Magnetic circular dichroism studies of cytochrome P-450cam. Characterization of axial ligands of ferric and ferrous low-spin complexes

MCD was applied to ferric and ferrous low-spin complexes of cytochrome $\text{P-450}{}_{\mathrm{<mtext>cam</mtext>}}$ to elucidate the electronic states and the nature of the axial ligands of the heme in cytochrome $\text{P-450}{}_{\mathrm{<mtext>cam</mtext>}}$. (1) Low-spin complexes of ferric cytochrome $\text{P-450}{}_{\mathrm{<mtext>cam</mtext>}}$, 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 $\text{P-450}{}_{\mathrm{<mtext>cam</mtext>}}$, 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, O₂ and NO-bound forms of cytochrome P-450_cam, which have two π-type axial ligands, showed the smallest α-MCD bands ($\text{[θ]}{}_{\mathrm{M}}\text{= 5.2–7.5}$) among complexes, while ferrous cytochrome $\text{b}{}_5$ and cytochrome $\text{c}$, which have two σ-electron-donating axial ligands, showed the largest magnitude ($\text{[θ]}{}_{\mathrm{M}}\text{= 120–176}$). 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.