Solution NMR determination of the anisotropy and orientation of the paramagnetic susceptibility tensor as a function of temperature for metmyoglobin cyanide: Implications for the population of excited electronic states

Comprehensive H-1 NMR assignments of the heme cavity proton resonances of s perm whale metmyoglobin cyanide have provided the dipolar shifts for nonlig ated residues which, together with the crystal coordinates of carbonyl myog lobin, allow accurate determination of both the anisotropies and orientatio n of the paramagnetic susceptibility tensor, chi, in the molecular framewor k. The resulting axial, Delta chi(ax) = 2.48 x 10(-8) m(3)/mol, and rhombic anisotropy, Delta chi(rh) = -0.58 x 10-8 m(3)/mol, values at 25 degrees C determined from the most complete set of dipolar shifts are determined to 2 % and 6% uncertainty, respectively, and agree well with theoretical estimat es (Horrocks, W. D., Jr. and Greenberg, E. S. Mol. Phys. 1974, 27, 993-999) . Numerically and spatially restricted input data sets lead to larger uncer tainties in Delta chi(ax) and Delta chi(rh), but do not systematically bias the orientation of the tensor. Determination of the anisotropies and orien tation over the temperature range 5-50 degrees C shows that the susceptibil ity tensor orientation is minimally influenced, with both anisotropies well -behaved, and with Delta chi(ax), exhibiting a temperature behavior close t o that predicted for the system. The quantitative determination of the magn etic anisotropies over temperature allows the quantitative separation of co ntact and dipolar shifts for the iron ligands. The heme contact shifts refl ect the expected dominant pi spin density at pyrrole positions, but the mes o-protons exhibit low-field contact shifts indicative of unpaired spin in a sigma orbital. Such delocalized sigma spin density could arise from either deformation of the heme from planarity or the loss of sigma/pi separation for the d(xz), d(yz) orbitals when the major magnetic axis is Lilted strong ly from the heme normal as is experimentally observed. The observed anomalo us temperature dependencies of the heme methyl and axial His ring contact s hifts, as well as that of the rhombic anisotropy, are all consistent with t hermal population of the excited orbital state. The limitations for quanita tively determining the excited orbital state energy separation from the ava ilable NMR data are discussed.