Electronic structure of low-spin ferric porphyrins: C-13 NMR studies of the influence of axial ligand orientation

Heteronuclear multiple quantum NMR is used to measure the paramagnetic C-13 shifts Of the alpha substituents of the hemes in five different tetraheme ferricytochromes c(3). The shifts of the 20 bis-histidine ligated hemes are assigned and then analyzed in terms of a model based on the pi molecular o rbitals of the heme under perturbed D-4 symmetry, which yields the orientat ion of the rhombic perturbation, theta, and an energy splitting parameter, Delta E. Comparison of these parameters with crystal structures provides a test of the nature and extent of the influence of axial ligand orientation on the electronic structure of the heme. Despite possible differences betwe en structures in solution and in the crystal, a clear correlation is found between theta and the resultant of the normals to the imidazole planes, and between Delta E and the angle between the normals. A weaker dependence of Delta E upon theta is also apparent. This is analogous to the results of lo w-temperature EPR studies of model compounds, which have been attributed to pseudo-Jahn-Teller distortion of the porphyrin. However, the effect is als o predicted by extended Huckel calculations made with undistorted geometrie s. This work demonstrates that the variation in the electronic structure of bis-histidinyl hemes c is dominated by the geometry of the axial ligands a nd that other perturbations, such as asymmetric substitution of the porphyr in or low symmetry of the surrounding protein, are relatively minor. The co rrelations with theta and Delta E can, therefore, be used to determine the ligand geometry with sufficient accuracy to detect differences between stru ctures in solution and in the crystal. The analysis can also be used to loc ate the principal axes of the magnetic susceptibility tensors of ferrihemes as well as providing orientational constraints for the axial ligands for t he calculation of solution structures of paramagnetic proteins. This is par ticularly important since paramagnetic relaxation may make it impossible to observe NOE effects to the imidazole protons, leaving the geometry of the heme pocket poorly defined.