Arthur S. Brill

Energy distributions at the high-spin ferric sites in myoglobin crystals.

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.

Epr characterization of alcohol complexes of ferric myoglobin and hemoglobin

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.

Influence of the freezing process upon fluoride binding to hemeproteins

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.

Magnetic circular dichroism of low symmetry cupric sites

General properties of the MCD of cupric ions in ligand fields of low symmetry are described. C terms arising from spin‐orbit coupling are dominant at liquid helium temperatures If spin‐orbit coupling of the excited states to the ground state is negligible compared with spin‐orbit coupling of the excited states among themselves, then the C values for the transitions sum to zero. It follows that nonvanishing of the sum of the C values over all transitions is due to spin‐orbit coupling with the ground state. This net rotational offset is calculated for hybrid orbital ‘‘united atom’’ models of the electronic states of the metal–ligand complexes. Simulations of C term spectra from hybrid atomic orbital models illustrate relations between MCD features and properties of low symmetry cupric sites.

Stability of H, D, 14N, and 15N atoms in solid ammonia above 100 K

The measurements reported below quantify the stability and decay of hydrogen, deuterium, and nitrogen atoms in frozen ammonia above 100 K. The decay of H atoms is observed on a time scale of minutes in the range of 100–110 K and follows first-order kinetics. Analogous decays of D and N atoms are observed in the ranges 105–120 and 140–160 K, respectively. Activation energies for the decay processes range from 0.1 to 0.4 eV.

Power saturation of electron paramagnetic resonances from high-spin ferric haemproteins at 4·2 K

The power saturation behaviour of first derivative electron paramagnetic resonance signals from the highly anisotropic high-spin haem group is formulated. A relation between the power at which the maximum signal is obtained and the quotient of the spin-lattice relaxation time and fundamental (intrinsic spin-packet) linewidth is shown to hold for certain experimental conditions. At 4·2 K the power saturation of ferrimyoglobin was studied at several modulation field frequencies between 80 Hz and 100 kHz, and ferrihaemoglobin and catalase at 100 kHz. Results are given in terms of the ratio of the powers for maximum signal with the scanning field perpendicular to the haem plane and in the plane, multiplied by the ratio of the corresponding transition probabilities. The observed product of ratios at low modulation frequencies is quantitatively explained by a proposed relation between iron out-of-planarity and the angular dependence of the linewidth.

ELECTRON PARAMAGNETIC RESONANCE IN SINGLE CRYSTALS OF CUPRIC INSULIN.

THE binding of divalent metal ions to insulin has been the subject of several investigations, both chemical and physical in nature1–6. In describing here the application of electron paramagnetic resonance (EPR) spectroscopy to this problem, we wish to emphasize the richness of the data obtainable from orientation investigations of single crystals. This information is of the following kinds: (1) the number of distinct magnetic centres; (2) the orientation with respect to the crystal axes of the symmetry axes of the magnetic centres; (3) the symmetries of the protein environments of the magnetic centres, and the ground-states of the transition metal ions; (4) the delocalization of the electrons in metal ion–protein bonds; (5) identification of protein atoms bound to metal ions when the former have nuclear magnetic moments; (6) an estimate of the distance between metal ions.

Water of co-ordination in insulin†

Single-crystal electron paramagnetic resonance orientation studies, hydrogenion titration, and X-ray diffraction data have established that, in the rhombohedral insulin crystals which form at neutral pH, the two metal ions in a unit cell are located on a trigonal axis, each co-ordinated to three imidazole nitrogens. We have now obtained electron paramagnetic resonance spectra of deuterium oxide-substituted cupric insulin crystals. (The magnetic moment of the deuteron is about one-third that of the proton.) Resonance line narrowing is observed which, together with previous data, indicates that water molecules occupy the remaining co-ordination positions about each metal ion. We have used the measured decrease in line-width to calculate the average proton-to-metal distance. The copper to water-oxygen bond length was found to be in the range of corresponding values in the structures of small complexes determined by X-ray and neutron diffraction. This calculation of interatomic separation shows that it is essential to take into account the effect on the radial factor of the unpaired cupric spin delocalization onto the water oxygen. This delocalization was quantitated from the ligand hyperfine splitting arising from the co-ordinated nitrogens.

Mechanical control of redox enthalpy

The change in elastic energy which accompanies transfer of an electron to an acceptor site in a protein is formulated. The sign and magnitude of this energy is found to depend upon the structural geometry to which the site is constrained by the stiffness of the protein. Through this enthalpy component, conditions which alter protein structure can contribute to the control of the oxidation-reduction equilibrium at the site. The difference between the total redox enthalpy and the elastic component is the electrostatic contribution. The formulation is applied to the electron acceptor site in the blue cupric protein azurin.

Iron out-of-planarity and tetrapyrrole nitrogen nuclear spin state mixing in high-spin ferric haem

The spin system of the haem group is first characterized by diagonalization of the contact terms, the tetrapyrrole nitrogen nuclear spin state then being specified by four individual states. The tetrapyrrole states are not pure, anisotropic dipole-dipole and quadrupole interactions connecting any one such state to a number of others. At second order there is an admixture of ‘flip-flop’ states in which opposite unit changes in the spin projections of a pair of nitrogens have occurred such that the total projection is unchanged. When the scanning magnetic field in a resonance experiment is normal to the haem plane, (a) only the ionic dipole-dipole interaction admixes flip-flop states, and then only when the iron is displaced from the average plane of the tetrapyrrole nitrogens; (b) quadrupole interactions give rise to resonance shifts among the flip-flop states but anisotropic dipole-dipole interactions do not; (c) the contribution from state mixing to the linewidth of the resonance is proportional to the s...