ABSTRACT
The experimental data available shows that the change in enthalpy accompanying electron transfer to cupric blue proteins decreases as the ratio of the strengths of two visible light absorption bands increases. A compact mathematical expression for this inverse relation is formulated, the derivation of which demonstrates that the unusual geometry imposed by the protein upon the redox site is responsible both for the optical band intensity ratio and for a significant fraction of the enthalpy change.
ABSTRACT
During rapid cooling of ferric heme protein solutions containing fluoride, locally concentrated ligand cannot fully equilibrate with heme before the temperature drops below 200 K and into the range where energy is insufficient for exchange with iron-bound water. When temperature is then jumped above 200 K, exchange of fluoride for bound water is activated. Between 200 and 240 K, further fluoride complex formation takes place over several minutes; its extent is measured along the kinetic curve by reimmersing the sample into liquid nitrogen and taking EPR spectra. Kinetic curves for replacement of iron-bound water by fluoride in horse aquo-ferrimyoglobin and human aquo-ferrihemoglobin, and corresponding equilibrium constants have been obtained at temperatures between 200 and 240 K. The reaction rates are affected by sucrose. Results indicate that the kinetics of exchange of fluoride for heme-bound water at subfreezing temperatures is protein specific and not diffusion-controlled, and is not affected by the phase transition of ice which takes place at subfreezing temperature. Free energy changes accompanying these reactions are largely continuous as the systems pass from above to below freezing.
Subject(s)
Freezing , Hemeproteins/chemistry , Animals , Horses , Kinetics , Protein Conformation , Temperature , Thermodynamics , ViscosityABSTRACT
Fluoride association with ferric myoglobins and hemoglobins in aqueous buffers above freezing has been well studied. We chose this reaction to investigate the feasibility of observing titration intermediates and estimating dissociation constants at the freezing temperature by electron paramagnetic resonance spectroscopy at cryogenic temperatures. Dependence of apparent dissociation constant upon protein concentration was observed, a factor of four decrease in protein accompanied by about a fourfold increase in the apparent tightness of binding in the range of protein concentration studied. Binding was also found to depend upon cooling rate and concentration of additives (serum albumin, sucrose, glycerol). These effects appear to be associated with freezing-induced concentration of ligand, a process described in the literature. Bands of high concentration of electrolyte accompany solute rejection during ice growth, sweeping by slowing moving macromolecules. Thus, just before being trapped in the solid, the protein can experience a greater concentration of salt than in the original liquid. A mathematical model of this process, based upon simplifying assumptions about nucleation and ice-crystal growth rates in super-cooled solution, shows how the average concentration of mobile solute species can depend upon the concentration of all species present. Semiquantitative computer simulations of the actual, more complex, freezing are also presented and lead to estimates of ice particle size which are then compared with estimates from the former model.
Subject(s)
Fluorides/metabolism , Hemeproteins/metabolism , Hemoglobins/metabolism , Myoglobin/metabolism , Animals , Electron Spin Resonance Spectroscopy , Freezing , Horses , Humans , Kinetics , Mathematics , Methemoglobin/metabolism , Models, Theoretical , Protein BindingABSTRACT
The orientation and temperature dependence (4.2-2.5 K) of electron paramagnetic resonance (EPR) power saturation and spin-lattice relaxation rate, and the orientation dependence of signal linewidth, were measured in single crystals of the aquo complex of ferric sperm whale skeletal muscle myoglobin. The spin-packet linewidth was found to be temperature independent and to vary by a factor of seven within the heme plane. An analysis is presented which enables one to arrive at (a) hyperfine component line-widths and, from the in-plane angular variation of the latter, at (b) the widths of distributions in energy differences between low-lying electronic levels and (c) the angular spread in the in-plane principal g-directions. The values of the energy level distributions in crystals obtained from the measurements and analysis reported here are compared with those obtained by a different method for the same protein complex in frozen solution. The spread in the rhombic energy splitting is significantly greater in solution than in the crystal.
Subject(s)
Models, Theoretical , Myoglobin/metabolism , Electron Spin Resonance Spectroscopy , Heme/metabolism , Iron , Mathematics , Microwaves , Protein Conformation , ThermodynamicsABSTRACT
Frozen solution electron paramagnetic resonance spectra of the aquo, methanol, and ethanol complexes of ferric myoglobin and hemoglobin are quantitatively analyzed in terms of the rhombic to tetragonal symmetry ratio and the admixture of quartet states, both with regard to central values of these parameters and the widths of their distributions. In both the methanol and ethanol complexes of ferric myoglobin the main change from the aquo complex is a narrowing of the spread in the rhombic to tetragonal symmetry ratio (reduction in structural variation). The alcohol complexes of both the alpha- and beta-chains within the tetramer of ferric hemoglobin are characterized by a lowering of symmetry (as compared with the aquo complex). Qualitative differences in distribution widths among the complexes are consistent with an origin in molecular structure and dynamics rather than in ice matrix-induced strain.
Subject(s)
Ethanol/metabolism , Hemeproteins/metabolism , Methemoglobin/metabolism , Metmyoglobin/metabolism , Animals , Electron Spin Resonance Spectroscopy/methods , Horses , Humans , Kinetics , Protein Binding , WhalesABSTRACT
At concentrations lower than those causing denaturation, methanol, ethanol, and 1-propanol produce changes in optical absorption of alkaline ferricytochrome c. These changes arise from weak equilibrium associations characterized by dissociation constants at 25 degrees C of about 4 and 2 M, respectively, for the methanol- and 1-propanol-ferricytochrome c complexes. The difference spectra and temperature dependence of enthalpy and entropy changes accompanying formation of methanol and 1-propanol complexes, as well as changes induced in the EPR spectra, are different and suggest distinct binding modes. Considered in conjunction with related parameters from ferrihemoglobin and ferrimyoglobin, the spectral and thermodynamic data are consistent with models in which methanol is bound directly to the ferric ion of cytochrome c, methanol and ethanol are bound directly to the ferric ions of hemoglobin and myoglobin, and 1-propanol is bound to a hydrophobic region of cytochrome c. Both the absolute and alcohol-induced optical difference spectra of these proteins have been simulated, the former through summation of Gaussian bands and the latter as the difference between two such summations, one with parameters slightly altered from the other. This analysis reveals and characterizes previously unresolved structure, which is discussed in terms of electronic transitions of the heme group and changes caused by differing interactions of the heme with surroundings. Similarity between the difference spectra produced by IHP perturbation of ferrihemoglobin and that from the difference between absolute spectra of ferrimyoglobin and ferrihemoglobin suggests that, with ferrihemoglobin as reference, the conformations about the hemes of ferrimyoglobin and of ferrihemoglobin-IHP are in some way similar.
Subject(s)
Alcohols/metabolism , Hemeproteins/metabolism , Electron Spin Resonance Spectroscopy , Models, Biological , Protein Binding , Spectrophotometry , ThermodynamicsABSTRACT
In X-band electron paramagnetic resonance spectra from single crystals of horse ferric hemoglobin, observed line widths at the low- and high-field extrema are 30 and 24 g, and as much as 400 G in the intermediate region. This behavior is similar to that of ferric myoglobin. Due to large anisotropy in the g-tensors, the line width variation can be accounted for on the basis of heme orientation disorder. This disorder is characterized by an angle, determined here by two independent methods. In these computations Gaussian disorder on a sphere is assumed. The disorder angle is found to be constant on the sphere and about 4 degrees for both alpha- and beta- chains. Treatment of crystals with heavy water (buffer) increases the disorder. Since ligand nitrogen hyperfine couplings are available from hemoglobin electron nuclear double resonance, single crystal electron paramagnetic resonance spectra can be simulated by superimposing hyperfine bands, where the line width of the component bands is a variable and the disorder model above is employed. Comparison with observed resonances fixes the hyperfine component line widths. These component line widths from ferric hemoglobin in the crystalline state are found to be smaller than those in frozen solution.
Subject(s)
Methemoglobin , Animals , Chemical Phenomena , Chemistry, Physical , Electron Spin Resonance Spectroscopy , Horses , Mathematics , Protein ConformationABSTRACT
The contributions to the dipolar broadening of ferric magnetic resonances, from crystals of hemoglobin for which the atomic coordinates are known, have been calculated. The total second moment of the g = 2 resonance so determined is about 50 (MHz)2 or 5.0 G (peak-to-trough), figures consistent with the range of values found from analysis of experimental data. Two-thirds of this second moment comes from the two protons of the H2O molecule coordinated to the iron. Treatment with D2O is predicted to reduce the total second moment at g = 2 to about 25 (MHz)2, whereas the experimental measurements on single crystals show no decrease. If the structure of the tetramer is assumed to be the same when in solution as in the crystal, the total second moment is readily redetermined for hemoglobin in solution; the value so obtained is found to be significantly smaller than that from analysis of the g = 2 resonance measured in frozen solution. These two unexpected observations can be explained in terms of distributions in spin Hamiltonian parameters, the spread depending upon the nature of the sample--crystal or solution, ordinary or heavy water-treated. This distribution in H2O and D2O solutions appears to be about the same, since the measured differences in component line width agree with the calculated difference in dipolar contributions.
Subject(s)
Methemoglobin , Animals , Deuterium , Electron Spin Resonance Spectroscopy , Horses , Mathematics , Protein ConformationABSTRACT
Thermal activation of electron transfer reactions of the blue proteins is considered in terms of vibronic coupling. Use of the electron paramagnetic resonance spectral distribution to obtain an estimate of the force constant for the relevant protein mode is proposed and demonstrated. This analysis leads to a model in which the configurations of the cupric and cuprous sites are displaced only a few degrees from each other, both being close to the configuration midway between planar and tetrahedral.
Subject(s)
Azurin , Bacterial Proteins , Copper , Models, Chemical , Oxidation-ReductionSubject(s)
Metals , Animals , Binding Sites , Copper , Heme , Humans , Iron , Iron-Sulfur Proteins , Mathematics , Metalloproteins , Metals/metabolism , Molecular Conformation , Molybdenum , Oxidation-Reduction , Protein Binding , Protein ConformationABSTRACT
The behavior of ferrihemoglobin and ferrimyoglobin in widely varying concentrations of the lowest four alcohols has been studied by optical and electron paramagnetic resonance absorption spectroscopy. Methanol and ethanol, at concentrations too low to cause general conformational destabilization of the protein, produce both optical and electron paramagnetic resonance absorption spectral changes in ferrihemoglobin. These changes arise from equilibrium associations, characterized by dissociation constants at 25 degrees C of about 40 and 200 mM, respectively, for the methanol-ferrihemoglobin and ethanol-ferrihemoglobin complexes so formed. Other optical spectral changes appear when the methanol concentration exceeds 3.5 M and the ethanol, 1.0 M. At concentrations lower than 0.5 M, 1- and 2-propanol produce spectral changes of this second kind. At room temperature no optical evidence has been found that the propanols associate with ferrihemoglobin in the manner of methanol and ethanol. Methanol and ethanol at low concentration have specific effects, characterized by electron paramagnetic resonance spectral differences, upon ferric alphaSH chains. All four alcohols, over a wide range of concentrations, reduce the symmetry of electron paramagnetic resonance spectra from frozen solutions of ferrihemoglobin; even at the high end of this concentration range, none of the alcohols reduces the symmetry of electron paramagnetic resonance spectra from frozen ferrimyoglobin. Ferrimyoglobin and catalase association with methanol is measurable optically; the binding is about five and sixty times weaker, respectively, for these two proteins as compared with ferrihemoglobin.
Subject(s)
Ethanol , Hemoglobins , Methanol , Myoglobin , Animals , Binding Sites , Electron Spin Resonance Spectroscopy , Horses , Humans , Kinetics , Macromolecular Substances , Protein Binding , Protein Conformation , Protein Denaturation , Spectrophotometry , Thermodynamics , WhalesSubject(s)
Catalase , Iron , Methemoglobin , Myoglobin , Peroxidases , Chemical Phenomena , Chemistry , Humans , Micrococcus/enzymology , Oxidation-Reduction , Peroxides , Plants/enzymology , Porphyrins , SpectrophotometrySubject(s)
Insulin , Animals , Binding Sites , Copper , Electron Spin Resonance Spectroscopy , Hydrogen-Ion Concentration , Nitrogen , Pancreas , Spectrum Analysis , Swine , X-Ray Diffraction , ZincABSTRACT
In order to evaluate the feasibility of observing the spectral behavior of protein groups in the coordination sphere of the iron in hemeproteins, criteria are developed to determine whether or not the application of difference absorption spectroscopy to the study of complex formation will be successful. Absolute absorption spectra, 300-1100 mmu, from bacterial catalase complexes are displayed, and the infrared bands correlated with magnetic susceptibility values of similar complexes of other hemeproteins. Dissociation constants for the formation of cyanide and azide complexes of metmyoglobin, methemoglobin, bacterial catalase, and horseradish peroxidase are given. Difference spectra, 210-280 mmu, are displayed for cyanide and azide complexes of these hemeproteins. A band at 235-241 mmu is found in the difference spectra of all low-spin vs. high-spin complexes. The factors which favor the assignment of this band to a transition involving a histidine residue are presented.