Nitrogenous ligation at the sixth coordination position of the Thr-301 to Lys-mutated P450IIC2 heme iron.
Threonine-301 of P450IIC2 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 beta band was more intense than the alpha 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 epsilon-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 epsilon-amino group may be located at just the right position for coordination as the internal 6th ligand.
Imai
Y
Y
Department of Veterinary Science, University of Osaka Prefecture.
Nakamura
M
M
D001483:Base Sequence; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics; D015342:DNA Probes; D006418:Heme / Q000737:chemistry* / Q000235:genetics; D007501:Iron / Q000737:chemistry; D008024:Ligands; D008239:Lysine / Q000737:chemistry; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009584:Nitrogen / Q000737:chemistry; D010084:Oxidation-Reduction; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013053:Spectrophotometry; D013912:Threonine / Q000737:chemistry
D015342:DNA Probes; D008024:Ligands; D013912:Threonine; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D007501:Iron; D006899:Mixed Function Oxygenases; D042926:Cytochrome P-450 CYP4A; D008239:Lysine; D009584:Nitrogen