Abstract

Studies on the spectral change (type I) accompanying the formation of the enzyme-substrate complex of the hepatic microsomal mixed-function oxidase have been made to ascertain the relationship of the heme of cytochrome P-450 to this spectral change. Previously work has suggested that the type II spectral change was due to ferrihemochrome formation. Using ferriheme solutions as a model system, we have shown that spectral changes could be induced by alteration of the electronegativity, or polarity, of one ligand of the heme by variation of pH between 5.0 and 8.0. The spectral changes observed were similar with respect to wavelengths of the peak, the trough, and the isosbestic point, as well as in magnitude in relation to the heme concentration, when compared with the type I spectral change of microsomes containing cytochrome P-450 as the only heme constituent. These findings suggest that the two phenomena result from the same process: an alteration of the electronegativity of the sixth ligand of the heme. It is suggested that in microsomes the spectral changes are due to the displacement of the sixth ligand from a hydrophobic region of the apoenzyme (possibly the active site) by the substrate. It is suggested that two forms of cytochrome P-450 exist: one form is the unreacted enzyme, and the other is the substrate-bound enzyme. In the course of the metabolism of the substrate, the first form is converted to the second form in the presence of substrate, and this transition is accompanied by the type I spectral change.