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

Abstract

Comprehensive 1H NMR assignments of the heme cavity proton resonances of sperm whale metmyoglobin cyanide have provided the dipolar shifts for nonligated residues which, together with the crystal coordinates of carbonyl myoglobin, allow accurate determination of both the anisotropies and orientation of the paramagnetic susceptibility tensor, χ, in the molecular framework. The resulting axial, Δχax = 2.48 × 10-8 m3/mol, and rhombic anisotropy, Δχrh = −0.58 × 10-8 m3/mol, values at 25 °C determined from the most complete set of dipolar shifts are determined to 2% and 6% uncertainty, respectively, and agree well with theoretical estimates (Horrocks, W. D., Jr. and Greenberg, E. S. Mol. Phys. 1974, 27, 993−999). Numerically and spatially restricted input data sets lead to larger uncertainties in Δχax and Δχrh, but do not systematically bias the orientation of the tensor. Determination of the anisotropies and orientation over the temperature range 5−50 °C shows that the susceptibility tensor orientation is minimally influenced, with both anisotropies well-behaved, and with Δχax exhibiting a temperature behavior close to that predicted for the system. The quantitative determination of the magnetic anisotropies over temperature allows the quantitative separation of contact and dipolar shifts for the iron ligands. The heme contact shifts reflect the expected dominant π spin density at pyrrole positions, but the meso-protons exhibit low-field contact shifts indicative of unpaired spin in a σ orbital. Such delocalized σ spin density could arise from either deformation of the heme from planarity or the loss of σ/π separation for the dxz, dyz orbitals when the major magnetic axis is tilted strongly from the heme normal as is experimentally observed. The observed anomalous temperature dependencies of the heme methyl and axial His ring contact shifts, as well as that of the rhombic anisotropy, are all consistent with thermal population of the excited orbital state. The limitations for quanitatively determining the excited orbital state energy separation from the available NMR data are discussed.