IDENTIFICATION OF A MEANDER REGION PROLINE RESIDUE CRITICAL FOR HEME-BINDING TO CYTOCHROME-P450 - IMPLICATIONS FOR THE CATALYTIC FUNCTION OF HUMAN CYP4B1

Alignment of xenobiotic-metabolizing P450 protein sequences highlights an invariant proline residue in the meander region two amino acids N- terminal to the distal arginine of the putative ERR triad thought to b e important for heme binding. This occurs as a serine in the sequences derived from human CYP4B1 gDNA and both human lung and placental CYP4 B1 cDNAs. Reversion of this serine to the conserved proline residue (S er427 --> Pro) by site-directed mutagenesis conferred the ability to i ncorporate heme on the human placental enzyme. Mutation of the corresp onding proline in rabbit CYP4B1 (Pro422 --> Ser) abolished heme incorp oration. Membrane preparations of human CYP4B1(Pro) and rabbit CYP4B1( Pro), but not the corresponding CYP4B1(Ser) variants, supported lauric acid hydroxylation preferentially at the omega-position. Purified, re constituted human CYP4B1(Pro) and rabbit CYP4B1(Pro) formed 12-hydroxy lauric acid at rates of 17-21 min(-1), and both enzymes were also C-8 to C-10 fatty acid omega-hydroxylases preferentially, with total rates of hydroxylation decreasing in the order C-12 > C-10 > C-9 > C-8. Fin ally, neither human nor rabbit CYP4B1(Pro) formed detectable levels of any hydroxylated testosterone metabolites. Therefore, the presence of a consensus Pro-X-Arg motif is critical for incorporation of the heme prosthetic group in human and rabbit CYP4B1 proteins expressed in ins ect cells. Native human CYP4B1, expressed in vivo, is likely to be fun ctionally impaired if Pro427 is required for holoenzyme expression in mammalian cells.