Nitrogenous Ligation at the Sixth Coordination Position of the Thr-301 to Lys-Mutated P450I1C2 Heme Iron

Abstract

Threonine-301 of P450I1C2 was replaced by lysine via site-directed mutagenesis. The Lys-mutated P450 exhibited absorption spectra that were characteristic of the nitrogenous-ligand-bound form of P450. In the oxidized form, the Soret band was red-shifted as compared with the typical ferric low-spin form of P450 and the β band was more intense than the α band. In the reduced form, two Soret peaks were observable at 447 and 423 nm and their relative heights were dependent on pH, indicating the existence of two interconvertible states of ferrous Lys-mutated P450 which are in equilibrium. In addition, the interaction of external ligands with the P450 heme iron was profoundly inhibited both in the oxidized and reduced forms. These findings suggest that ε-amino nitrogen of Lys-301, which was introduced by amino acid substitution, occupies the 6th coordination position with the heme iron of the Lys-mutated P450, because, owing to conformation of the P450 protein, the ε -amino group may be located at just the right position for coordination as the internal 6th ligand.