Identification of the binding site on cytochrome P450 2B4 for cytochrome b5 and cytochrome P450 reductase.
A model of cytochrome P450 2B4, which was constructed by homology modeling with the four known crystal structures of the cytochromes P450 (Chang, T.-T., Stiffelman, O. B., Vakser, I. A., Loew, G. H., Bridges, A., and Waskell, L. (1997) Protein Eng. 10, 119-129), was used to select amino acids predicted, by computer docking studies and numerous previous biochemical and site-directed mutagenesis studies, to be involved in binding the heme domain of cytochrome b5. Twenty-four amino acid residues located on both the distal and the proximal surface of the molecule were chosen for mutagenesis. These 24 mutant proteins were expressed in Escherichia coli, purified, and characterized with respect to their ability to bind cytochrome b5 and support substrate oxidation. Seven mutants, R122A, R126A, R133A, F135A, M137A, K139A, and K433A, all on the proximal surface of cytochrome P450 2B4 near the heme ligand, were identified that exhibited decreased ability to bind cytochrome b5. All of the mutants except K433A are located in either the C or C* helices or their termini. In addition, these seven mutants and two additional mutants on the proximal surface of cytochrome P450, R422A and R443A, were shown to exhibit decreased binding to cytochrome P450 reductase. These studies indicate that the binding sites for cytochrome b5 and cytochrome P450 reductase are, as predicted, located on the proximal surface of cytochrome P450 2B4 and are partially overlapping but not identical.
Bridges
A
A
Department of Anesthesia, Veterans Affairs Medical Center, San Francisco, California 94121, USA.
Gruenke
L
L
Chang
Y T
YT
Vakser
I A
IA
Loew
G
G
Waskell
L
L
D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015786:Cytochromes b5 / Q000378:metabolism*; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics; D008861:Microsomes / Q000201:enzymology; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase; D010084:Oxidation-Reduction; D013250:Steroid Hydroxylases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D004274:DNA, Recombinant; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C050356:steroid 15-alpha-hydroxylase; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase