Analysis of interactions among purified components of the liver microsomal cytochrome P-450-containing monooxygenase system by second derivative spectroscopy.

Second derivative spectroscopy together with the respective difference spectroscopy offers an effective methodical tool to resolve overlapping bands and shoulders into distinct bands at eliminated background absorption. The improved resolution allows attribution of the distinct bands to individual amino acid residues. Both methods have been utilized to analyze interactions between the three essential components of the liver microsomal cytochrome P-450-containing monooxygenase system. The improved resolution of the aromatic amino acid residues in the derivative spectra of cytochrome P-450LM2 and reductase allows one to determine that in the interactions of the essential components tyrosine residue(s) are involved. The participation of phenylalanine is likely and the participation of tryptophan residues is excluded. The pH-dependent decrease of the tyrosine absorption bands in the medium ultraviolet region with increasing pH is accompanied by a concurrent decrease of the heme absorption in the Soret region. Based on this concurrence, the existence of a heme-linked tyrosine as one of the axial heme iron ligands in cytochrome P-450 is postulated.