The Soret magnetic circular dichroism of ferric high-spin myoglobins. A probe for the distal histidine residue.

To find a simple criterion for the presence of the distal (E7) histidine residue in myoglobins and hemoglobins, the Soret magnetic-circular-dichroic spectra were examined for ferric metmyoglobins from various species. A distinct and symmetric dispersion-type curve was obtained for myoglobins containing the distal histidine, whereas a relatively weak and unsymmetric pattern was observed for myoglobins lacking this residue, such as those from three kinds of gastropodic sea molluscs, a shark and the African elephant. The magnetic-circular-dichroic spectra obtained would thus be a direct reflection of the presence or absence of a water molecule at the sixth coordinate position of the heme iron(III), this axial water ligand being stabilized by hydrogen-bond formation to the distal histidine residue. On the basis of these Soret magnetic-circular-dichroic signals, we also examined the structure of a protozoan myoglobin (or a monomeric hemoglobin) from Paramecium caudatum of particular interest for the evolution of these proteins from protozoa to higher animals.     

Modification of the heme distal side in myoglobin by cyanogen bromide. Heme environmental structures and ligand binding properties of the modified myoglobin

Met, deoxy, and CO forms of myoglobin (Mb) react with a stoichiometric amount of cyanogen bromide (BrCN) to cause substantial changes in the 1H NMR, optical absorption, and infrared spectra. These spectral changes were interpreted as arising from the substantial alterations in the heme environments, most probably due to the modification of the histidine residue at the heme distal side. It is also revealed that the modified Mb does not combine with some exogenous ligands such as CN-, CH3NH2, and O2, although it does with N-3 or CO. These unique ligand binding properties are also discussed with relevance to a role of the distal histidine in stabilizing the coordinated ligand through a hydrogen bond and to a steric constraint.     

Effect of the distal histidine modification (Cyanation) of myoglobin on the ligand binding kinetics and the heme environmental structures

The kinetics of carbon monoxide (CO) binding to myoglobin (Mb) modified at the distal histidine (His) by cyanogen bromide (BrCN) has been studied. The CO association and dissociation rates of BrCN-modified Mb were obtained as 1.8 x 10(3) M-1 s-1 and 0.13 s-1, respectively (20 degrees C and pH 7.0). Thermodynamic parameters were obtained as well. These values are notable, compared with those for other hemoproteins, the slowest association and the fastest dissociation rates among various hemoproteins examined so far. On the basis of the available structural data obtained from the absorption, 1H NMR, and IR spectral measurements, these unique kinetic and thermodynamic properties were reasonably explained in terms of the steric restriction at the modified distal side.     

Coordination structure of the ferric heme iron in engineered distal histidine myoglobin mutants.

Recombinant human myoglobin mutants with the distal His residue (E7, His64) replaced by Leu, Val, or Gln residues were prepared by site-directed mutagenesis and expression in Escherichia coli. Electronic and coordination structures of the ferric heme iron in the recombinant myoglobin proteins were examined by optical absorption, EPR, 1H NMR, magnetic circular dichroism, and x-ray spectroscopy. Mutations, His-->Val and His-->Leu, remove the heme-bound water molecule resulting in a five-coordinate heme iron at neutral pH, while the heme-bound water molecule appears to be retained in the engineered myoglobin with His-->Gln substitution as in the wild-type protein. The distal Val and distal Leu ferric myoglobin mutants at neutral pH exhibited EPR spectra with g perpendicular values smaller than 6, which could be interpreted as an admixture of intermediate (S = 3/2) and high (S = 5/2) spin states. At alkaline pH, the distal Gln mutant is in the same so-called "hydroxy low spin" form as the wild-type protein, while the distal Leu and distal Val mutants are in high spin states. The ligand binding properties of these recombinant myoglobin proteins were studied by measurements of azide equilibrium and cyanide binding. The distal Leu and distal Val mutants exhibited diminished azide affinity and extremely slow cyanide binding, while the distal Gln mutant showed azide affinity and cyanide association rate constants similar to those of the wild-type protein.     

Novel circular dichroism spectroscopic approach for detection of ligand binding of proteins: Avidin as example

Various globular proteins having low or no α-helical content exhibit atypical far-ultraviolet (UV) circular dichroism (CD) spectra due to aromatic-aromatic residue and aromatic residue-peptide bond exciton coupling interactions. As a representative example of such proteins, far-UV CD spectra of chicken avidin were recorded before and after the addition of different small molecules. Intensity increase-decrease and/or wavelength shift of the positive CD peak of avidin at 228 nm were observed in the presence of various drugs, dyes, and natural compounds. The results were interpreted by exciton interactions between the aromatic residues of the biotin binding site and the substances bound to it. This novel, fast, microgram (μg) scale approach can be applied for detection of ligand binding of additional proteins displaying avidin-like far-UV CD spectra.     

Magnetic circular dichroism spectroscopy as a probe of axial heme ligand replacement in semisynthetic mutants of cytochrome c.

Horse heart cytochrome c with either histidine or cysteine replacing the endogenous axial methionine ligand at position 80 has been characterized with magnetic circular dichroism (MCD) spectroscopy in the UV-visible region. Comparison of the MCD spectra of the mutant proteins in the ferric state to those of authentic bis-imidazole- and imidazole/thiolate-ligated ferric heme proteins clearly shows that the histidine-imidazole and cysteine-thiolate groups of the replacement amino acids at position 80 are coordinated to the heme iron in the mutant proteins. This study demonstrates the power of MCD spectroscopy in identifying axial ligands in mutant heme proteins. Accurate axial ligand assignment is essential for proper interpretation of the altered properties of such novel proteins.     

Assignment of the heme axial ligand(s) for the ferric myoglobin (H93G) and heme oxygenase (H25A) cavity mutants as oxygen donors using magnetic circular dichroism.

UV-visible absorption and magnetic circular dichroism (MCD) data are reported for the cavity mutants of sperm whale H93G myoglobin and human H25A heme oxygenase in their ferric states at 4 degreesC. Detailed spectral analyses of H93G myoglobin reveal that its heme coordination structure has a single water ligand at pH 5.0, a single hydroxide ligand at pH 10.0, and a mixture of species at pH 7.0 including five-coordinate hydroxide-bound, and six-coordinate structures. The five-coordinate aquo structure at pH 5 is supported by spectral similarity to acidic horseradish peroxidase (pH 3.1), whose MCD data are reported herein for the first time, and acidic myoglobin (pH 3.4), whose structures have been previously assigned by resonance Raman spectroscopy. The five-coordinate hydroxide structure at pH 10.0 is supported by MCD and resonance Raman data obtained here and by comparison with those of other known five-coordinate oxygen donor complexes. In particular, the MCD spectrum of alkaline ferric H93G myoglobin is strikingly similar to that of ferric tyrosinate-ligated human H93Y myoglobin, whose MCD data are reported herein for the first time, and that of the methoxide adduct of ferric protoporphyrin IX dimethyl ester (FeIIIPPIXDME). Analysis of the spectral data for ferric H25A heme oxygenase at neutral pH in the context of the spectra of other five-coordinate ferric heme complexes with proximal oxygen donor ligands, in particular the p-nitrophenolate and acetate adducts of FeIIIPPIXDME, is most consistent with ligation by a carboxylate group of a nearby glutamyl (or aspartic) acid residue.     

H93G myoglobin cavity mutant as versatile template for modeling heme proteins: magnetic circular dichroism studies of thiolate- and imidazole-ligated complexes

Recent ligand binding and spectroscopic investigations of the myoglobin H93G cavity mutant are reviewed, revealing it to be a versatile template for the preparation of model heme complexes of defined structure. The H93G myoglobin cavity mutant is shown to be capable of forming mixed ligand adducts because of the difference in accessibility of the two sides of the ferric heme iron. With imidazole bound in the proximal cavity, H93G myoglobin also forms reasonably stable oxyferrous and oxoferryl derivatives, thereby providing a potential system to use for the study of such complexes with proximal ligands other than imidazole. In addition, thiolate-ligated ferric H93G derivatives are described that serve as spectroscopic models for the high-spin ferric state of cytochrome P450. All of the complexes described are characterized with magnetic circular dichroism spectroscopy, and they are compared to the appropriate derivatives of native myoglobin and P450.     

Aromatic side-chain cluster of biotin binding site of avidin allows circular dichroism spectroscopic investigation of its ligand binding properties

Promiscuous ligand binding by hen egg-white avidin has been demonstrated and studied by using circular dichroism (CD) spectroscopy complemented by molecular docking calculations. It has been shown that the biotin-binding pocket of avidin is able to accommodate a wide variety of chemical compounds including therapeutic drugs (e.g., thalidomide, NSAIDs, antihistamines), natural compounds (bilirubin, myristic acid), and synthetic agents (xanthenone dyes). The cluster of aromatic residues located at the biotin-binding pocket renders the intrinsic CD spectrum of avidin sensitive to ligand binding that results in the increase of the vibronic components of the (1) L(b) transition of the Trp residues. Extrinsic (induced) CD bands measured with chemically diverse avidin ligands are generated by intramolecular coupled oscillator (e.g., bilirubin) or by intermolecular ligand-Trp exciton coupling mechanism [e.g., 2-(4'-hydroxyazobenzene)-benzoic acid (HABA)]. Among the compounds of which avidin-binding affinity constants have been calculated, two novel high-affinity ligands, flufenamic acid and an enzyme inhibitor thiazole derivative have been identified (K(d) ≈ 1 μM). Avidin binding mode of the ligand molecules has been discussed in the light of docking results. The induced CD profile of the thiazole derivative has been correlated with the stereochemistry of its docked conformation. The important role in the ligand binding of a polar side-chain cluster at the bottom of the biotin-binding cavity as well as the analogous avidin-binding mode of HABA and fenamic acid type NSAIDs have been proposed.     

Effects of formylation of vinyl side chains of heme on optical and ligand binding properties of horse heart ferric myoglobin.

Effects of substitution of vinyl groups of hemin with formyl groups on the optical and ligand binding properties of horse heart ferric myoglobin were investigated. The peak positions as well as the line shapes of the absorption spectra of the ferric derivatives of three kinds of formylmyoglobin, 2-vinyl-4-formyl-, 2-formyl-4-vinyl-, and 2,4-diformylmyoglobins depend on the number and the position of the formyl groups. Absorption maxima in the Soret region of the acid forms of these ferric formylmyoglobins in 0.1 M potassium phosphate buffer, pH 6.0, at 20 degrees were 415.2, 422, and 429 nm, respectively. The acid forms of these formylmyoglobins exhibit absorption spectra of the mixture of high- and low spin states at ambient temperature. Since proto-, deutero- and mesomyoglobins have a high spin state under the same condition, the increase of the low spin iron in these formylmyoglobins may be due to the strong electron withdrawal by the formyl groups toward the periphery of the porphyrin ring. The affinities of these ferric formylmyoglobins and protomyoglobin for N3-, F-, OCN-, and SCN- increased in the order of proto-, monoformyl-monovinyl-, 2,4-diformyl-myoglobin, which corresponds to the increasing order of electron-withdrawing power of the porphyrin side chains. The pKa values of the acid-alkaline transition decreased in the same order. Although the ferric forms of the two isomeric monoformyl-monovinylmyoglobins exhibited different optical spectra, the dissociation constants of the complexes of these isomers for various ligands were similar to each other. The pKa values of the acid-alkaline transition were also similar. These results indicate that affinities of ferric myoglobin for ligands, in contrast to those of the ferrous form for oxygen and carbon monoxide (Sono, M., and Asakura, T. (1975) J. Biol. Chem. 250, 5527-5232 and Sono, M., Smith, P.D., McCray, J.A., and Asakura, T. (1976) J. Biol. Chem 251, 1418-1426), are not affected by the position of modifications at the two vinyl groups, but are determinedby the number of the formyl groups and that two vinyl groups at position 2 and 4 are equivalent in the binding of various ligands by ferric myoglobin. The electron density of the ferric iron appears to be similar for the two isomeric monoformyl-monovinylmyoglobins.     

Low temperature magnetic circular dichroism spectroscopy as a probe for the optical transitions of paramagnetic nickel in hydrogenase

A partially-purified sample of hydrogenase from Methanobacterium thermoautotrophicum (delta H strain) has been investigated by optical absorption, magnetic circular dichroism and electron paramagnetic resonance spectroscopy. Variable temperature magnetic circular dichroism studies reveal, for the first time, the optical transitions associated with the Ni(III) center in the oxidized enzyme. Low temperature magnetic circular dichroism spectroscopy provides a new method of assessing both the coordination environment of Ni in hydrogenase and the appropriateness of inorganic model complexes.     

Studies on the charge transfer band in high spin state of ferric myoglobin and hemoglobin by low temperature optical and magnetic circular dichroism spectroscopy.

The behavior of charge transfer band, appearing at 600-650 nm in ferric high spin derivatives of myoglobin and hemoglobin, was studied under various conditions by low temperature optical and magnetic circular dichroism spectroscopy. Optical absorption spectra have demonstrated that: (1) The charge transfer band at 630 nm of myoglobin (Fe3+)-H2O (pH 7.0) at room temperature split into three bands, 627 nm, 645 nm and 664 nm (shoulder) at 77 degrees K, whereas that of hemoglobin (Fe3+)-H2O showed no splitting. (2) By lowering the pH value from 7.5 to 4.3 this splitting in myoglobin was observed to disappear only in the presence of a small amount of phosphate ion, accompanying a midpoint at pH 6.7 +/- 0.1. This does not originate from the released hemin. (3) Hemin (pH 7.55) showed no splitting of the charge transfer band at 77 degrees K. (4) This splitting depended on the species of 6th ligand. For myoglobin-F- the splitting could scarcely be observed, whereas the proton-donating ligands such as HCOOH and CH3OH exhibit the splitting as well as H2O. Magnetic circular dichroism spectra have demonstrated that: (5) The charge transfer band at 600-500 nm indicated Faraday A term and B term. (6) A negative B term band was observed at 650 nm for myoglobin-H2O in the glassic solvent of potassium glycerophosphate-glycerol, whereas it was not observed for hemoglobin-H2O. Several discussions were performed on the origin of splitting of the charge transfer band in myoglobin-H2O. It is now concluded that the hydrogen bond between the 6th ligand and the distal histidine contributes to the splitting of the charge transfer band around 630 nm for myoglobin Fe3+)-H2O at low temperature and that disappearance of the splitting at low pH is originated from the presence of phosphate ion.     

Characterization of the coral allene oxide synthase active site with UV-visible absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopy: evidence for tyrosinate ligation to the ferric enzyme heme iron

Coral allene oxide synthase (AOS), a hemoprotein with weak sequence homology to catalase, is the N-terminal domain of a naturally occurring fusion protein with an 8R-lipoxygenase. AOS converts 8R-hydroperoxyeicosatetraenoic acid to the corresponding allene oxide. The UV--visible absorption and magnetic circular dichroism spectra of ferric AOS and of its cyanide and azide complexes, and the electron paramagnetic resonance spectra of native AOS (high-spin, g = 6.56, 5.22, 2.00) and of its cyanide adduct (low-spin, g = 2.86, 2.24, 1.60) closely resemble the corresponding spectra of bovine liver catalase (BLC). These results provide strong evidence for tyrosinate ligation to the heme iron of AOS as has been established for catalases. On the other hand, the positive circular dichroism bands in the Soret region for all three derivatives of ferric AOS are almost the mirror image of those in catalase. In addition, the cyanide affinity of native AOS (K(d) = 10 mM at pH 7) is about 3 orders of magnitude lower than that of BLC. Thus, while these results conclusively support a common tyrosinate-ligated heme in AOS as in catalase, significant differences exist in the interaction between their respective heme prosthetic groups and protein environments, and in the access of small molecules to the heme iron.     

On the use of iron octa-alkylporphyrins as models for protoporphyrin IX-containing heme systems in studies employing magnetic circular dichroism spectroscopy.

The magnetic circular dichroism (MCD) properties of numerous oxidation and ligation state derivatives of myoglobin and horseradish peroxidase reconstituted with an iron octa-alkylporphyrin (mesoheme IX) have been investigated in order to establish the utility of such porphyrins as models for protoporphyrin IX-containing systems. The MCD spectra of the mesoheme-reconstituted proteins are blue-shifted (4-12 nm) and are somewhat more intense (1.5-2.5 fold) when compared to the spectra of analogous derivatives of native myoglobin and horseradish peroxidase. However, the spectral band patterns of the mesoheme-reconstituted proteins closely resemble those of the native proteins in essentially all cases. These data demonstrate that octa-alkylporphyrins can be productively used as models for protoporphyrin IX in studies of heme proteins with MCD spectroscopy.     

Modification of the distal histidyl imidazole in myoglobin to N-tetrazole-substituted imidazole and its effects on the heme environmental structure and ligand binding properties.

The mechanism of N-tetrazole ring formation at the distal histidyl imidazole of sperm whale myoglobin (Mb) has been studied by nitrogen-15 (15N) NMR spectroscopy by utilizing 15N-labeled cyanogen bromide (BrCN) and azide ion (N3-). The 15N-NMR spectrum of BrC15N-modified Mb + N3- afforded two hyperfine-shifted 15N resonances, both of which are identical with the resonance positions of two of the three 15N resonances for BrCN-modified Mb + 15NN2-. This unusual spectral feature is due to the formation of the N-tetrazole ring attached to the distal histidyl imidazole and the scrambling of the labeled nitrogen originated from N3- or BrCN over the tetrazole ring upon coordination to the ferric heme iron. The ferric iron-bound N-tetrazole ring comes off upon reduction to the ferrous state, and the stable CO complex of tetrazole-modified Mb (tetrazole-Mb) is formed. Electronic absorption and 1H-NMR spectra of deoxy and carbonmonoxy forms of tetrazole-Mb are slightly altered from those of native Mb by the modification, while the most significant effect is exerted on the C-O stretching frequency of iron-bound CO. The C-O stretching band for tetrazole-MbCO is observed at 1966 cm-1 in contrast to 1945 cm-1 for native MbCO, suggesting that the geometry of iron-bound CO in tetrazole-Mb is relatively upright which is characteristic of the "open" conformer. This result corresponds to the 15-fold increase of the CO association rate constant by the N-tetrazole modification of the distal His. The oxy form of tetrazole-Mb is readily autoxidized to its ferric state, indicating that hydrogen bonding between the distal His and iron-bound oxygen is essential for stable O2 binding to the heme iron.     

Modulating carbon monoxide binding affinity and kinetics in myoglobin: the roles of the distal histidine and the heme pocket docking site

 Myoglobin has long served as a model system for understanding the relations between protein structure, dynamics, and function. Its ability to discriminate between toxic CO and vital O₂, two small ligands that are almost equivalent in size and dipole moment, has attracted much attention. To understand discrimination and reversible ligand-binding in Mb, both the bound state and the "docked" state that leads to binding need to be studied. We have reported previously the nearly linear Fe–C–O geometry of bound CO and the nearly orthogonal geometry of docked CO [Lim et al. (1995), Science 269 : 962]. With the exception of X-ray structures, a preponderance of evidence points to a nearly linear Fe–C–O geometry and calls into question the proposal that the highly conserved distal histidine forces CO to bind in a nonoptimal geometry. The differences between the bound CO structures determined using IR and X-ray methods might arise from a water molecule hydrogen bonded to the distal histidine in some of the unit cells. Discrimination by Mb is manifested not only thermodynamically but also kinetically. Time-resolved CO rebinding studies that compare Mb with microperoxidase suggest that the heme pocket docking site in Mb exerts steric control of the ligand rebinding rate, slowing the rate of CO binding by a factor of more than 10⁴. Received, accepted: 23 May 1997     

A comparison of the heme electronic states in equilibrium and nonequilibrium protein conformations of high-spin ferrous hemoproteins. Low temperature magnetic circular dichroism studies

The visible and near infrared magnetic circular dichroism (MCD) spectra of equilibrium high-spin ferrous derivatives of myoglobin, hemoglobin, horseradish peroxidase and mitochondrial cytochrome c oxidase at 15 K are compared with those of the corresponding proteins in nonequilibrium conformations produced by low-temperature photodissociation of CO-complexes of these proteins as well as of O2-complexes of myoglobin and hemoglobin. Over all the spectral region (450-800 nm) the intensities of MCD bands of hemoproteins studied in equilibrium conformation are shown to be strongly temperature-dependent, including a negative band at ca. 630 nm and positive bands at ca. 690 nm and at ca. 760 nm. In contrast to the absorption spectra, the low-temperature MCD spectra of high-spin ferrous hemoproteins differ significantly, reflecting the peculiarities in the heme iron coordination sphere which are created by a protein conformation. The MCD spectra reveal clearly the structural changes in the heme environment which occur on ligand binding. On the basis of assignment of d leads to d and charge-transfer transitions in the near infrared region the correlation is suggested between the wavelength position of the MCD band at approx. 690 nm and the value of iron out-of-plane displacement as well as between the location of the band at approx. 760 nm and the Fe-N epsilon (proximal histidine) bond strength (length) in equilibrium and nonequilibrium conformations of the hemoproteins studied. The high sensitivity of low-temperature MCD spectra to geometry at heme iron is discussed.     

Single-crystal EPR study of novel azide complex of cyanogen bromide-modified myoglobin. Influence of specific modification of the heme distal histidyl residue on the ligand-binding structure

Stable azide complex of cyanogen bromide-modified met-myoglobin (metMb) was prepared and crystallized. The principal values and eigen vectors of g-tensor were determined by single-crystal EPR spectroscopy at 77 K: gxx = 1.50, gyy = 2.32, and gzz = 2.91. These g values were similar to those of tetrazole derivative rather than azide derivative of native metMbs, suggesting that tetrazole derivative might be formed from N-cyanoimidazole of distal histidyl residue via nucleophilic attack of azide ion by 1,3-dipolar cycloaddition reaction. The orientation of the maximal g value (gzz) of the novel product was found to deviate about 13 degrees from the heme normal of native aquometMb. Thus, the orientation of the heme plane might be altered in passing from native metMb to cyanogen bromide-mediated metmyoglobin. The present EPR results demonstrated that the modification of the histidyl residue at the heme distal side causes the changes in the stereochemical and electronic natures of the ligand binding to the heme.