Effects of cyanogen bromide modification of the distal histidine on the spectroscopic and ligand binding properties of myoglobin: magnetic circular dichroism spectroscopy as a probe of distal water ligation in ferric high-spin histidine-bound heme proteins

Magnetic circular dichroism (MCD) spectroscopy has been utilized to characterize the change in coordination structure in native ferric sperm whale myoglobin upon cyanogen bromide-modification. Comparison of the MCD properties of the ferric high-spin state of cyanogen bromide-modified myoglobin (BrCN-Mb) with those of native ferric horseradish peroxidase and Aplysia myoglobin suggests that ferric BrCN-Mb is a potential MCD model for the pentacoordinate state of ferric high-spin histidine-ligated heme proteins. These five-coordinate heme proteins afford a relatively weak and unsymmetric signal in the Soret region of the MCD spectrum. In contrast, native ferric myoglobin and the benzohydroxamic acid adduct of ferric horseradish peroxidase show a strong and symmetric derivative-shaped Soret MCD signal which is indicative of hexacoordination with water and histidine axial ligands. Therefore it seems that MCD spectroscopy could be used to probe the presence of water ligated to the distal side of ferric high-spin heme proteins. The MCD spectra of the ferric-azide, ferrous-deoxy and ferrous-CO forms of BrCN-Mb have also been measured and compared to those of analogous native myoglobin complexes. The present MCD study has been extended to include new ligands, NO, thiocyanate and cyanate, which bind to ferric BrCN-Mb. With exogenous ligands such as CO, NO and thiocyanate, the coordination structures of the BrCN-Mb complexes are similar to those of the respective native myoglobin adducts. In the case of ferrous-deoxy and ferric-cyanate BrCN-Mb, however, the altered MCD spectra (and EPR for the latter) reveal changes in electronic structure which likely correlate with alterations of the coordination environment of these BrCN-Mb derivatives. Data are also presented which support the proposed tetrazole-bound structure for azide-treated BrCN-Mb (Hori, H., Fujii, M., Shiro, Y., Iizuka, T., Adachi, S. and Morishima, I. (1989) J. Biol. Chem. 264, 5715-5719) and the inability of the distal histidine of BrCN-Mb to stabilize the ferric ligand-bound state.     

Spectral properties unique to the myoglobins lacking the usual distal histidine residue

Myoglobins can be divided into two groups. One group contains the usual myoglobins that have histidine at the distal (E7) position, and the other contains a few, but interesting myoglobins that lack the usual distal histidine residue. Spectroscopic examinations have shown that there is a remarkable difference in the Soret band between the two types of myoglobin, and an absorbance ratio of the Soret peak of the acidic met-form to that of the oxy-form seems to be very useful as a simple criterion for predicting whether or not a myoglobin has the usual distal histidine residue.     

The distal residue-CO interaction in carbonmonoxy myoglobins: a molecular dynamics study of two distal histidine tautomers.

Four independent 90 ps molecular dynamics simulations of sperm-whale wild-type carbonmonoxy myoglobin (MbCO) have been calculated using a new AMBER force field for the haem prosthetic group. Two trajectories have the distal 64N delta nitrogen protonated, and two have the 64N epsilon nitrogen protonated; all water molecules within 16 A of the carbonyl O are included. In three trajectories, the distal residue remains part of the haem pocket, with the protonated distal nitrogen pointing into the active site. This is in contrast with the neutron diffraction crystal structure, but is consistent with the solution phase CO stretching frequencies (upsilon CO) of MbCO and various of its mutants. There are significant differences in the "closed" pocket structures found for each tautomer: the 64N epsilon H trajectories both show stable distal-CO interactions, whereas the 64N delta H tautomer) has a weaker interaction resulting in a more mobile distal side chain. One trajectory (a 64N delta H tautomer) has the distal histidine moving out into the "solvent", leaving the pocket in an "open" structure, with a large unhindered entrance to the active site. These trajectories suggest that the three upsilon CO frequencies observed for wild-type MbCO in solution, rather than representing significantly different Fe-C-O geometries as such, arise from three different haem pocket structures, each with different electric fields at the ligand. Each pocket structure corresponds to a different distal histidine conformer: the A3 band to the 64N epsilon H tautomer, the A1,2 band to the 64N delta H tautomer, and the A0 band to the absence of any significant interaction with the distal side chain.     

Solvent isotope effects on NMR spectral parameters in high-spin ferric hemoproteins: an indirect probe for distal hydrogen bonding

The influence of solvent isotope composition on 1H-NMR resonance position and linewidth of heme methyls has been investigated for a variety of high-spin ferric hemoproteins for the purpose of detecting hydrogen-bonding interactions in the heme cavity. Consistently larger hyperfine shifts and paramagnetic linewidths in 2H2O than 1H2O are observed for metmyoglobins and methemoglobin possessing a coordinated water molecule. The analysis of the dynamics of labile proton exchange in sperm whale metmyoglobin, and the absence of any isotope effects in the five-coordinate Aplysia metmyoglobin, indicate that the significant axial modulation of heme electronic structure by solvent isotope is consistent with arising from distal hydrogen-bonding interactions. The presence or absence of similarly large isotope effects on shifts and linewidths in other hemoproteins, depending on the presence of a bound water in the distal heme pocket, suggests that this isotope effect can serve as a probe for the presence of such bound water. The absence of any detectable isotope effect on either shifts or linewidths in resting-state horseradish peroxidase supports a five-coordinate structure with bound water absent from the vicinity of the iron.     

Hemoglobin M: effect of the proximal or distal histidine replacement on circular dichroism in the visible region

To examine the effects of a replacement of the proximal or the distal histidine on the structure of hemoglobin (Hb), absorption and circular dichroic (CD) spectra of five species of Hbs M in the visible region were measured. Four Hbs M had a characteristic but a similar absorption spectrum upon amino acid substitution, however, the proximal histidine replaced Hbs M (Hb M Iwate and Hb M Hyde Park) showed considerably different CD spectra from those of the distal histidine replaced ones (Hb M Boston and Hb M Saskatoon). The former exhibited large positive CD but the latter gave a complex CD spectrum with positive and negative extrema. On the other hand, absorption and CD spectra of Hb M Milwaukee did not changed very much from those of Hb A.     

Near-infrared magnetic circular dichroism of cytochrome c'.

The near-infrared magnetic circular dichroism (MCD) of Rhodospirillum rubrum, Chromatium vinosum, and Rhodopseudomonas palustris cytochromes c' are reported. The spectra of the reduced protein are very similar to those of deoxymyoglobin. The spectra of the oxidized proteins in the pD range 1-13 can be analyzed on the basis of four species A, B, C, and D. The existence of nine species, reported in a recent electron paramagnetic resonance study, is not substantiated. The MCD spectra support the assignment of B as high spin and C and D as low spin. The MCD of species A is close to that of high-spin proteins and does not support the recently proposed assignment of a mixed high- and intermediate-spin ground state for this species. The energies of the near-IR electronic transitions of all four oxidized species point to axial ligation via oxygen, assuming histidine to be the opposite axial ligand. Unfortunately, insufficient model compounds with ligation by carboxyl or hydroxyl moieties exist to enable more precise assignments.     

Infrared magnetic circular dichroism of myoglobin derivatives.

By use of a newly constructed CD instrument, infrared magnetic circular dichroism (MCD) spectra were observed for various myoglobin derivatives. The ferric high spin myoglobin derivatives such as fluoride, water and hydroxide complexes, commonly exhibited the MCD spectra consisting of positive A terms. Therefore, the results reinforced the assignment that the infrared band is the charge transfer transition to the degenerate excited state (eg (dpi)). Since the fraction of A term estimated was approximately 80% for myoglobin fluoride and approximately 35% for myoglobin water, the effective symmetry for myoglobin fluoride is determined to be as close as D4h, while that for myoglobin water seems to have lower symmetry components. The ferric low spin derivatives such as myoglobin cyanide, myoglobin imidazole and myoglobin azide showed positive MCD spectra which are very similar to the electronic absorption spectra. These MCD spectra were assigned to the charge transfer transitions from porphyrin pi to iron d orbitals on the ground that they were observed only for the ferric low spin groups and insensitive to the axial ligands. The lack of temperature dependence in the MCD magnitude indicated that the MCD spectra are attributable to the Faraday B terms. Deoxymyoglobin, the ferrous high spin derivative, had fairly strong positive MCD around 760 nm with an anisotropy factor (delta epsilon/epsilon) of 1.4-10(-4). It shows some small MCD bands from 800 to 1800 nm. Among the ferrous low spin derivatives, carbonmonoxymyoglobin did not give any observable MCD in the infrared region while oxymyoglobin seemed to have significant MCD in the range from 700 to 1000 nm.     

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.     

The swinging movement of the distal histidine residue and the autoxidation reaction for midge larval hemoglobins.

Some insects have a globin exclusively in their fast-growing larval stage. This is the case in the 4th-instar larva of Tokunagayusurika akamusi, a common midge found in Japan. In the polymorphic hemoglobin comprised of 11 separable components, hemoglobin VII (Ta-VII Hb) was of particular interest. When its ferric met-form was exposed to pH 5.0 from 7.2, the distal histidine was found to swing away from the E7 position. As a result, the iron(III) was converted from a hexacoordinate to a pentacoordinate form by a concomitant loss of the axial water ligand. The corresponding spectral changes in the Soret band were therefore followed by stopped-flow and rapid-scan techniques, and the observed first-order rate constants of k(out) = 25 s(-1) and kin = 128 s(-1) were obtained for the outward and inward movements, respectively, of the distal histidine residue in 0.1 m buffer at 25 degrees C. For O2 affinity, Ta-VII Hb showed a value of P50 = 1.7 Torr at pH 7.4, accompanied with a remarkable Bohr effect (deltaH+ = -0.58) almost equal to that of mammalian hemoglobins. We have also investigated the stability property of Ta-VII HbO2 in terms of the autoxidation rate over a wide range of pH from 4 to 11. The resulting pH-dependence curve was compared with those of another component Ta-V HbO2 and sperm whale MbO2, and described based on a nucleophilic displacement mechanism. In light of the O2 binding affinity, Bohr effect and considerable stability of the bound O2 against acidic autoxidation, we conclude that T. akamusi Hb VII can play an important role in O2 transport and storage as the major component in the larval hemolymph.     

Characterization of equilibrium intermediates in denaturant-induced unfolding of ferrous and ferric cytochromes c using magnetic circular dichroism, circular dichroism, and optical absorption spectroscopies

Protein unfolding during guanidine HCl denaturant titration of the reduced and oxidized forms of cytochrome c is monitored with magnetic circular dichroism (MCD), natural CD, and absorption of the heme bands and far-UV CD of the amide bands. Direct MCD spectral evidence is presented for bis-histidinyl heme ligation in the unfolded states of both the reduced and oxidized protein. For both redox states, the unfolding midpoints measured with MCD, which is an indicator of tertiary structure, are significantly lower than those measured with far-UV CD, an indicator of secondary structure. The disparate titration curves are interpreted in terms of a compound mechanism for denaturant-induced folding and unfolding involving a molten globulelike intermediate state (MG) with near-native secondary structure and nonnative tertiary structure and heme ligation. A comparison of the dependence of the free energy of formation of the MG intermediate on the redox state with the known contributions from heme ligation and solvation suggests that the heme is significantly more accessible to solvent in the MG intermediate than it is in the native state.     

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.     

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.     

Assignment of exchangeable proximal histidine resonances in high-spin ferric hemoproteins: substrate binding in horseradish peroxidase.

Single-proton, exchangeable resonances have been detected in the high spin ferric hemoproteins, met-aquo myoglobin and horseradish peroxidase, which can be assigned to the proximal histidyl imidazole by virtue of their very large hyperfine shifts. While this proton is relatively labile in myoglobin, it is exchangeable in HRP only at extreme pH values, indicating a buried heme pocket. The insensitivity of the imidazole peak of HRP to substrate binding argues against direct interaction of imidazole and substrate.     

The circular dichroism of lysozyme.

The circular dichroism spectra of hen egg white lysozyme, and of lysozyme derivatives in which tryptophan residues 62 or 108, or both, are selectively oxidized, have been measured as a function of pH over the range of 200 to 310 nm. Neither Trp-62 nor Trp-108 is principally responsible for the positive rotational strength in the 280 to 300 nm region. The spectrum in the 200 to 230 nm region is nearly the same in the native protein and in the derivatives, and is little affected by binding of saccharide. These results are used to reinterpret the circular dichroism spectra of the lysozymes and alpha-lactalbumins.     

Circular dichroism and magnetic circular dichroism spectra of chlorophylls a and b in nematic liquid crystals. II. Magnetic circular dichroism spectra

Absorption and magnetic circular dichroism (MCD) spectra are reported for chlorophyll (Chl) a and Chl b dissolved in nematic liquid crystal solvents. The spectra were measured with the dye molecules oriented uniaxially along the direction of. the magnetic field and measuring light beam. It is significant that under such conditions the MCD spectra recorded in the wavelength region of the Q and Soret bands of the chlorophyll are essentially unchanged with respect to rotation of the sample cell around this axis, even though there is almost complete orientation of the chlorophyll molecules by the liquid crystals. The MCD spectra of Chl a and b in the nematic liquid crystal solvents used in this study are surprisingly similar to the spectra obtained under isotropic conditions. These results illustrate an important technique with which to examine the optical spectra of dyes oriented in liquid crystal matrices in which the anisotropic effects can be reduced the negligible proportions by the application of a strong magnetic field parallel to the direction of the measuring light beam. The first deconvolution calculations are reported that describe the deconvolution of pairs of absorption and MCD spectra, in the Q and B band regions, for both Chl a and b. The spectral analysis to obtain quantitative estimates of transition energies was accomplished by carrying out detailed deconvolution calculations in which the both the absorption and MCD spectral envelopes were fitted with the same number of components; each pair of components had the same hand centres and bandwidth values. This procedure resulted in an assignment of each of the main transitions in the absorption spectra of both Chl a and b. Chl a is clearly monomeric, with Qy, Qx, By and Bx located at 671, 582, 439 and 431 nm, respectively. Analysis of the spectral data for Chl b located Qy, By and Bx, at 662, 476 and 464 nm, respectively.     

Time-resolved circular dichroism in carbonmonoxy-myoglobin: the central role of the proximal histidine

A calculation of the circular dichroism (CD) spectra of carbonmonoxy- and deoxy-myoglobin is carried out in relation to a time-resolved CD experiment. This calculation allows us to assign a dominant role to the proximal histidine in the definition of the electronic normal modes and to interpret the transient CD structure observed in a strain of the proximal histidine. This strain builds up in 10 ps and relaxes in 50 ps as the protein evolves towards its deoxy form.     

A high-pressure sample cell for circular dichroism studies.

The construction and operation of a preparative polyacrylamide-gel electrophoresis system is described. Emerging protein bands are collected by an intermittent pumping system which is based on the design of Brownstone ((1969) Anal. Biochem.27, 25–46). The original pressure-sensitive operation was, however, simplified to time-volume operation. Cooling of the gel by a central cooling finger, essentially according to Jovin et al. ((1964) Anal. Biochem.9, 351–369), has also been added. To accomodate the polyethylene tubing needed for intermittent collection of protein and also the central cooling finger, it is necessary to polymerize the gel in a mold before it is installed in the gel housing compartment of the electrophoresis cell. Gel concentrations of 5% and higher can be used in this system. Dilution of emerging protein samples by the intermittent collection system is kept to a minimum. This fact, together with simplicity of design makes it suitable for general preparative work with polyacrylamide-gel electrophoresis. Operation of the apparatus and resolution of protein bands are demonstrated by separation of bovine serum albumin polymers and thyroxine-binding proteins in human serum.