Ligand binding studies of engineered cytochrome P-450d wild type, proximal mutants, and distal mutants.
Interactions of various axial ligands with cytochrome P-450d wild type, proximal mutants (Lys453Glu, Ile460Ser), and putative distal mutants (Glu318Asp, Thr319Ala, Thr322Ala) expressed in yeast were studied with optical absorption spectroscopy. P-450d wild type and all five mutants were purified essentially as the high-spin form, but the putative distal mutants contained about 5% low-spin form. Bindings of metyrapone and 4-phenylimidazole to the wild type and all mutants formed nitrogen-bound low-spin forms. In contrast, binding of 2-phenylimidazole to the wild type and most of mutants formed oxygen-bond low-spin forms except for the mutant Glu318Asp in which the nitrogen-bound low-spin form was formed. By analogy with the distal structure of P-450cam, it was thus suggested that Glu318 of P-450d, which corresponds with Asp251 of P-450cam, somehow interacts with 2-phenylimidazole over the heme plane. Addition of 1-butanol and acetanilide, a substrate of P-450d, to the wild type and mutants caused the spin change to the low-spin form. The order of dissociation constants of these oxygen ligands to P-450d was wild type greater than proximal mutants greater than putative distal mutants. Spectral analyses showed that the binding of acetanilide is the same as that of another substrate, 7-ethoxycoumarin, in the putative distal mutants but is not the same in the wild type and proximal mutants. From these findings together with other spectral data, it was suggested that the region from Glu318 to Thr322 is located at the distal region of the heme in membrane-bound P-450d as suggested from the X-ray crystal structure of water-soluble P-450cam and amino acid alignments of P-450s.
Shimizu
T
T
Chemical Research Institute of Non-Aqueous Solutions, Tohoku University, Sendai, Japan.
Sadeque
A J
AJ
Sadeque
G N
GN
Hatano
M
M
Fujii-Kuriyama
Y
Y
D000595:Amino Acid Sequence; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D007093:Imidazoles / Q000378:metabolism; D008024:Ligands; D008797:Metyrapone / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013053:Spectrophotometry
D007093:Imidazoles; D008024:Ligands; C059194:2-phenylimidazole; D003577:Cytochrome P-450 Enzyme System; D008797:Metyrapone