotinate, 19.2 and 37.8 nanomoles for nicotinamide, and 170 to 280 nanomoles for leghemoglobin were measured. Even if all the nicotinate were bound to ferrous leghemoglobin, only 11% or less of the total leghemoglobin would be unavailable for binding O(2). Using the measured levels of nicotinate and a pH of 6.8 in the cytosol of presenescent soybean nodules, we estimate that the proportion of ferrous leghemoglobin bound to nicotinate in such nodules would be less than 1%. These levels of nicotinate are too low to interfere with the reaction between ferrous leghemoglobin and O(2) in soybean root nodules.

Journal Article↗
PubMed1976

Leghemoglobin. An electron paramagnetic resonance and optical spectral study of the free protein and its complexes with nicotinate and acetate.

Electron paramagnetic resonance (EPR) and optical spectra are used as probes of the heme and its ligands in ferric and ferrous leghemoglobin. The proximal ligand to the heme iron atom of ferric soybean leghemoglobin is identified as imidazole by comparison of the EPR of leghemoglobin hydroxide, azide, and cyanide with the corresponding derivatives of human hemoglobin. Optical spectra show that ferric soybean leghemoglobin near room temperature is almost entirely in the high spin state. At 77 K the optical spectrum is that of a low spin compound, while at 1.6 K the EPR is that of a low spin form resembling bis-imidazole heme. Acetate binds to ferric leghemoglobin to form a high spin complex as judged from the optical spectrum. The EPR of this complex is that of high spin ferric heme in a nearly axial environment. The complexes of ferrous leghemoglobin with substituted pyridines exhibit optical absorption maxima near 685 nm, whose absorption maxima and extinctions are strongly dependent on the nature of the substitutents of the pyridine ring; electron withdrawing groups on the pyridine ring shift the absorption maxima to lower energy. A crystal field analysis of the EPR of nicotinate derivatives of ferric leghemoblobin demonstrates that the pyridine nitrogen is also bound to the heme iron in the ferric state. These findings lead us to picture leghemoglobin as a somewhat flexible molecule in which the transition region between the E and F helices may act as a hinge, opening a small amount at higher temperature to a stable configuration in which the protein is high spin and can accommodate exogenous ligand molecules and closing at low temperature to a second stable configuration in which the protein is low spin and in which close approach of the E helix permits the distal histidine to become the principal sixth ligand.

Acetates↗
PubMed1991

Computational studies of ligand diffusion in globins: I. Leghemoglobin.

The thermally assisted diffusion of a small ligand (carbon monoxide) through a protein matrix (lupine leghemoglobin) is investigated computationally. The diffusion paths are calculated by a variant of the time-dependent Hartree approximation which we call LES (locally enhanced sampling). The variant which was recently introduced by Elber and Karplus is based on the classical TDSCF approximation of Gerber et al. The simulation enables more significant search for diffusion pathways than was possible before. This is done by increasing the number of ligand trajectories using a single trajectory for the protein. We compare qualitatively diffusion rates in leghemoglobin and in myoglobin. The calculation shows that the diffusion in leghemoglobin is much faster than the diffusion in myoglobin, in agreement with experiment. The gate in leghemoglobin is opened by fluctuations at a close contact between the B/C and the G helices. The most relevant fluctuation is the rigid shift of the C helix with respect to the G helix. This path is not observed in a comparable calculation for myoglobin. This finding is rationalized by the lack of the D helix in leghemoglobin and a significantly more flexible CE loop. Supporting experimental evidence for the importance of the CE loop in leghemoglobin can be found in the kinetics studies of Gibson et al.

Amino Acid Sequence↗
PubMed1980

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 Gd3+ 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 (pK approx. 5.0). A further change in the conformation of this group occurs in leghemoglobin nicotinate upon deprotonation of the distal histidine. The pK 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.

Azides↗
PubMed1997

Absence of synproportionation between oxy and ferryl leghemoglobin. off.

The synproportionation reaction between ferryl leghemoglobin and oxyleghemoglobin does not occur, at least under conditions where this process could be clearly demonstrated with myoglobin and hemoglobin. In contrast, a cross synproportionation can occur between oxyleghemoglobin and ferryl myoglobin or between ferryl leghemoglobin and oxymyoglobin. The non-exposure, at the surface of the leghemoglobin molecule, of the nearest tyrosine residue to the heme group could explain this behaviour. Thus leghemoglobin per se does not appear to be able to act as an antioxidant in removing H2O2 by synproportionation. However, in the presence of ascorbate and/or glutathione which can reduce ferryl leghemoglobin, this hemoprotein could act as an H2O2-removing antioxidant, in a process similar to that described for myoglobin. This could also explain why, despite the absence of synproportionation, ferryl leghemoglobin is not detected in nodule extracts.

Antioxidants↗
PubMed1977

Studies on ligand binding of kidney bean leghemoglobin.

Absorption spectra of different ligand derivative;s of kidney bean leghemoglobin alpha have been recorded. The effect of pH on the absorption spectra of kidney bean leghemoglobin alpha has been studied. The pK of the acid-alkaline transition of the heme-linked water molecule is 8.25 and the pK for the acid dissociation of the heme group is 4.03. Affinities of kidney bean leghemoglobin for two different types of ligands have been studied in comparison with soybean leghemoglobins alpha and c and sperm whale myoglobin. All these leghemoglobins have similar affinities for the small anionic ligand fluoride ion, and they are only slightly more accessible to this ligand than is sperm whale myoglobin. Differences in the primary structure or in conformation of these proteins are reflected in the affinity for the bulky ligand imidazole. The accessibility to imidazole increases in the order sperm whale myoglobin less than soybean Lbalpha less than soybean Lbc less than kidney bean Lbalpha, and also low spin Lbalpha less than high spin Lbalpha. The results are discussed with respect to the amino acid sequences of the leghemoglobins.

Binding Sites↗
PubMed1990

Kinetics and thermodynamics of oxygen, CO, and azide binding by the subcomponents of soybean leghemoglobin.

Leghemoglobin shows extreme high affinity behavior in the binding of both oxygen and CO. We have determined the temperature dependence of the rate constants for ligation of oxygen and CO and from these data the thermodynamics (delta G0, delta H0, delta S0) of ligation for the purified components of soybean leghemoglobin. X-ray crystallography has shown that the heme cavity can easily accommodate ligands the size of nicotinate, and analysis of extended x-ray absorption fine structure data has shown that the Fe atom is in the mean plane of the heme in the leghemoglobin-CO complex. Ligation of oxygen and CO are in accord with this picture in that the Ea for oxygen binding is that expected for a diffusion controlled reaction and delta S0 for the ligation of both CO and oxygen is consistent with the simple immobilization of the ligand at the Fe, with no evidence for significant conformational changes in the protein or changes in solvation. At 20 degrees C the rate constants for oxygen and CO binding vary by 26-44% among the eight leghemoglobin components. For azide binding the variation is a factor of 2. These variations appear to arise from amino acid substitutions outside either the heme cavity or the two major paths for ligand entry to the heme. The distribution of leghemoglobin components varies with the age of the soybean nodule during the growing season. The changes in composition alone, however, would only allow the concentration of free oxygen to vary by about 3%. This finding calls into question models that ascribe a significant functional role to changes in the distribution of leghemoglobin components in regulating oxygen concentration in the nodule.

Azides↗
PubMed2004

Tyrosine B10 inhibits stabilization of bound carbon monoxide and oxygen in soybean leghemoglobin.

Detailed comparisons of the carbon monoxide FTIR spectra and ligand-binding properties of a library of E7, E11, and B10 mutants indicate significant differences in the role of electrostatic interactions in the distal pockets of wild-type sperm whale myoglobin and soybean leghemoglobin. In myoglobin, strong hydrogen bonds from several closely related conformations of the distal histidine (His(E7)) side chain preferentially stabilize bound oxygen. In leghemoglobin, the imidazole side chain of His(E7) is confined to a single conformation, which only weakly hydrogen bonds to bound ligands. The phenol side chain of Tyr(B10) appears to "fix" the position of His(E7), probably by donating a hydrogen bond to the Ndelta atom of the imidazole side chain. The proximal pocket of leghemoglobin is designed to favor strong coordination bonds between the heme iron and axial ligands. Thus, high oxygen affinity in leghemoglobin is established by a favorable staggered geometry of the proximal histidine. The interaction between His(E7) and Tyr(B10) prevents overstabilization of bound oxygen. If hydrogen bonding from His(E7) were as strong as it is in mammalian myoglobin, the resultant ultrahigh affinity of leghemoglobin would prevent oxygen transport in root nodules.

Animals↗
PubMed1997

The Vicia faba leghemoglobin gene VfLb29 is induced in root nodules and in roots colonized by the arbuscular mycorrhizal fungus Glomus fasciculatum.

To investigate similarities between symbiotic interactions of broad bean (Vicia faba) with rhizobia and mycorrhizal fungi, plant gene expression induced by both microsymbionts was compared. We demonstrated the exclusive expression of 19 broad bean genes, including VfENOD2, VfENOD5, VfENOD12 and three different leghemoglobin genes, in root nodules. In contrast, the leghemoglobin gene VfLb29 was found to be induced not only in root nodules, but also in broad bean roots colonized by the mycorrhizal fungus Glomus fasciculatum. In uninfected roots, none of the 20 nodulin transcripts investigated was detectable. VfLb29 has an unusually low sequence homology with all other broad bean leghemoglobins as well as with leghemoglobins from other legumes. It can be regarded as a novel kind of leghemoglobin gene not described until now and the induction of which is common to symbiotic interactions of broad bean with both Rhizobium and a mycorrhizal fungus.

Amino Acid Sequence↗
PubMed1976

Intracellular site of synthesis and localization of leghemoglobin in root nodules.

A majority of the total protein synthesis in host cell cytoplasm is inhibited by cycloheximide. Because leghemoglobin represents a large proportion of total cellular protein in the nodule, this observation suggests that leghemoglobin may be translated on the 80S-type ribosomes. Analysis of the nascent peptides isolated from free and membrane-bound polysomes showed that free polysomes contain more immunoreactive material against antibodies to leghemoglobin as compared to that of membrane-bound polysomes. When free and membrane-bound polysomes were incubated in a wheat embryo cell-free protein-synthesizing system, a larger percentage of the released polypeptides from free polysomes was found to be immunoreactive. Similar results were obtained by translation in vitro of the poly(A)-containing mRNA isolated from free and membrane-bound polysomes. For immunocytochemical localization of leghemoglobin, the antibodies were conjugated with ferritin. Antibody conjugates were strictly localized in the host cell cytoplasm and adjacent to the outer surface of the membrane surrounding the bacteroids. Ferritin was not found on the inner surface of the membrane or within the membrane sac. These data suggest that leghemoglobin is synthesized preferentially on the free polysomes in the host cell cytoplasm, directed by a poly(A)-containing 9S mRNA that is most likely of plant origin, and that its location is restricted to the host cell cytoplasm.

Journal Article↗
PubMed1989

Leghemoglobin in Lupin Plants (Lupinus albus cv Multolupa).

Leghemoglobin was localized by immunogold techniques in nodules of Lupinus albus cv Multolupa inoculated with Bradyrhizobium sp. (Lupinus) strain ISLU 16. The protein localization was performed in nodules embedded in Spurr's and Araldite epoxy resins and Lowycryl K4M. A very good preservation of both the ultrastructure and antigenicity was obtained with the tissues embedded in Araldite following glutaraldehyde fixation and unpostfixed in osmium tetroxide. Lupin leghemoglobin is a stable and abundant protein which allows a conventional method to be safely used for localization of leghemoglobin. Labeling of leghemoglobin was specifically confined to the cytosol matrix and nuclei. Gold particles were never observed in the peribacteroidal spaces nor in the cytoplasmic organelles of the infected cells. Decrease of leghemoglobin was observed when the plants were grown with 10.7 micromolar and 21.4 micromolar of nitrate.

Journal Article↗
PubMed1997

Structure of ferric soybean leghemoglobin a nicotinate at 2.3 A resolution.

Soybean leghemoglobin a is a small (16 kDa) protein facilitating the transport of O(2) to respiring N(2)-fixing bacteria at low free-O(2) tension. The crystal structure of soybean ferric leghemoglobin a nicotinate has been refined at 2.3 A resolution. The final R factor is 15.8% for 6877 reflections between 6.0 and 2.3 A. The structure of soybean leghemoglobin a (143 residues) is closely similar to that of lupin leghemoglobin II (153 residues), the proteins having 82 identical residues when the sequences are aligned. The new structure provides support for the conclusion that the unique properties of leghemoglobin arise principally from a heme pocket considerably larger and more flexible than that of myoglobin, a strongly ruffled heme group, and a proximal histidine orientation more favourable to ligand binding.

Journal Article↗
PubMed1985

Discrepancies among published amino acid sequences of soybean leghemoglobins: experimental evidence against cultivar differences as the sources of the discrepancies.

Despite discrepancies among charged amino acid residues in published amino acid sequences, isoelectric focusing experiments failed to detect varietal differences in soybean leghemoglobins a, c1, c2, or c3. Leghemoglobins from 69 domesticated soybean (Glycine max) cultivars and plant introductions and 18 wild soybean (Glycine soja) plant introductions were compared; the sources included soybean cultivars used by research groups in obtaining amino acid sequences and most of the ancestors of North American soybean cultivars. Thus, at least some of the discrepancies among published amino acid sequences of soybean leghemoglobins are due to sequencing difficulties rather than structural differences among the leghemoglobins used by different research groups.

Amino Acid Sequence↗
PubMed1977

Purification and properties of soybean leghemoglobin messenger RNA.

Poly(A)-containing leghemoglobin mRNA from soybean root nodules has been purified 84-fold, as judged by its ability to direct the in vitro synthesis of leghemoglobin in a wheat germ system. It has a poly(A) content of 8.6% and a molecular weight, estimated by formamide gel electrophoresis, of 260 000. mRNA with a molecular weight of around 143 000 would be sufficient to code for leghemoglobin. Thus, with respect to both its poly(A) content and its unexpectedly high molecular weight, leghemoglobin mRNA is similar to mRNAs isolated from animal tissues.

Hemeproteins↗
PubMed1978

Study of the pseudoperoxidatic activity of soybean leghemoglobin and sperm whale myoglobin.

The compound formation between soybean leghemoglobins a and c and H2O2 or ethyl hydroperoxide has been studied and compared with the hydrogen peroxide compound of sperm whale myoglobin. the titration data show that the hydrogen peroxide compounds of leghemoglobins are formed in a 1:1 molar ratio. The kinetics of the formation of the compounds follow first-order kinetics and the compounds are formed considerably faster than the myoglobin peroxide compound. The pseudoperoxidatic activity of leghemoglobins a and c and myoglobin was studied using guaiacol as electron donor. The maximal reaction velocities of leghemoglobins are greater than that of myoglobin. The results indicate that the peroxidatic activity of ferrileghemoglobin may be biologically important for instance in aging root nodules.

Animals↗
PubMed1978

Heme sulfuric anhydrides as soybean leghemoglobin structure probes.

Mesoheme monosulfuric anhydride reacts at three distinct sites in soybean apoleghemoglobin a, at lysine-6, lysine-19 and lysine-57, the last one being the major site of reaction. The heme peptides obtained from thermolytic and pronase hydrolysates of the anhydride-leghemoglobin a were purified and correlated with the known amino acid sequence of the protein. Mesoheme bissulfuric anhydride also reacts with soybean apoleghemoglobin a giving a complex mixture of hemepeptides after hydrolysis with pronase. The visible spectrum of anhydride leghemoglobin is that of low spin heme. This suggests that anhydride leghemoglobin has a conformation with a covalent attachment via propionic acid side chain to lysine-57 and the sixth coordination position of the heme iron occupied by the distal histidine at position 61. Native leghemoglobin is assumed to exist in a similar type of configuration at low temperature, but with the heme propionate side chain being involved in a salt bridge with lysine-57.

Affinity Labels↗
PubMed1987

Nonenzymatic reduction of ferric leghemoglobin.

Ferric leghemoglobin isolated from soybean root nodules was reduced nonenzymatically to ferrous leghemoglobin in vitro at pH 5.2 using either 1.0 mM NADH or NADPH as the reductant. In the pH range of 5.2 to 7.0, the highest rates of reduction occurred below pH 6.5 with a maximum rate observed at pH 5.2. Rates of nonenzymatic ferric leghemoglobin reduction above pH 6.5 or at reduced-pyridine nucleotide concentrations below 0.4 mM were insignificant. Oxygen was required for the nonenzymatic reduction. Inhibition of ferric leghemoglobin reduction by superoxide dismutase and catalase indicated that superoxide and hydrogen peroxide may be intermediates in the reaction.

Buffers↗