Magnetic circular dichroism studies of cytochrome [formula omitted]. Characterization of axial ligands of ferric and ferrous low-spin complexes

Abstract

MCD was applied to ferric and ferrous low-spin complexes of cytochrome P-450 cam to elucidate the electronic states and the nature of the axial ligands of the heme in cytochrome P-450 cam . (1) Low-spin complexes of ferric cytochrome P-450 cam , produced either by ligation of external ligands such as pyridine and imidazole derivatives or by being freed of (−)-camphor, showed sinusoidal Soret and α-MCD bands. The magnitude ratio of the Soret vs. α-MCD bands was quite sensitive to the nature of axial ligands of the ferric low-spin complexes. The ratio (2.7) for the camphor-free form of cytochrome P-450 cam , thus, was the smallest among those (2.7–9.0) for low-spin forms of cytochrome P-450 cam and other corresponding low-spin hemoproteins (ratios 7.8–13.9). The ratio (4.2) for the α-picoline-bound form of cytochrome P-450 cam, however, was the closest to that (2.7) for the camphorfree form of cytochrome P-450 cam among those (4.2–9.0) for the external ligand-bound form of cytochrome P-450 cam. The ratio for the 2-methylimidazole-bound form of cytochrome P-450 cam was the smallest among those of cytochrome P-450 cam bound with imidizole derivatives. Thus, among the nitrogen-bound low-spin forms, the low-spin form with a sterically hindered nitrogen ligand trans to the thiolate anion (−S −) most reproduced spectral characteristics of the native low-spin ferric form. Low-temperature absorption studies offered the same results. (2) It was found that MCD magnitudes of α-bands of ferrous low-spin complexes are intimately related to the electronic character of axial ligands. Thus, the CO, O 2 and NO-bound forms of cytochrome P-450 cam, which have two π-type axial ligands, showed the smallest α-MCD bands ( [θ] M = 5.2–7.5 ) among complexes, while ferrous cytochrome b 5 and cytochrome c, which have two σ-electron-donating axial ligands, showed the largest magnitude ( [θ] M = 120–176 ). The data for the ferrous low-spin complexes of other hemoproteins so far available were well rationalized in consideration of the property of the axial ligands.