An enzymatically active chimeric protein containing the hydrophilic form of NADPH-cytochrome p450 reductase fused to the membrane-binding domain of cytochrome b(5)

The microsomal flavoprotein NADPH-cytochrome P450 reductase (CPR) contains an N-terminal hydrophobic membrane-binding domain required for reconstituti on of hydroxylation activities with cytochrome P450s. In contrast, cytochro me b(5) (b(5)) contains a C-terminal hydrophobic membrane-binding domain re quired for interaction with P450s. We have constructed, expressed and purif ied a chimeric flavoprotein (hdb5-CPR) where the C-terminal 45 amino acid r esidues of b(5) have replaced the N-terminal 56 amino acid domain of CPR. T his hybrid flavoprotein retains the catalytic properties of the native CPR and is able to reconstitute fatty acid and steroid hydroxylation activities with CYP4A1 and CYP17A. However hdb5-CPR is much less effective than CPR f or reconstituting activity with CYP3A4, We conclude that differences on the surface of the P450s reflect unique and specific information essential for the recognition needed to establish reactions of intermolecular electron t ransfer from the flavoprotein CPR. (C) 2001 Academic Press.