Visible Spectra of Type II Cytochrome P450-Drug Complexes: Evidence that “Incomplete” Heme Coordination Is Common

Abstract

ABSTRACT: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 because 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, although 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 seemed to be more sensitive to the steric properties of the ligand because they facilitated comparison of the spectral amplitudes achieved with the drugs versus those 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.When studying substrate binding to the cytochromes P450 (P450s), the heme prosthetic group often serves as a very useful chromophore that can be exploited for purposes of characterization. With P450s, the heme is bound as in a b-type cytochrome except that the iron atom is liganded to a single Cys side chain as found in only a few other heme-containing proteins (e.g., chloroperoxidase, nitric oxide-synthase, and prostacyclin synthase). Because oxidation of P450-bound substrate occurs at the heme, certain aspects of substrate binding can be monitored readily with a spectrophotometer. Most commonly, ligand titration experiments are carried out to determine a spectral dissociation constant (K S ) for a P450 substrate or inhibitor, but little attention is paid to the signature manners in which the P450 heme spectra are altered (reviewed by Jefcoate, 1978). In particular, there seems to be an oversimplification regarding coordination of P450 substrates to the heme iron. This interaction stabilizes the low-spin iron configuration and thus is assumed to prevent catalysis since it is the high-spin iron that is more conducive to reduction by P450 reductase. [More specifically, P450 isoform differences arise because the rate of reduction is more dependent on reduction potential than spin state (Ost et al., 2003).]Generally, nitrogen-containing heme ligands, which bind more avidl...

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