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.     

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.     

The structure of hemoglobin reconstituted with deuteroheme

The structure of horse methemoglobin reconstituted with deuteroheme, in which the heme vinyl substituents are replaced by hydrogens, has been compared with that of native horse methemoglobin by X-ray difference Fourier techniques. The tertiary structures of the two molecules are almost completely identical, with the exception of small perturbations in the globin which occur in direct response to the missing vinyls. These perturbations are, however, highly localized and do not propagate beyond the immediate vicinity of the hemes. Contrary to expectation, complete removal of these heme vinyls results in much less drastic structural changes than does replacement by ethyl substituents, as in methemoglobin reconstituted with mesoheme.     

Interactions of solvent with the heme region of methemoglobin and fluoro-methemoglobin.

It is now more than 20 years since Davidson and collaborators (1957, Biochim. Biophys, Acta. 26:370-373; J. Mol. Biol. 1:190-191) applied the theoretical ideas of Bloembergen et al. (1948. Phys. Rev. 73:679-712) on outer sphere magnetic relaxation of solvent protons to studies of solutions of methemoglobin. From then on, there has been debate regarding the relative contributions to paramagnetic solvent proton relaxation by inner sphere (ligand-exchange) effects and by outer sphere (diffusional) effects in methemoglobin solutions. Gupta and Mildvan (1975. J. Biol. Chem 250:146-253) extended the early measurements, attributed the relatively small paramagnetic effects to exchange with solvent of the water ligand of the heme-Fe3+ ion, and interpreted their data to indicate cooperativity and an alkaline Bohr effect in the presence of inositol hexaphosphate. They neglected the earlier discussions entirely, and made no reference to outer sphere effects. We have measured the relaxation rate of solvent protons as a function of magnetic field for solutions of methemoglobin, under a variety of conditions of pH and temperature, and have given careful consideration to the relatively large diamagnetic corrections that are necessary by making analogous measurements on oxyhemoglobin, carbonmonoxyhemoglobin, and cyano- and azide-methemoglobin. (The latter two, because of their short electronic relaxation times, behave as though diamagnetic). We show that the paramagnetic contribution to solvent relaxation can be dominated by outer sphere effects, a result implying that many conclusions, including those of Gupta and Mildvan, require reexamination. Finally, we present data for fluoro-methemoglobin, which relaxes solvent protons an order of magnitude better than does methemoglobin. Here one has a startling breakdown of the dogma that has been the basis for interpreting many ligand-replacement studies; in contrast to the prevailing view that replacement of a water ligand of a protein-bound paramagnetic ion by another ligand should decrease relaxation rates, replacement of H2O by F- increases the relaxation rate drastically. The data can all be reconciled, however, with what is anticipated from knowledge of ligand interactions in the heme region.     

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.     

A proposed mechanism for the catalatic action of catalase

A detailed mechanism for catalatic action has been proposed which includes the formation of Chance's catalase compound I in the first step and hydride ion transfer in the second step. The first (oxidative) step involves direct reaction of hematin iron with an ionized H2O2 molecule, followed by an oxidation of the iron to Fe IV. The second step is assumed to depend upon the reductive action of a second H2O2 molecule on Chance's compound I through a catalyzed hybride ion transfer, resulting in the regeneration of uncomplexed catalase. Differences between the catalatic and peroxidative actions of catalase are discussed briefly in respect to the proposed mechanism for catalatic action. The rationale of the proposed mechanism is based to a considerable extent upon the type of ligand binding by the hematin iron of catalase, and this type of ligand bonding is contrasted with ligand binding in methemoglobin, which does not show catalatic activity. Finally, the dispositions of electrons in the outer electronic orbitals of the hematin iron of catalase and methemoglobin are discussed, as a means of justifying formulae presented for catalase and methemoglobin and their derivatives. One of the features of the proposed catalatic mechanism is the assumption, based on electron spin number, that the sixth coordination position around the hematin iron of uncomplexed catalase is unoccupied.     

Relaxation amplitude analysis of thiocyanate and formate binding to human aquomethemoglobin A

The kinetics of the reaction of thiocyanate and formate ions with aquomethemoglobin can be adequately accounted for by a scheme in which the ligand-binding step in both the alpha and beta subunits is preceded by a fast transition of the iron atom from high to low spin (Okonjo, K.O. (1980) Eur. J. Biochem. 105, 329-334). Amplitude expressions derived from this scheme are used to analyse the relaxation amplitude data for alpha and beta subunits within the methemoglobin tetramer. The mean of the reaction enthalpies for ligand binding by the subunits within the tetramer is in good agreement with the reaction enthalpy for ligand binding by the methemoglobin tetramer obtained from a Van't Hoff plot of equilibrium titration data.     

Investigations of cyanide as an infrared probe of hemeprotein ligand binding sites

The measurement of infrared spectra for cyanide liganded to hemeproteins and hemins has been investigated. The hemeproteins included human methemoglobin A, lamprey methemoglobin, metchlorocruorin, horse metmyoglobin, and horseradish peroxidase. The hemins were dicyanide and monopyridine monocyanide species of deuteroporphyrin IX iron(III) and its 2,4-divinyl(proto) and 2,4-diacetyl derivatives. C-N stretch bands of low intensity detected near 2100 cm-1 exhibit changes in frequency, width, intensity, and isotope shift with changes in cyanide compound structure. Infrared band parameters are particularly sensitive to a change in oxidation state (Fe2+ versus Fe3+) and are affected to a lesser extent by changes in porphyrin ring substituent, ligand trans to the cyanide, and protein structure. Evidence of multiple conformers (i.e. multiple C-N stretch bands) was found for several hemeproteins. The cyanide infrared spectra provide direct evidence for cyanide binding as a metal cyanide (Fe--C identical to N) and against HCN being the ligand in nitrile-like bonding (Fe--N identical to C--H) in all the hemeprotein and hemin cyanides studied. With the reduced horseradish peroxidase cyanide, differences between infrared spectra for D2O and H2O solutions can result from hydrogen bonding between a protein amino acid residue and the distal atom of the cyanide (Fe--C identical to N...H+--R). The binding of cyanide to reduced iron (Fe2+) of a hemeprotein was only observed in the case of the reduced peroxidase. These findings demonstrate that cyanide infrared spectra can not only determine when cyanide is bound to a metalloprotein but can also provide information on how the cyanide is bonded to metal and on characteristics of the ligand binding site.     

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.     

Effective immobilization of subunit protein in mesoporous silica modified with ethanol

Ethoxylated FSM-type mesoporous silica (folded-sheet mesoporous material) with a pore diameter of 6.2 nm (FSM6.2) remarkably enhances rigidly of the structure in aqueous solutions. The esterified material could be used successfully as an adsorbent to accommodate subunit protein, methemoglobin (Fe(3+)). Furthermore, methemoglobin (Fe(3+)) in the pores of ethoxy-FSM is maintained a peroxidase activity similar to the native, indicating methemoglobin retains its fore subunit structure in the pores of FSM6.2.     

Analysis of cooperativity in hemoglobin. Valency hybrids, oxidation, and methemoglobin replacement reactions.

An allosteric model proposed previously for structure-function relations in hemoglobin is applied to the analysis of low- and high-spin valency hybrids. By assuming that the low-spin oxidized chains have the tertiary structure of oxygenated chains while the high-spin oxidized chains have a tertiary structure intermediate between that of deoxygenated and oxygenated chains, the model parameters associated with the different valency hybrids can be obtained, and their equilibrium properties can be estimated. The hybrid results are used also to provide an interpretation of methemoglobin and its ligand replacement reactions and of the oxidation-reduction equilibrium of normal hemoglobin. For the various systems studied it is found that the effects of pH and 2,3-diphosphoglycerate are in agreement with the model.     

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.     

The uptake of protons by heme-linked ionizable groups on azide binding to methemoglobin

When azide ion reacts with methemoglobin in unbuffered solution the pH of the solution increases. This phenomenon is associated with increases in the pK values of heme-linked ionizable groups on the protein which give rise to an uptake of protons from solution. We have determined as a functional of pH the proton uptake, delta h+, on azide binding to methemoglobin at 20 degrees C. Data for methemoglobins A (human), guinea pig and pigeon are fitted to a theoretical expression based on the electrostatic effect of these sets of heme-linked ionizable groups on the binding of the ligand. From these fits the pK values of heme-linked ionizable groups are obtained for liganded and unliganded methemoglobins. In unliganded methemoglobin pK1, which is associated with carboxylic acid groups, ranges between 4.0 and 5.5 for the three methemoglobins; pK2, which is associated with histidines and terminal amino groups, ranges from 6.2 to 6.7. In liganded methemoglobin pK1 lies between 5.8 and 6.3 and pK2 varies from 8.1 to 8.5. The pH dependences of the apparent equilibrium constants for azide binding to the three methemoglobins at 20 degrees C are well accounted for with the pK values calculated from the variation of delta h+ with pH.     

Temperature-jump studies on formate binding to methemoglobin and metmyoglobin.

The binding of formate ion to sperm whale metmyoglobin after a temperature-jump is monophasic and not affected by organic phosphate; the Hill coefficient obtained from equilibrium measurements is unity, and there is internal consistency between equilibrium and kinetic results. Formate binding to stripped human methemoglobin, on the other hand, is biphasic. The two relaxation phases can be attributed, on the basis of their equal relaxation amplitudes, to the different kinetic properties of both types of chains. Equilibrium measurements yield a single binding constant. Thus, formate belongs to the class of high-spin ligands which show no binding specificity but strong kinetic heterogeneity for α- and β-chains. There is, however, a lack of consistency between equilibrium and kinetic results, indicating that a reaction scheme which considers only ligand binding to α- and β-chains appears not to be fully adequate. Organic phosphates exert a drastic influence on the kinetics but not on the thermodynamics of ligand binding. In the presence of inositol hexaphosphate the relaxation spectrum is characterized by more than two relaxation processes: A very fast phase—about an order of magnitude faster than the fast process in stripped methemoglobin—appears with high amplitude. The slow relaxation process, however, is only slightly affected. The binding constant of formate obtained from equilibrium measurements is only little changed and the Hill coefficient is 0.97 both in the presence and absence of the phosphate. The phosphate-induced kinetic changes indicate that functionally significant structural changes are introduced in the tertiary structure of one type of chains, presumably the β-chains, to which inositol hexaphosphate is bound.     

Mechanisms of induced processes in aqueous hemoglobin solutions at 77 K

ESR spectra of gamma-irradiated and frozen at 77 K human oxyhemoglobin and partially denaturated methemoglobin solutions were analysed. The quartet signal ascribed to the anion-radical of proximal histidine was shown to dominate in the spectra of both solutions. The spectra of methemoglobin solution irradiated with relatively small doses have an intensive singlet ascribed to the stabilized electron. The formation mechanism of free radicals is discussed.     

The effect of methemoglobin and ferricytochrome c on the lipid bilayer structure

The effect of ferricytochrome c and methemoglobin on the model phospholipid membrane structure has been investigated using fluorescent probes: 1-anilinonaphthalene-8-sulphonate, 4-dimethylaminochalcone and 3-methoxybenzanthrone. Ferricytochrome c and methemoglobin is found to cause the disordering of the phospholipid bilayer surface region.     

Subunit iron spin heterogeneity in human aquomethemoglobin A

On the basis of a reaction scheme in which the ligand binding steps are preceded by fast iron spin transitions (Okonjo, K.O. (1980) Eur. J. Biochem. 105, 329-334; Iwuoha, E.I. and Okonjo, K.O. (1985) Biochim. Biophys. Acta 829, 327-334), the spin equilibrium constants of methemoglobin subunits are calculated from kinetic and equilibrium binding parameters with azide ion as ligand. The results demonstrate the existence of thermodynamic spin heterogeneity within the tetramer.     

Structural states and transitions of carp hemoglobin.

The wide ligand affinity range previously observed for carp hemoglobin is bounded at both extremes by regions of constant affinity. Within these regions, pH, organic phosphates, and the extent of ligand binding have no effect on the measured affinity and the cooperativity of ligand binding is greatly reduced or absent. The rates of CO recombination to fully and partially unliganded carp hemoglobin, under various organic phosphate and pH conditions, are shown to reflect this behavior. Constant kinetic rates are seen to directly correspond to the regions of constant affinity. Therefore, these are taken to be single protein conformations, one of high and one of low ligand affinity. In the simplest view, these conformations represent the R and T states of a two-state model, and most of the properties of carp hemoglobin are explained quite well within this framework. Increases in either hydrogen or phosphate ion concentrations favor the stabilization of the low affinity structure of even fully liganded carp hemoglobin. We have studied the structural transition from high to low affinity by monitoring the absorption spectra of carp hemoglobins at constant pH as a function of organic phosphate concentration. We find that different spectra are induced in both carp methemoglobin and cyanomethemoglobin by inositol hexaphosphate addition. Furthermore, the dependence of the magnitude of the spectral changes on pH and organic phosphate concentration is the close agreement with that predicted from studies of the ligand binding properties of the molecule.     

Quaternary structure has little influence on spin states in mixed-spin human methemoglobins

A key feature of the Perutz stereochemical model for cooperativity in hemoglobin is a strong coupling between quaternary structure and the spin state of the heme iron [Perutz, M. F. (1979) Annu. Rev. Biochem. 48, 327-386]. While this coupling appears to be present for carp azide methemoglobin, it should also be present for all liganded forms of human methemoglobin that exhibit a thermal high-spin in equilibrium low-spin equilibrium. To test this hypothesis, we have measured the changes in spin equilibria upon conversion of six mixed-spin forms of human methemoglobin from the R (high-affinity) to the T (low-affinity) quaternary structure by addition of inositol hexaphosphate. These experiments were done with a sensitive superconducting magnetic susceptibility instrument on solutions at 20 degrees C in 20 mM maleate buffer, pH 6. The data show zero or small increases in high-spin content upon switching from R to T, changes that are equivalent to a relative stabilization of the high-spin form by only 0-300 cal mol-1 heme-1. These changes in energy are far less than the 1200 cal mol-1 heme-1 predicted from the Perutz stereochemical model [Cho, K. C., & Hopfield, J. J. (1979) Biochemistry 18, 5826-5833]. That is, these data do not support a view that the low affinity of the T state is due to restraints acting through the iron-proximal histidine linkage. The mechanistic implications of these results and the differences between species and ferric ligands are discussed.     

Cooperative ligand binding by ferrihemoglobin: An experimental artifact?

We have carried out a detailed study of ligand binding of ferrihemoglobins under various conditions. Our results show that n varies with time and that this variation is paralleled by changes in the spectrum of methemoglobin. This suggests some form of structural perturbation. The time-dependent value of n is discussed in terms of the observed spectral changes accompanying prolonged equilibration.