Heme ligand replacement reactions of cytochrome P-450. Characterization of the bonding atom of the axial ligand trans to thiolate as oxygen.
Evidence of several types has accumulated that cytochrome P-450 has a thiolate anion as one of the axial ligands to heme (the fifth ligand). On the other hand, there is as yet no general agreement on the nature of the axial ligand trans to thiolate (the sixth ligand), although nitrogen and oxygen have been proposed. To resolve the controversy, the ligand exchange reactions of cytochrome P-450 were investigated by the use of optical spectroscopy. Two isozymes of rabbit liver microsomal cytochrome P-450 were examined: the isozyme induced by phenobarbital (P-450LM2), which has a sixth ligand and is low spin and the isozyme induced by 5,6-benzoflavone (P-450LM4), which is without a sixth ligand and is high spin. A series of artificial ligands was chosen to model the coordination of each of the potential native ligands, including water and certain amino acid residues. When the artificial ligand coordinated through an oxygen atom, the spectrum of pentacoordinate P-450LM4 changed to one closely resembling that of native, hexacoordinate P-450LM2. The spectrum of P-450LM2 was unchanged in the presence of oxygen-coordinating ligands. However, when artificial ligands which coordinate through nitrogen or sulfur were added to either P-450LM2 or P-450LM4, the induced spectra did not resemble the native spectrum and, in fact, were distinctive and characteristic of the particular ligand type. With three of the artificial ligands, 1-butanol, 1-benzylimidazole, and diethylphenylphosphine, the binding was found to be reversible by dilution, ultrafiltration, or gel filtration. The binding of 1-pentanol and 1-benzylimidazole was competitive, as expected for heme ligands, and the number of 1-benzylimidazole binding sites per molecule of P-450LM2 was estimated as 1.1. These results provide strong evidence that the native sixth ligand in P-450LM2 is oxygen rather than nitrogen.
D000818:Animals; D000838:Anions; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism; D010634:Phenobarbital / Q000494:pharmacology; D011190:Potassium Cyanide; D011485:Protein Binding; D011817:Rabbits; D013053:Spectrophotometry
D000838:Anions; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D011190:Potassium Cyanide; D010634:Phenobarbital