Heme symmetry, vibronic structure, and dynamics in heme proteins: ferrous nicotinate horse myoglobin and soybean leghemoglobin

We report the visible and Soret absorption bands, down to cryogenic temperatures, of the ferrous nicotinate adducts of native and deuteroheme reconstituted horse heart myoglobin in comparison with soybean leghemoglobin-a. The band profile in the visible region is analyzed in terms of vibronic coupling of the heme normal modes to the electronic transition in the framework of the Herzberg-Teller approximation. This theoretical approach makes use of the crude Born-Oppenheimer states and therefore neglects the mixing between electronic and vibrational coordinates; however, it takes into account the vibronic nature of the visible absorption bands and allows an estimate of the vibronic side bands for both Condon and non-Condon vibrational modes. In this framework, an x-y splitting of the Q transition for native and deuteroheme reconstituted horse myoglobin is clearly assessed and attributed to electronic perturbations that, in turn, are caused by a reduction of the typical D(4h) symmetry of the system due to heme distortions of B(1g)-type symmetry and/or to an x-y asymmetric position of the nicotinate ring; in deuteroheme reconstituted horse myoglobin the asymmetric heme peripheral substituents add to the above effect(s). On the contrary, in leghemoglobin-a no spectral splitting upon nicotinate binding is observed, pointing to a planar heme configuration in which only distortions of A(1g)-type symmetry are effective and to which the nicotinate ring is bound in an x - y symmetric position. The local dynamic properties of the heme pocket of the three proteins are investigated through the temperature dependence of spectral line broadening. Leghemoglobin-a behaves as a softer matrix with respect to horse myoglobin, thus validating the hypothesis of a looser heme pocket conformation in the former protein, which allows a nondistorted heme configuration and a symmetric binding of the bulky nicotinate ligand.     

Structure of azide methemoglobin

We have compared the structures of horse azide methemoglobin and methemoglobin (MetHb) at 2.8 Å resolution by X-ray difference Fourier analysis. Of four low-spin liganded Hb derivatives (nitric oxide Hb, carbon monoxide Hb, cyanide MetHb, and azide MetHb), azide MetHb is closest in structure to MetHb. In azide MetHb the ligands are co-ordinated end-on at angles of about 125 ° to the heme axes, which is similar to the stereochemistry assumed by azide in binding to free heme. Because of its bent binding geometry, azide encounters less interference in binding and perturbs the protein structure less than carbon monoxide and cyanide, which are smaller, but prefer linear axial co-ordination to heme. Steric interactions between ligand and protein are greater on the β chain, where the E helix is pushed away from the heme relative to MetHb, than on the α chain. Iron position is the same and heme stereochemistry and position are very similar in azide MetHb and MetHb.     

Ligand binding properties of horse hemoglobins containing deutero- and mesoheme.

The reactions of horse globin reconstituted with proto-, deutero-, and mesoheme have been examined by equilibrium and kinetic methods. In virtually all reactions studied, mesohemoglobin displays the more extreme functional behavior, whereas deuterohemoglobin exhibits behavior which is either very similar to native hemoglobin or intermediate between the two. Our kinetic and equilibrium results indicate that the primary effect of heme modification on the functional properties of hemoglobin is to alter the intrinsic reactivities of the deoxy and liganded conformations. Heme modification does not, however, result in substantial alterations in the conformational equilibrium between the two states. Simple inductive electronic effects of the 2- and 4-substituents of the heme moiety in deutero- and mesohemoglobin are apparently not sufficient to explain the observed equilibrium and kinetic properties completely, which indicates that steric effects of these substituents may also play a role in determining the functional behavior of the hemoglobin molecule.     

The oxidation of hemins by microsomal heme oxygenase. Structural requirements for the retention of substrate activity

The substrate specificity of microsomal heme oxygenase from rat liver was studied by introducing systematic structural changes in the array of substituents of the protohemin IX rings. Replacement of the vinyls by methyl groups resulted in hemins which were excellent substrates of the heme oxygenase. Replacement of the 4-vinyl group by a propionic acid chain (harderohemin), decreased substrate activity to 40%. The replacement of the vinyls by formyl residues strongly decreased substrate activity but the hemins were still substrates of heme oxygenase. The oxidation rates of Spirographis hemin and of 2,4-diformyldeuterohemin IX showed a time lag which was absent when isoSpirographis hemin was used as a substrate. This lag could be attributed to the formation of a transient hemiacetal between the 2-formyl group and the alpha-mesohydroxy residue. The isomeric protohemins I, XI, and XIV (Fischer's notation) were examined as possible substrates of microsomal heme oxygenase. In these protohemins the array of substituents of rings A and B was the same as in protohemin IX, but the methyl and propionic acid residues of rings C and D were at different positions from those of protohemin IX. None of them had substrate activity, indicating that the presence of two vicinal propionic acid side-chains at C6 and C7 was necessary for substrate activity. A hemin with only one propionic acid residue at C5 was not a substrate of the enzyme, either. When the propionic acid residues of protohemin IX were replaced by butyric acid residues, substrate activity decreased to 50% (as compared to protohemin IX), while when they were replaced by acetic acid residues, the substrate activity was entirely suppressed. The addition of dimethyl sulfoxide (25 mM) to the incubation mixture enhanced the oxidation of hemins with non-polar substituents in rings A and B by about 35%, while it was without effect on hemins with polar substituents in the same rings.     

Proton NMR study of myoglobin reconstituted with 3, 7-diethyl-2, 8-dimethyl iron porphyrin: remarkable influence of peripheral substitution on heme rotation

The iron complex of 3,7-diethyl-2,8-dimethylporphyrin was incorporated into horse heart apomyoglobin to investigate the influence of peripheral substitution on artificial heme rotation. The hyperfine-shifted 1H NMR spectrum of the reconstituted deoxymyoglobin (rMb) revealed the proximal imidazole N-H resonance at 82.5 ppm to indicate the formation of the Fe--N (His93) bond. The pyrrole-protons of the hemin of myoglobin in the absence of external ligand appeared as four resonances between -10 and -18 ppm, indicating a mainly low-spin ferric hemin, with a ligated distal histidine (His64). This also indicates the lost of the symmetry of the hemin, according to an absence of free rotation of the prosthetic group. The 1H NMR spectrum of reconstituted rMbCO revealed a set of four pyrrole-protons and a set of four meso-protons. Accordingly, the prosthetic group without acid side chains interacts specifically with the surrounding globin showing a unique heme orientation in the 1H NMR time-scale, despite the presence of only four alkyl substituents on the porphine ring. This also suggests that two ethyl groups are large enough to avoid the free rotation movement of the heme.     

A pH-dependent aquomet-to-hemichrome transition in crystalline horse methemoglobin

In 1947, Perutz and co-workers reported that crystalline horse methemoglobin undergoes a large lattice transition as the pH is decreased from 7.1 to 5.4. We have determined the pH 7.1 and 5.4 crystal structures of horse methemoglobin at 1.6 and 2.1 A resolution, respectively, and find that this lattice transition involves a 23 A translation of adjacent hemoglobin tetramers as well as changes in alpha heme ligation and the tertiary structure of the alpha subunits. Specifically, when the pH is lowered from 7.1 to 5.4, the Fe(3+) alpha heme groups (but not the beta heme groups) are converted from the aquomet form, in which the proximal histidine [His87(F8)alpha] and a water molecule are the axial heme ligands, to the hemichrome (bishistidine) form, in which the proximal histidine and the distal histidine [His58(E7)alpha] are the axial heme ligands. Hemichrome formation is coupled to a large tertiary structure transition in the eight-residue segment Pro44(CD2)alpha-Gly51(D7)alpha that converts from an extended loop structure at pH 7.1 to a pi-like helix at pH 5.4. The formation of the pi helix forces Phe46(CD4)alpha out of the alpha heme pocket and into the interface between adjacent hemoglobin tetramers where it participates in crystal lattice contacts unique to the pH 5.4 structure. In addition, the transition from aquomet alpha subunits to bishistidine alpha subunits is accompanied by an approximately 1.2 A movement of the alpha heme groups to a more solvent-exposed position as well as the creation of a solvent channel from the interior of the alpha heme pocket to the outside of the tetramer. These changes and the extensive rearrangement of the crystal lattice structure allow the alpha heme group of one tetramer to make direct contact with an alpha heme group on an adjacent tetramer. These results suggest possible functional roles for hemichrome formation in vivo.     

Horse heart myoglobin reconstituted with a symmetrical heme. A circular dichroism study

Proton NMR studies on myoglobins and hemoglobins reconstituted with non-natural hemes, possessing different side chains in the pyrrolic rings, have provided interesting information for the understanding of the mechanism governing heme reorientation in the globin pocket, during synthesis of the native protein in vivo or in the reconstitution process in vitro. More recently, circular dichroism (CD) studies have been reported as a qualitative, alternative tool, with respect to 1H-NMR for detecting heme disorder in a reconstituted myoglobin or hemoglobin. In this paper, a CD study is reported on the reconstitution of horse heart myoglobin with protoheme XIII, a heme possessing true rotational symmetry about its alpha, gamma-meso axis. The results obtained show that the reconstitution product with this heme, which binds to the apoprotein with high affinity, not dissimilar from that of the natural heme, is characterized by a CD spectrum with bands possessing rotational strengths much lower than in the native protein. Furthermore, the CD changes detected as a function of time, during heme reorientation, in the case of natural heme, are absent when the apoprotein is reconstituted with protoheme XIII. These data provide independent evidence for reorientation of the natural heme, which follows its insertion into the protein matrix.     

Structure of cyanide methemoglobin

X-ray difference Fourier analysis at 2.8 Å resolution shows that the tertiary structures of horse cyanide methemoglobin and methemoglobin differ significantly. The conformations of the heme groups and their interactions with the globin are altered. Short contacts with globin side chains affect cyanide binding to the hemes, and the changes in globin-ligand contact upon substitution of cyanide for water in turn directly affect globin structure. Although the ligand peaks lie off the heme axes, the atoms FeCN may still lie on a straight line (as they do in small iron cyanide complexes), with this line not normal to the mean heme plane. This linear binding configuration is consistent with the observed motion and deformation of the porphyrin. Although motion of the iron atoms is not directly apparent, there is evidence that some changes in tertiary structure are induced by shortening of the iron-pyrrol nitrogen bond lengths. This and other studies suggest that the structural changes responsible for co-operativity in hemoglobin may be initiated not merely by an alteration in the covalent porphyrin-proximal histidine linkage, but by changes in the noncovalent interactions of the globin with the ligand and porphyrin as well.     

Proton magnetic resonance study of the influence of heme 2,4 substituents on the exchange rates of labile protons in the heme pocket of myoglobin.

Four exchangeable protons with large hyperfine shifts are assigned in the heme pocket of sperm whale met-cyano myoglobin reconstituted with heme possessing acetyl groups, ethyl groups, bromines, and hydrogens at the 2,4 position, using both relaxation and chemical-shift data. The four protons arise from the ring NH's of the proximal (F8), distal (E7), and FG2 histidines, and the peptide NH of His F8. The similarity of all chemical shifts to those of the native protein as well as the invariance of the relaxation rates of the distal histidyl ring NH dictate essentially the same structure for the heme cavity of both native and reconstituted proteins. The exchange rates with bulk water of the four labile proteins in each modified protein were determined by saturation-transfer and line width methods. All four labile protons were found to have the same exchange rate as in the native protein for acetyl and ethyl 2,4 substituents; the two resolved labile protons in the derivative with 2,4 bromine were also unchanged. The reconstituted protein with hydrogens at the 2,4 position exhibited slower exchange rates for three of the four protons, indicating an increased dynamic stability of the heme pocket in the absence of bulky 2,4 substituents.     

Structure of imidazole methemoglobin

Crystals of horse methemoglobin shatter when soaked in crystallization buffer containing high concentrations of imidazole. By using less than saturating concentrations of imidazole, a stable imidazole derivative of crystalline methemoglobin was prepared and analyzed by X-ray difference Fourier techniques. Both subunits of imidazole methemoglobin show extensive, but different, changes in tertiary structure. Many of the tertiary structural changes observed in the transition from deoxyhemoglobin to methemoglobin are amplified in the transition from methemoglobin to imidazole methemoglobin. Unlike all other ligands that have been examined, imidazole only partially enters the ligand pocket and does not occupy the usual ligand site distal to pyrrole II. The position of the imidazole is on a possible pathway for entrance of smaller diatomic ligands from the solvent into the heme pocket. The extent of imidazole binding of the α-hemes and β-hemes is about 25% and 45%, respectively. An explanation for this difference in occupancy is suggested, involving steric interaction of the distal histidine and phenylalanine CD4 in each subunit. This structural hypothesis may have implications for the kinetics of ligand binding.     

Interaction of human adult methemoglobin in low-spin state with inositol hexaphosphate. A proton magnetic resonance study

The hyperfine-shifted proton nuclear magnetic resonance (NMR) spectra of the low-spin complexes of human adult methemoglobin were found to be much altered by the addition of inositol hexaphosphate (IHP). The stoichiometry and pH-dependence of IHP binding, and the spin equilibrium of azide methemoglobin are parallel to those of high-spin human methemoglobin and of carp methemoglobin, both of which are proposed to be switched from the R to T states with IHP. The present NMR results show that IHP affects the structure of human methemoglobin regardless of the spin state of the heme iron, suggesting that there is no correspondence between quaternary structure and the spin state of ferric heme iron.     

Electron paramagnetic resonance of single crystal deoxycobaltohemoglobin

Single crystals of horse ^CoHb were obtained by reduction of ^CoHb⁺ crystals with dithionite. Epr measurements showed that the g? and ^Coà tensors are both axial and share the same principal axis systems. Of the four subunits, the “heme” normals of C? and d? subunits ãb?plane 29 ± 1° from b?; they have the same orientation as the hemes in methemoglobin. The normals of “hemes” à and B? are 47 above the ãb? plane as compared to 16° in methemoglobin.     

1H NMR study of the influence of hydrophobic contacts on protein-prosthetic group recognition in bovine and rat ferricytochrome b5

The proton nuclear magnetic resonance spectra of the soluble fragment of native bovine and genetically engineered wild-type rat ferricytochrome b5 reconstituted with a wide variety of hemes chemically modified at 2- and/or 4-positions have been recorded and analyzed. While all but one nonsymmetric heme yielded comparable amounts of the two heme orientations immediately after reconstitution, the relative proportion of the two orientations at equilibrium varied widely. The unpaired spin density distribution in the heme pi system leads to substituent hyperfine shift patterns in these paramagnetic complexes that are completely diagnostic of the heme orientation in the protein matrix. An empirical assignment strategy is outlined and applied which allows unequivocal assignment of the absolute orientation of a derivatized heme within the protein matrix. Using a series of hemes lacking 2-fold symmetry solely due to a single substitution, the preferences for localized site occupation of vinyls, methyls, and hydrogens are developed. The large differences in relative stability of the two orientations of native protohemin in the two cytochromes b5 is shown to result from the additivity of localized effects for the bovine protein and the near cancellation of competing effects in the rat protein. The major determinant of the heme orientation is judged to be a repulsive interaction between a vinyl and a hydrophobic cluster of amino acids including positions 23 and 25. The differences in this heme orientational preference among bovine, rat, and chicken ferricytochromes b5 could be correlated with the relative steric bulk of the residues at positions 23 and 25.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solution 1H NMR investigation of the seating and rotational "hopping" of centrosymmetric etioheme-I in myoglobin: effect of globin origin and its oxidation/spin state on heme dynamics

Solution 1H NMR spectroscopy was used to investigate the heme active-site structure and dynamics of rotation about the Fe-His bond of centrosymmetric etioheme-I reconstituted into sperm whale and horse myoglobin (Mb). Comparison of the NOESY cross-peak pattern and paramagnetic relaxation properties of the cyanomet complexes confirm a heme pocket that is essentially the same as Mb with either native protoheme or etioheme-I. Dipolar contacts between etioheme and the conserved heme pocket residues establish a unique seating of etioheme that conserves the orientation of the N-Fe-N vector relative to the axial His plane, with ethyl groups occupying the vinyl positions of protoheme. Saturation transfer between methyls on adjacent pyrroles in etioheme-reconstituted horse Mb in all accessible oxidation/spin states reveals rotational hopping rates that decrease dramatically with either loss of ligands or reduction of the heme, and correlate qualitatively with expectations based on the Fe-His bond strength and the rate of heme dissociation from Mb. The rate of hopping for etioheme in metMbCN, in contrast to hemes with propionates, is the same in the sperm whale and horse proteins.     

Structure of nitric oxide hemoglobin

We have compared the structure of horse nitric oxide hemoglobin (HbNO) and methemoglobin in the oxy quaternary structure by difference Fourier analysis at 2.8 Å resolution. Both nitric oxide and oxygen assume bent co-ordination geometry and form low-spin complexes in binding to heme; on the basis of preferred ligand and heme stereochemistry, HbNO is the closest analog of HbO₂ (oxyhemoglobin) examined to date. To the resolution of the X-ray data, the stereochemistry of the heme-NO complex in hemoglobin and the corresponding free heme complex appears similar. In contrast, the ligand pockets in hemoglobin hinder binding of cyanide and carbon monoxide in their preferred linear axial co-ordination modes and force them to assume a strained off-axis binding stereochemistry. The structural similarity between HbNO and HbO₂ is reflected in their kinetic behavior, which is similar, and distinct from that of carboxyhemoglobin.     

Properties and reactivity of myoglobin reconstituted with chemically modified protohemin complexes

The synthetic complexes protohemin-6(7)-L-arginyl-L-alanine (HM-RA) and protohemin-6(7)-L-histidine methyl ester (HM-H) were prepared by condensation of suitably protected Arg-Ala or His residues with protohemin IX. HM-RA and HM-H were used for reconstitution of apomyoglobin from horse heart, yielding the Mb-RA and Mb-H derivatives, respectively, of the protein. The spectral, binding and catalytic properties of Mb-RA and Mb-H are significantly different from those of Mb. As shown by MM and MD calculations, these differences are determined by some local structural changes around the heme which are generated by increased mobility of a key peptide segment (Phe43-Lys47), containing the residue (Lys45) that in native Mb interacts with one of the porphyrin carboxylate groups. In the reconstituted Mbs this carboxylate group is bound to the Arg-Ala or His residue and is no longer available for electrostatic interaction with Lys45. The mobility of the peptide segment near the active site allows the distal histidine to come to a closer contact with the heme, and in fact Mb-RA and Mb-H exist as an equilibrium between a high-spin form and a major low-spin, six-coordinated form containing a bis-imidazole ligated heme. The two forms are clearly distinguishable in the NMR spectra, that also show that each of them consists of a mixture of the two most stable isomers resulting from cofactor reconstitution, as also anticipated by MM and MD calculations. Exogenous ligands such as cyanide, azide, or hydrogen peroxide can displace the bound distal histidine, but their affinity is reduced. On the other hand, mobilization of the peptide chain around the heme in the reconstituted Mbs increases the accessibility of large donor molecules at the heme periphery, with respect to native Mb, where a rigid backbone limits access to the distal pocket. The increased active site accessibility of Mb-RA and Mb-H facilitates the binding and electron transfer of phenolic substrates in peroxidase-type oxidations catalyzed by the reconstituted proteins in the presence of hydrogen peroxide.     

Heme orientational disorder in human adult hemoglobin reconstituted with a ring fluorinated heme and its functional consequences

A ring fluorinated heme, 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2-fluoro-7,12,18-trimethyl-porphyrinatoiron(III), has been incorporated into human adult hemoglobin (Hb A). The heme orientational disorder in the individual subunits of the protein has been readily characterized using (19)F NMR and the O(2) binding properties of the protein have been evaluated through the oxygen equilibrium analysis. The equilibrated orientations of hemes in alpha- and beta- subunits of the reconstituted protein were found to be almost completely opposite to each other, and hence were largely different from those of the native and the previously reported reconstituted proteins [T. Jue, G.N. La Mar, Heme orientational heterogeneity in deuterohemin-reconstituted horse and human hemoglobin characterized by proton nuclear magnetic resonance spectroscopy, Biochem. Biophys. Res. Commun. 119 (1984) 640-645]. Despite the large difference in the degree of the heme orientational disorder in the subunits of the proteins, the O(2) affinity and the cooperativity of the protein reconstituted with 2-MF were similar to those of the proteins reconstituted with a series of hemes chemically modified at the heme 3- and 8-positions [K. Kawabe, K. Imaizumi, Z. Yoshida, K. Imai, I. Tyuma, Studies on reconstituted myoglobins and hemoglobins II. Role of the heme side chains in the oxygenation of hemoglobin, J. Biochem. 92 (1982) 1713-1722], whose O(2) affinity and cooperativity were higher and lower, respectively, relative to those of native protein. These results indicated that the heme orientational disorder could exert little effect, if any, on the O(2) affinity properties of Hb A. This finding provides new insights into structure-function relationship of Hb A.     

Electron spin resonance study on peroxidase- and oxidase-reactions of horse radish peroxidase and methemoglobin.

The intermediate free radicals generated from phenols, naphthols and benzoate, in the peroxidase- and oxidase-reactions of horse radish peroxidase and in the peroxidase-reaction of methemoglobin, were studied by electron spin resonance spectroscopy.The difference between the peroxidase- and oxidase-reactions of HRP are demonstrated, i.e., the ferro horse radish peroxidase-O₂ system attacks both phenols and benzoate yielding unidentified radicals, which may be hydroxy-cyclohexadienyl radicals, while the horse radish peroxidase-H₂O₂ system reacts only with phenols and naphthols producing the phenoxy-and naphthoxy-radicals.Phenoxy-radical formation from phenols, in the reactions horse radish peroxidase-H₂O₂ and methemoglobin-H₂O₂, occurs independently of the molecular sizes of phenols but dependently on their redox-potentials.On the basis of kinetic studies on methemoglobin-H₂O₂ system, the existence of a reactive intermediate complex between methemoglobin and H₂O₂ is proposed, which may be similar to compound-I or -II of horse radish peroxidase and which further degenerates to MetHb radical. The oxidation of phenols and naphthols takes place outside of the hemepocket of methemoglobin.     

Activity, stability, and unfolding of reconstituted horseradish peroxidase with modified heme

Heme-propionates of horseradish peroxidase (HRP) were esterified by p-nitrophenol, phenol and p-methylphenol to change its electron character and to increase its hydrophobicity. These synthetic hemes were inserted apo-HRP to give a novel HRP, respectively. Of the three reconstituted HRPs, reconstituted HRP with p-nitrophenol-modified heme derivative had a larger initial rate, affinity, catalytic efficiency and substrate-binding efficiency than native HRP in aqueous buffer and some solvents. The reconstituted HRPs showed higher thermostability and tolerance of DMF because of the increase of the hydrophobicity of the active site. Changing the electron character of the aromatic moieties linked at each terminal of the two heme-propionates can control activity and stability of HRP. The initial rate, affinity, catalytic efficiency and substrate-binding efficiency increased with the increases of electron-withdrawing efficiency of substituents at 4-position of the phenolic used to synthesize the heme derivatives, contrariwise, the stability decreased. The modifications resulted in the increase in the temperature (T_m) at the midpoint of thermal denaturation and the decreases in both enthalpy and entropy change at T_m. The changes of catalytic properties and stabilities are related to the changes of the conformation of HRP. The modification changed the environment of heme and tryptophan, increased α-helix content of HRP. The present work demonstrates that enhancement of the hydrophobicity and the electron-withdrawing efficiency of heme improves the activity and stability of HRP.     

Influence of dinitroglycerol and nitroethyleneglycol lipid derivatives on spectral parameters of human hemoglobin

The interaction of organic nitrates (nitroethyleneglycol, dinitroglycerol, and their esters with arachidonic acid) with oxyhemoglobin and methemoglobin has been studied. Addition of nitroethyleneglycol and dinitroglycerol to oxyhemoglobin is accompanied by a modest but significant increase in oxidation rate of the heme protein to the high-spin ferri-form--methemoglobin. Arachidonoylglycerol dinitrate exerts a similar but more pronounced effect on hemoglobin: a molar excess of this dinitrate induces the transformation of a significant portion of oxyhemoglobin to methemoglobin, whereas arachidonoylnitroethyleneglycol is inactive. Arachidonoylglycerol dinitrate also induces changes in the spectral characteristics of methemoglobin; this may be due to disintegration of the methemoglobin with the loss of heme. The data demonstrate that some organic nitrates can interact with hemoglobin; this should be taken into account when using the oxyhemoglobin technique for measuring nitric oxide generation from these compounds.