ANALYSIS OF THE TEMPERATURE-DEPENDENCE OF THE H-1 CONTACT SHIFTS IN LOW-SPIN FE(III) MODEL HEMES AND HEME-PROTEINS - EXPLANATION OF CURIE AND ANTI-CURIE BEHAVIOR WITHIN THE SAME MOLECULE

The reasons that the temperature dependence of the NMR isotropic shift s of model ferrihemes and ferriheme proteins deviate from Curie behavi or have been analyzed by considering the energies of the valence orbit als of the metal and the porphyrinate. For low-spin Fe(III), overlap o f the e-symmetry pi orbitals of a symmetrical porphyrin ring and the d (pi) orbitals of the metal produces two low-energy molecular orbitals that are mainly porphyrin in character and are filled and two high-ene rgy (valence) molecular orbitals that are mainly metal in character an d contain three electrons. The odd electron in the valence set thus gi ves rise to the spin delocalization that results in the observed conta ct shift pattern of these systems. Unsymmetrical substitution and/or p resence of a planar axial ligand that is prevented from rotation remov es the degeneracy of these e(pi) orbitals, producing a system in which the energy separation between the two formerly degenerate pi orbitals , Delta E(pi), is of the order of only tens of cm(-1) for the former o r quite large (several times k(B)T) for the latter. In either case, bo th orbitals are utilized for spin delocalization to a significant exte nt as the temperature is varied, according to their varying Boltzmann populations. Such a two-level system obeys a modified Curie law that t akes into account the thermal population of the two levels as a functi on of temperature. In fact, the temperature dependence of some of the contact shifts of model hemes or heme proteins may show anti-Curie beh avior if Delta E(pi) is large compared to k(B)T at ambient temperature s. Such anti-Curie behavior has been observed for two of the heme meth yl resonances of several cytochromes c and b(5) and cyanometmyoglobins or -hemoglobins, where the axial methionine pi-symmetry lone pair or histidine imidazole plane orientation, respectively, is believed to be the important factor in determining Delta E(pi). Assuming reasonable energy separations of the two valence e(pi) orbitals, from very small to quite large (similar to 1000 cm(-1)), the expected temperature depe ndence of the contact shifts has been calculated for an assumed set of valence MO coefficients. These results have then been compared to the experimental isotropic shifts of several model heme systems having un symmetrical substitution patterns and/or one fixed axial ligand and to several heme proteins. Using a computer program developed to fit the observed isotropic shifts to the two-level equation, Delta E(pi) was e stimated from the temperature dependence of the isotropic shifts of th e protons of the beta-pyrrole substituents of the above-mentioned syst ems. In the case of the proteins investigated, Aplysia cyanometmyoglob in and cytochrome b(5), the values of Delta E(pi), obtained from analy sis of proton isotropic shifts are similar to those calculated from EP R g values measured at low temperatures, while for model hemins, the v alues of Delta E(pi) obtained are smaller than those predicted and var y in accord with the expectations as to the rigidity, or lack thereof, of the orientation of at least one planar axial ligand, indicating th at thermal averaging of the two levels due to rapid rotation (or libra tion) of the axial ligand is fast on the NMR time scale. This same two -level approach could be applied to any system in which there is a the rmal equilibrium between two states separated by an energy within seve ral factors of k(B)T.