Visible spectra of type-II P450-drug complexes: evidence that “incomplete” heme coordination is common

The visible spectrum of a ligand-bound cytochrome P450 is often used to determine the nature of the interaction between the ligand and the P450. One particularly characteristic form of spectra arises from the coordination of nitrogen-containing ligands to the P450 heme iron. These type-II ligands tend to be inhibitors since they stabilize the low reduction potential P450 and prevent oxygen binding to the heme. Yet, several type-II ligands containing aniline, imidazole, and triazole moieties are also known to be substrates of P450, though P450 binding spectra are not often scrutinized to make this distinction. Therefore, the three nitrogenous ligands aniline, imidazole, and triazole were used as binding spectra standards with purified human CYP3A4 and CYP2C9, because their small size should not present any steric limitations in their accessing the heme prosthetic group. Next, the spectra of P450 with drugs containing the three nitrogenous groups were collected for comparison. The absolute spectra demonstrated that the red-shift of the low spin Soret band is mostly dependent on the electronic properties of the nitrogen ligand since they tended to match their respective standards, aniline, imidazole, and triazole. On the other hand, difference spectra appeared to be more sensitive to the steric properties of the ligand because they facilitated comparison of the spectral amplitudes achieved with the drugs versus with the standard nitrogen ligands. Therefore, difference spectra may help reveal “weak” coordination to the heme that results from suboptimal orientation or ligand binding to more remote locations within the P450 active sites.