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.     

First-principles calculations of protein circular dichroism in the near ultraviolet

Electronic transitions in aromatic side chains are responsible for the characteristics of proteins in the near UV. We present the first systematic study of a large number of proteins focused on the accurate calculation of near-UV circular dichroism (CD) spectra. We report new parameter sets derived from ab initio calculations for benzene, phenol, and indole that describe the valence electronic transitions to the (1)L(b), (1)L(a), (1)B(b), and (1)B(a) states in the side chains of amino acids phenylalanine, tyrosine, and tryptophan. CD spectra were calculated, using the matrix method with the new side-chain parameters, for 30 proteins whose CD spectra and crystal structures have been made publicly available. The new parameter sets are fully self-consistent and yield near-UV spectra better than those obtained using previous parameter sets. The mean absolute errors for computed wild-type spectra in the near UV are reduced by a factor of approximately 2. A similiar reduction is found for the near-UV spectra (and difference spectra) of mutants involving aromatic amino acids. Empirical modifications to model vibronic coupling in the side-chain chromophore of phenylalanine offer no overall improvement. Protein CD calculations from first principles coupled with atomic-level modeling enhance the utility and interpretability of CD measurements in the near UV.     

Magnetic circular dichroism studies of myoglobin,hemoglobin and peroxidase at room and low temperatures. Ferrous high spin derivatives

The magnetic circular dichroism spectra (MCD) recorded for the visible and near-UV regions of high-spin ferrous derivatives of myoglobin, hemoglobin, hemoglobin dimers and isolated chains as well as of horseradish peroxidase at pH 6.8 and 11.4 have been compared at the room and liquid nitrogen temperatures. The MCD of the Q ₀₀- and Q_v-bands have been shown to be sensitive to structural differences in the heme environment of these hemoproteins. The room temperature visible MCD of native hemoglobin differs from that of myoglobin, hemoglobin dimers and isolated chains as well as from that of model pentacoordinated complex. The MCD of hemoglobin is characterized by the greater value of the MCD intensity ratio of derivative shape A-term in the Q ₀₀-band to the A-term in the Q _v-band. The evidences are presented for the existence of two pH-dependent forms of ferroperoxidase, the neutral peroxidase shows the hemoglobin-like MCD, while the alkaline ferroperoxidase is characterized by the myoglobin-like MCD spectrum in the visible region. The differences in the MCD of deoxyhemoglobin and neutral ferroperoxidase as compared with other high-spin ferrous hemoproteins are considered to result from the constraints on heme group imposed by quaternary and/or tertiary protein structure. The differences between hemoproteins which are seen at the room temperature become more pronounced at liquid nitrogen temperature. Except the peak at 580 nm in the MCD of deoxymyoglobin and reduced peroxidase at pH 11.4 the visible MCD does not show appreciable temperature dependent C-terms. The nature of the temperature dependent effect at 580 nm is not clear. The Soret MCD of all hemoproteins studied are similar and are predominantly composed of the derivative-shaped C-terms as revealed by the increase of the MCD peaks approximately in accordance with Boltzmann distribution. The interpretation of temperature-dependent MCD observed for the Soret band has been made in terms of porphyrin to Fe-ion charge-transfer electronic transition which may be assigned as b() 3d. This charge-transfer band is strongly overlapped with usual B( - *) band resulting in diffuse Soret band. Adopting that only two normal vibrations are sinphase with charge-transfer transition the extracted C-terms of the Soret MCD have been fitted by theoretical dispersion curves.     

Magnetic circular dichroism studies of myoglobin, hemoglobin and peroxidase at room and low temperatures. Ferrous high spin derivatives.

The magnetic circular dichroism spectra (MCD) recorded for the visible and near-UV regions of high-spin ferrous derivatives of myoglobin, hemoglobin, hemoglobin dimers and isolated chains as well as of horseradish peroxidase at pH 6.8 and 11.4 have been compared at the room and liquid nitrogen temperatures. The MCD of the Q00- and QV-bands have been shown to be sensitive to structural differences in the heme environment of these hemoproteins. The room temperature visible MCD of native hemoglobin differs from that of myoglobin, hemoglobin dimers and isolated chains as well as from that of model pentacoordinated complex. The MCD of hemoglobin is characterized by the greater value of the MCD intensity ratio of derivative shape A-term in the Q00-band to the A-term in the QV-band. The evidneces are presented for the existence of two pH-dependent forms of ferroperoxidase, the neutral peroxidase shows the "hemoglobin-like" MCD, while the alkaline ferroperoxidase is characterized by the "myoglobin-like" MCD spectrum in the visible region. The differences in the MCD of deoxyhemoglobin and neutral ferroperoxidase as compared with other high-spin ferrous hemoproteins are considered to result from the constraints on heme group imposed by quaternary and/or tertiary protein structure. The differences between hemoporteins which are seen at the room temperature become more pronounced at liquid nitrogen temperature. Except the peak at approximately 580 nm in the MCD of deoxymyoglobin and reduced peroxidase at pH 11.4 the visible MCD does not show appreciable temperature dependent C-terms. The nature of the temperature dependent effect at approximately 580 nm is not clear. The Soret MCD of all hemoproteins studied are similar and are predominantly composed of the derivative-shaped C-terms as revealed by the increase of the MCD peaks approximately in accordance with Boltzmann distribution. The interpretation of temperature-dependent MCD observed for the Soret band has been made in terms of porphyrin to Fe-iron charge-transfer electronic transition which may be assigned as b( pi) leads to 3d. This charge-transfer band is strongly overlapped with usual B(pi --pi*) band resulting in diffuse Soret band. Adopting that only two normal vibrations are sinphase with charge-transfer transition the extracted C-terms of the Soret MCD have been fitted by theoretical dispersion curves.     

Sugar-induced change in near ultraviolet circular dichroism of α-crystallin

In order to study the effect of sugar molecules on the structure of α-crystallin, the protein from bovine lenses was isolated and treated with D-glucose and D-glucose-6-phosphate. Structural studies of the products were carried out using circular dichroism (CD) measurements. In the near-ultraviolet (UV) region, three positive and two negative CD bands are observed for α-crystallin. Upon incubation with the saccharides, the positive ellipticities of these near-UV CD bands increase greatly. The CD spectrum of α-crystallin in the far-UV region, which is typical of the β-conformation of a protein, does not change. The increase does not appear to be caused by glycosylation, or by sugar metabolites such as sorbitol or glycolytic products. When the concentration of sugar is higher than that has been used in this study, the protein tends to aggregate. The results strongly suggest that sugar, upon interaction with α-crystallin, induces a change in the tertiary structure of the protein while its secondary structure remains intact. The interaction of sugar molecules causing such change is weak, noncovalent, and temperature-dependent. The near-UV CD of α-crystallin appears to be a useful probe for the study of such structural changes, which may be significant in the pathogenesis of cataract.     

Domain specificity in metal binding to metallothionein. A circular dichroism and magnetic circular dichroism study of cadmium and zinc binding at temperature extremes

Rabbit liver Zn metallothionein-(MT) will bind cadmium readily between -26 degrees C and 70 degrees C. The binding reaction was monitored by recording the circular dichroism and magnetic circular dichroism spectra, in the region of the RS(-)----Cd2+ charge transfer transition at 250 nm, at intervals as aliquots of cadmium were added. For all temperatures, these data can be analyzed in terms of a distributed mechanism for cadmium binding when Zn-MT is used, and a domain-specific mechanism when apo-MT is used. The CD spectrum measured at -26 degrees C for Cd,Zn-MT, which was made by adding excess cadmium directly to Zn7-MT at -26 degrees C, is not the same as the CD spectrum of Cd-MT prepared at room temperature from the same Zn7-MT. Measurements of the stoichiometry of the cadmium and zinc bound to MT in the presence of excess cadmium at different temperatures indicates that below 5 degrees C at least one zinc atom remains bound to the protein. The mixed metal metallothionein, Cd/Zn-MT, that always forms below 5 degrees C, is characterized by a single maximum near 250 nm in the CD spectrum, rather than the derivative-shaped CD envelope that is diagnostic of the (Cd4-S11)alpha cluster, which indicates that the zinc occupies a site in the alpha domain. Rearrangement of the bound metals to the domain-specific distribution takes place if Cd,Zn-MT, prepared at subzero temperatures, is warmed above 30 degrees C.     

Circular dichroism and magnetic circular dichroism of Azotobacter vinelandii ferredoxin I

Room temperature circular dichroism (CD) and low temperature magnetic circular dichroism (MCD) spectra of air-oxidized and dithionite-reduced Azotobacter vinelandii ferredoxin I (FdI), a [( 4Fe-4S]2+/1+, [3Fe-4S]1+/0) protein, are reported. Unlike the CD of oxidized FdI, the CD of dithionite-reduced FdI exhibits significant pH dependence, consistent with protonation-deprotonation at or near the cluster reduced: the [3Fe-4S] cluster. The MCD of reduced FdI, which originates in the paramagnetic reduced [3Fe-4S]0 cluster, is also pH-dependent. Detailed studies of the field dependence and temperature dependence of the MCD of oxidized and reduced FdI, in the latter case at pH 6.0 and 8.3, are reported. The low-field temperature dependence of the MCD of oxidized FdI, which originates in the paramagnetic oxidized [3Fe-4S]1+ cluster, establishes the absence of a significant population of excited electronic states of this cluster up to 60 K. The low-field temperature dependence of the MCD of reduced FdI establishes that the ground-state manifold of the reduced [3Fe-4S]0 cluster possesses S greater than or equal to 2 at both pH 6.0 and 8.3. Analysis, assuming S = 2 and an axial zero-field splitting Hamiltonian, leads to D = -2.0 and -3.5 cm-1 at pH 6.0 and 8.3, respectively. The site of the (de)protonation affecting the spectroscopic properties of the [3Fe-4S] cluster remains unknown.     

Circular dichroism and magnetic circular dichroism of bismuth-induced,metallothionein-like proteins

Optical studies have been carried out on bismuth-containing proteins which were isolated from the livers and kidneys of rats following injections of BiCl₃. Absorption, circular dichroism and magnetic circular dichroism spectra of hepatic Bi,Zn-metallothionein 1 and 2 indicate that the spectra are dominated by transitions from the zinc thiolate chromophore. The data from the renal Bi,Cu-metallothionein 2 are quite different and it is suggested that these spectra involve a mixture of transitions from the bismuth and copper thiolate binding sites.     

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.     

Absorption, circular dichroism, magnetic circular dichroism and emission study of rat kidney Cd,Cu-metallothionein

Absorption, circular dichroism (CD), magnetic circular dichroism (MCD) and emission spectra of rat liver and rat kidney cadmium-, zinc- and copper-containing metallothioneins (MT) are reported. The absorption, CD and MCD data of native rat kidney Cd,Cu-MT protein closely resemble data recorded for the rat liver Cd,Zn-MT. This suggests that the major features in all three spectra of the native Cd,Cu-MT are dominated by cadmium-related bands. The CD spectrum of the Cd,Cu-MT recorded at pH 2.7 has the same band envelope that is observed for a Cd,Cu-MT formed in vitro by titration of Cd,Zn-MT with Cu(I), suggesting that the copper occupies the zinc sites in Cd,Cu-MT formed both in vivo and, at low molar ratios, in vitro. Remetallalion of the metallothionein from low pH in the presence of both copper and cadmium results in considerably less cadmium bound to the protein than was present in the native sample. It is suggested that this is due to the effect of the distribution of the copper amongst all available binding sites, thus inhibiting cluster formation by the cadmium. Emission spectra are reported for the first time for a cadmium- and copper-containing metallothionein. An emission band at 610 nm is shown to be a sensitive indicator of Cu(I) binding to metallothionein. Both the native Cd,Cu-MT and a Cd,Cu-MT formed in vitro exhibit an excitation spectrum with a band in the copper-thiolate charge-transfer region.     

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.     

Magnetic circular dichroism and spin equilibrium of methemoglobin and its subunits

The intensity of the Soret magnetic circular dichroism (MCD) spectra of various complexes of methemoglobin subunits (α⁺ and β⁺) as well as methemoglobin (metHb A) was correlated well with the spin states of ferric heme. Upon the subunit association, spin state transition toward higher spin was observed only in high spin derivatives and the changes in spin state were due to mainly those of β⁺ chains. The effect of an allostric effector, inositol hexaphosphate (IHP), on the MCD spectra of metHb A derivatives was observed much significantly for high spin forms than low spin ones.     

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.