Möessbauer Investigations of High-Spin Ferrous Heme Proteins. I. Cytochrome P-450

Anaerobically reduced samples of cytochrome P-450 from Pseudomonas pulida were studied by Móssbauer spectroscopy. In the presence of an applied magnetic field the high-spin ferrous heme iron showed an intricate pattern of electric and magnetic hyperfine interactions which could be parametrized successfully in terms of a spin Hamiltonian formalism. The results imply a very low (triclinic) symmetry of the heme iron. The effects of the ligand environment and of spin-orbit coupling result in a large zero-field splitting of the electronic ground state. The electric-field gradient tensor is characterized by a large asymmetry parameter, and its principal axes are rotated substantially from the frame that defines the zero-field splitting. This study shows that high-field Mössbauer spectroscopy provides a unique tool for structural investigations of highspin ferrous compounds and can substitute, under suitable conditions, for magnetic susceptibility measurements. The present paper focuses on the methodology and data analysis; in the subsequent paper the data obtained for P-450 are compared with new results obtained for hemoglobin, chloroperoxidase, and horseradish peroxidase.