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 orbitals of the heme under perturbed D-4 symmetry, which yields the orientation 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 between 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 low-temperature EPR studies of model compounds, which have been attributed to pseudo-Jahn-Teller distortion of the porphyrin. However, the effect is also predicted by extended Huckel calculations made with undistorted geometries. This work demonstrates that the variation in the electronic structure of bis-histidinyl hemes c is dominated by the geometry of the axial ligands and that other perturbations, such as asymmetric substitution of the porphyrin or low symmetry of the surrounding protein, are relatively minor. The correlations with theta and Delta E can, therefore, be used to determine the ligand geometry with sufficient accuracy to detect differences between structures in solution and in the crystal. The analysis can also be used to locate the principal axes of the magnetic susceptibility tensors of ferrihemes as well as providing orientational constraints for the axial ligands for the calculation of solution structures of paramagnetic proteins. This is particularly 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.