The linear electric field shift in paramagnetic resonance has, for the first time, been observed in frozen solutions. The magnitude of the shift parameters, measured at g.. of the electron paramagnetic resonance absorption envelope,_ has been used to characterize lowspin ferric heme mercaptide complexes where the nonmercaptide liganid was varied. The magnitude of the shift provides a measure of the difference between the crystal field contribution of the mercaptide and nonmercaptide ligands. In low-spin ferric cytochrome P-450 of rat liver, the presence of an electric field shift indicates a deviation from inversion symmetry and proves that the axial ligands to the heme are not the same. From the magnitude of the shift and its dependence upon the angle between applied electric and magnetic fields, it is suggested that the nonaxial ligand to the heme has a crystal field contribution greater than imidazole and smaller than guanidine.Cytochrome P450 represents a class of heme proteins characterized by an unusual optical absorption when reduced and reacted with CO (1, 2). Various members of this class have been isolated from bacteria (3, 4) and mammals (5, 6), but cytochrome P450 found in the endoplasmic reticulum of mammalian liver [and which is the terminal oxidase of the xenobiotic hydroxylating systems (7) ] has drawn the greatest attention.In liver slices and liver microsomes, cytochrome P450 is found largely in the low-spin ferric state and can therefore be studied by electron paramagnetic resonance (EPR) spectroscopy (7,8). On the basis of magnetic resonance comparison with model compounds, it has been suggested that a necessary z ligand to heme of all cytochromes P450, irrespective of their sources, is a mercaptide sulfur, endogenous to the protein structure and most likely from a cysteinyl residue (9, 10). The nature of the nonmercaptide z ligand in the various cytochromes P450 remains unknown, although EPR examination suggests a heterogeneity of structures, at least in soluble preparations from liver microsomes (11, 12).It is, however, the presence of the mercaptide ligand in cytochrome P450 that essentially determines the EPR behavior. It is difficult to make structural assignments on the basis of EPR, as was done for various low-spin heme proteins (9, 13), since the second z ligand has only a minor effect on the g tensor.In this preliminary report we describe a new technique that makes it possible to investigate cases of this kind. Specifically, we have observed for the first time a linear electric fieldinduced shift in the EPR frequency of low-spin ferric cytochrome P-450 from rat-liver microsomes of a phenobarbitaltreated animal and also of several heme mercaptide model compounds in frozen solution. In this way we were able to characterize the deviations from inversion symmetry of the center composed of the heme group and the ligands in these materials and to show that there is a marked difference in the bonding strengths of the two z ligands in the heme protein. In consideration of the magnitu...