LIGAND INTERACTIONS WITH HEMOPROTEIN P-450 .2. INFLUENCE OF PHENOBARBITAL AND METHYLCHOLANTHRENE INDUCTION PROCESSES ON P-450 SPECTRA

The absolute spectrum of submicrosomal particles (free of cytochrome b5) which have been prepared from liver microsomes shows the induction of a second type of P-450 (oxidized: λmax 395, 505, and 650 mμ) after pretreatment of rabbits with 3-methylcholanthrene. Correspondingly, electron paramagnetic resonance spectra of fresh 3-methylcholanthrene microsomes show a new signal at g = 6.6, suggesting the presence of a high-spin hemoprotein in addition to the usual triplet of low-spin P-450. Double integration of these signals has shown that the two hemoproteins are present in a ratio of about 1:1. Similar proportions are calculated from difference spectra of reduced P-450 with CO. By use of labeled 3-methylcholanthrene it has been shown that this high-spin P-450 cannot arise from a direct 1:1 binding of 3-methylcholanthrene and P-450. The difference in spin state probably arises from different interactions of protein ligands. Thus, investigation of ligand interaction with the heme center of metmyoglobin revealed a close similarity between the electron paramagnetic resonance spectrum of metmyoglobin-n-propylmercaptide and the low-spin form of P-450. Further, the relative affinities of organic ligands (alkylamines, imidazole, and propylmercaptan) for P-450 appeared in a reverse order to those for metmyoglobin. Based on light absorption and electron paramagnetic resonance spectroscopy and ligand binding a scheme is proposed for protein ligand binding to P-450 in the two oxidation states and the two spin states of iron. © 1969, American Chemical Society. All rights reserved.