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At least 19 recordsLinked to original sources
PubMed1978

The reaction of ferrous leghemoglobin with hydrogen peroxide to form leghemoglobin(IV).

Ferrous leghemoglobin reacts with hydrogen peroxide to form the stable product, leghemoglobin(IV). The reaction follows second order kinetics (k = 2.24 X 10(4) M-1 S-1 at 20 degrees C) and may be regarded as a single-step, two-electron oxidation. Ferric leghemoglobin is not an intermediate. The oxidation state of leghemoglobin(IV) is established by reductive titration with dithionite; 2 eq of dithionite are required to convert 1 mol of leghemoglobin(IV) to ferrous leghemoglobin. An outstanding property of leghemoglobin(IV) is its stability, little change is noted after 12 h at 25 degrees C. Leghemoglobin(IV) differs from the higher oxidation states of other hemoglobins and myoglobins in that it does not react with hydrogen peroxide to form the oxygenated protein.

Dithionite↗
PubMed1978

Leghemoglobin. Low temperature optical spectra of acid and alkaline forms of leghemoglobin(IV). Configuration of the heme.

Leghemoglobin(IV), the derivative of leghemoglobin at the formal oxidation state IV, when cooled to liquid nitrogen temperature exhibits radically different spectra at acid and alkaline pH. The acid and alkaline forms are freely interconvertible. The optical spectrum of the acid form is closely similar to optical spectra of the red higher oxidation states of horseradish and cytochrome c peroxidases, showing that the configuration of the heme iron is the same throughout this family of compounds. That configuration is believed to be Fe(IV) in a porphyrin environment. The optical spectrum of the alkaline form of leghemoglobin(IV) recalls that of alkaline low spin ferric leghemoglobin. Near infrared spectra of leghemoglobin(IV), myoglobin(IV), and the higher oxidation states of the peroxidases are featureless to 1300 nm, suggesting a common structural feature. The acid form of leghemoglobin(IV), seen in fluid buffer as a transient species at pH 5 or less, is conveniently generated by cooling a solution of the more stable alkaline form in borate buffer to liquid nitrogen temperature. At this temperature borate buffers become acid.

Freezing↗
PubMed1980

The heme environment of leghemoglobins. Absorption and circular dichroism spectra of artificial leghemoglobins and myoglobins.

Artificial leghemoglobins were reconstituted from apoleghemoglobin and meso-, deutero- and diacetyldeuteroheme. Absorption and circular dichroism spectra of their high-spin and low-spin derivatives in the ferrous and ferric forms were recorded in the ultraviolet and visible wavelength regions. The substitution of the 2,4-side-chains of heme induced changes in the optical activity, reflecting alterations in the heme environment. The effect on the conformation of aromatic amino acid residues around heme obviously correlates with the sixth axial ligand and the spin state of iron. Absorption and CD spectra of the aquoferric derivatives of artificial myoglobins were recorded in comparison. Strongly electron-withdrawing acetyl side-chains at the 2,4-positions of diacetyldeuteroheme caused a change in the absorption spectra of aquoferric leghemoglobin and myoglobin towards low spin. On the basis of the spectra it was suggested that the displacement of the ferric iron from the pyrrole plane in leghemoglobin derivatives would be smaller than in the corresponding myoglobin derivatives.

Animals↗
PubMed1979

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 c1 prepared by ion exchange chromatography are homogeneous by isoelectric focusing criteria. Leghemoglobin c2 prepared by ion exchange chromatography is an approximately 1:2 mixture of leghemoglobins c2 and c3. Leghemoglobin d consists of three components. The ratio of leghemoglobin a to leghemoglobin c3 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 O2 concentration only when the nodule structure is complex. The ratio of leghemoglobin c1 content to leghemoglobin c3 content increases somewhat during the early period of nodule development, while the ratio of leghemoglobin c2 content to leghemoglobin c3 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.

Hemeproteins↗
PubMed1980

Kinetic studies of the reaction of ferric soybean leghemoglobins with hydrogen peroxide, cyanide and nicotinic acid.

A kinetic study of the reaction of two soybean leghemoglobins (components a and c) with hydrogen peroxide to form the oxidized compound (leghemoglobin IV) has been carried out over the pH range 2.5--10. Three different ionization processes of leghemoglobins with pKa values of 3,4.7 +/- 0.2 and 8.2 +/- 0.1 are required to explain the rate/pH profiles. Protonation of the former group and ionization of the latter cause a decrease in the rate of reaction of the hemoproteins with H2O2. The results are compared to those obtained for the reactions of plant peroxidases and myoglobin with H2O2. The results obtained from the kinetic study of cyanide binding to soybean leghemoglobins indicate that CN- is the reactive species. Two ionization processes of leghemoglobins with pKa values of 4.7 +/- 0.2 and 8.2 +/- 0.1 affect the reaction rates. The association and dissociation rate constants corresponding to nicotinic acid binding to leghemoglobins a and c have been measured over the pH range 2.5--7. The dissociation rate constant is affected by ionization of a group with pKa less than 2.5 for both leghemoglobin-nicotinate complexes. In this pH range the association rate constant is only affected by ionization of a group with pKa value of 4.7 +/- 0.2. The analysis of these results shows that both ionization processes corresponding to ring nitrogen atom of the ligand (pKa approximately equal to 4.9) and to a heme-linked group (pKa approximately equal to 4.7 +/- 0.2) influence the association rate constant. Furthermore, it appears that in the binding site of leghemoglobins the pKa value corresponding to ionization of the ring nitrogen atom of nicotinic acid is shifted from the normal value of 4.9 to a value of less than 2.5. This pecularity might explain the exceptional reactivity of leghemoglobins for nicotinic acid, over a large pH range. For both cyanide and nicotinic acid binding reactions, the ionizable group of leghemoglobins with pKa value of 4.7 +/- 0.2 seems to act as an electrostatic gate. When the group is deprotonated, it restricts the access of anion ligands to the heme pocket. For all the three reactions studied, leghemoglobin a reacts about twice as fast as leghemoglobin c.

Cyanides↗
PubMed1979

CO and O2 complexes of soybean leghemoglobins: pH effects upon infrared and visible spectra. Comparisons with CO and O2 complexes of myoglobin and hemoglobin.

The effects of pH upon infrared spectra [CO stretching frequency (vco) region] and visible spectra of the CO complexes of soybean leghemoglobins a, c1, and c2, sperm whale myoglobin, and human hemoglobin A are reported. The vco for leghemoglobin--CO complexes was 1947.5 cm-1 at neutral pH. At acid pH myoglobin-- and hemoglobin--CO complexes developed vco bands at 1966--1968 cm-1, whereas leghemoglobin--CO complexes developed vco bands at approximately 1957 cm-1. All pKapp co values determined by pH-dependent variation of vco fell in the range 4.0--4.6. The pKapp co values determined from visible spectra were consistent with vco-determined values except for that of myoglobin--CO (visible pKapp co = 5.8). The pKapp co values in the 4.0--4.6 range appear to be pK values of the distal histidines, while the visible pKapp co of myoglobin--CO appears to be the pK of a group other than the distal and proximal histidines. The data are consistent with a model in which protonation of the distal histidine permits protein-free heme FeCO geometry in leghemoglobin--CO complexes but not in myoglobin-- or hemoglobin--CO complexes. Thus the heme pockets of leghemoglobins appear to be more flexible than the heme pockets of myoglobin and hemoglobin. The effects of pH upon visible spectra of the O2 complexes of soybean leghemoglobins a, c1, and c2, sperm whale myoglobin, and human hemoglobin A also are reported. pKapp o2 values of approximately 5.5 (leghemoglobins) and 4.4 (hemoglobin) are probably the pK values of the distal histidines. Comparisons of pKapp o2 values with pKapp co values indicate a more flexible heme pocket in leghemoglobins than in hemoglobin. The O2 complex of leghemoglobin c2 differed significantly from the O2 complexes of leghemoglobins a and c1 in visible spectra and titration behavior. These differences might be associated with the small structural differences in the region between the E and F helixes of leghemoglobins.

Animals↗
PubMed1988

NMR studies of the conformations of leghemoglobins from soybean and lupin.

Phase-sensitive two-dimensional NMR methods have been used to obtain extensive proton resonance assignments for the carbon monoxide complexes of lupin leghemoglobins I and II and soybean leghemoglobin a. The assigned resonances provide information on the solution conformations of the proteins, particularly in the vicinity of the heme. The structure of the CO complex of lupin leghemoglobin II in solution is compared with the X-ray crystal structure of the cyanide complex by comparison of observed and calculated ring current shifts. The structures are generally very similar but significant differences are observed for the ligand contact residues, Phe30, His63 and Val67, and for the proximal His97 ligand. Certain residues are disordered and adopt two interconverting conformations in lupin leghemoglobin II in solution. The proximal heme pocket structure is closely conserved in the lupin leghemoglobins I and II but small differences in conformation in the distal heme pocket are apparent. Larger conformational differences are observed when comparisons are made with the CO complex of soybean leghemoglobin. Altered protein-heme packing is indicated on the proximal side of the heme and some conformational differences are evident in the distal heme pocket. The small conformational differences between the three leghemoglobins probably contribute to the known differences in their O2 and CO association and dissociation kinetics. The heme pocket conformations of the three leghemoglobins are more closely related to each other than to sperm whale myoglobin. The most notable differences between the leghemoglobins and myoglobin are: (a) reduced steric crowding of the ligand binding site in the leghemoglobins, (b) different orientations of the distal histidine, and (c) small but significant differences in proximal histidine coordination geometry. These changes probably contribute to the large differences in ligand binding kinetics between the leghemoglobins and myoglobin.

Amino Acids↗
PubMed1980

The amino acid sequence of pea (Pisum sativum) leghemoglobin.

The amino acid sequence has been determined for leghemoglobin component I from root nodules of pea, Pisum sativum. Pea leghemoglobin is one polypeptide chain composed of 147 amino acids, it contains one methionine residue at position 144, and three histidines, which are at positions 60, 92 and 101. The sequence has at least seven polymorphic residues, but it was not possible to separate the polymorphic protein forms which had identical electric charge. The approximate molecular weight of pea leghemoglobin component I is 16,350. The other major leghemoglobin component (II) from pea has an amino acid composition very similar to that of leghemoglobin component I, suggesting that the gene has duplicated relatively recently. P. sativum leghemoglobin differs from that of Vicia faba by 22--23%, depending on the polymorphic form. The leghemoglobins from Phaseolus vulgaris and Glycine max differ from pea leghemoglobin by 35--44%, and Lupinus luteus leghemoglobins differ from it by 45--48%. The seven leghemoglobins so far sequenced have 50 residues (33%) which are common to all.

Amino Acid Sequence↗
PubMed1992

An alfalfa (Medicago sativa L.) cDNA encoding an acidic leghemoglobin (MsLb3).

We have found an alfalfa cDNA clone that encodes an acidic leghemoglobin. To date, 14 alfalfa leghemoglobin clones have been identified. Five different leghemoglobin 'components' have been biochemically defined on the basis of their pI. A higher-resolution comparison, provided by sequence data analysis, identifies six leghemoglobin 'classes'. All 14 leghemoglobins are assigned to the six 'classes', which can be distributed among the five leghemoglobin 'components'. The newly identified leghemoglobin is the only member of a sixth 'class' of leghemoglobins, and it also is the only member of one of the acidic leghemoglobin 'components' IV or V.

Amino Acid Sequence↗
PubMed1986

High-performance liquid chromatographic separation of leghemoglobins from soybean root nodules.

A crude fraction of soybean nodule ferri-leghemoglobin was absorbed onto a commercial DEAE HPLC column at pH 8.0, and resolved into eight isoprotein fractions. The identity of the leghemoglobins were determined by their order of elution from the DEAE column and by isoelectric focusing, using isoprotein standards isolated by conventional procedures. The three isoproteins of the c complex, c1, c2, c3, were not resolved. Unexpected heme containing proteins eluted just after leghemoglobin a and the c complex. These components possessed proteins similar to leghemoglobin a and the c complex, respectively, as judged by isoelectric focusing and absorbance spectra of the ferri and ferrous forms. The components designated leghemoglobin a' and leghemoglobin c' were also differentiated from leghemoglobin a and c by reverse-phase HPLC in a C18 column. Amounts of protein for the DEAE HPLC column ranged from 10 micrograms to 20 mg and sample volumes ranged from 2 to 250 microliters. The time required for chromatography varied depending on the gradient used, but never exceeded 40 min for samples up to 5 mg protein or 120 min for samples containing 5 to 20 mg protein. Due to the sensitivity of detection at 403 nm and leghemoglobins being the predominant chromophore at that wavelength, it was possible to quantitate levels of individual leghemoglobins in samples extracted from single nodules (ca. 15 to 65 mg fresh weight tissue). Quantitation was performed by interfacing the spectrophotometer output (10 mV) to a microcomputer and using commercially available software.

Chromatography, High Pressure Liquid↗
PubMed1975

Circular dichroism studies of myoglobin and leghemoglobin.

The circular dichroism spectra of leghemoglobin a from the root nodules of soybean have been compared with those for sperm whale myoglobin in the fat- and near-ultraviolet and the Soret and visible regions of the spectrum. Circular dichroism spectra in the far-ultraviolet show that the leghemoglobins all have a high alpha-helix content (soybean leghemoglobin a, 55%) regardless of the nature of bound ligands and oxidation or spin state of the heme iron. The known sequence homologies with mammalian hemoglobins may therefore be reflected in conformational homologies as suggested by the x-ray studies of Vainshtein et al. ((1975) Nature (London) 254, 163-164) on lupin leghemoglobin. Removal of the heme moiety decreases helicity by only 9% for leghemoglobins, compared with 23% for myoglobin. This, the much smaller heme contribution to the near-ultraviolet circular dichroism than in myoglobin, and the greater accessibility of the heme moiety to aqueous solvent (Nicola et al. (1974), Proc. Aust. Biochem. Soc. 7, 21) suggest that the association between heme and protein is much weaker in leghemoglobins than in myoglobin. The aromatic Soret and visible circular dichroism spectra for all derivatives of leghemoglobin are opposite in sense to those for myoglobin, showing that the patterns of protein side chain contacts with the heme are different in the two classes of heme proteins. There is strong evidence that one of the two tryptophans whose identity and structural role in myoglobin is known, is present also in plant leghemoglobins, hydrogen-bonded and in a similar nonpolar environment whether heme is present or not. The above findings help to explain the remarkably high oxygen affinity and some other ligand-binding properties of leghemoglobins which differ from those of myoglobin.

Animals↗
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↗