Reconstitution of the enzymatic activities of cytochrome P450s using recombinant flavocytochromes containing rat cytochrome b(5) fused to NADPH-cytochrome P450 reductase with various membrane-binding segments

The role of the hydrophobic membrane-binding segments of NADPH-cytochrome P 450 reductase (CPR) and cytochrome b(5) remain undefined, We have expressed four different recombinant flavocytochromes containing b(5) linked to CPR with different hydrophobic segments as linkers. These fusion proteins have been expressed in Escherichia coli and purified and some of their physical properties and electron transfer activities described in the accompanying p aper, Of interest is the presence of internal "membrane-binding" hydrophobi c segments in these flavocytochromes. This paper describes the ability of t hese flavocytochromes to reconstitute in vitro two P450 activities that hav e been reported to be stimulated by the addition of b(5) (the 17,20-lyase a ctivity of CYP17A and the 6 beta hydroxylation of testosterone catalyzed by CYP3A4) and two P450 reactions that do not respond to the presence of b(5) (the 17 alpha -hydroxylation of progesterone catalyzed by CYP17A and the o mega hydroxylation of lauric acid catalyzed by CYP4A1). The present study s hows that a hydrophobic "membrane-binding" segment must be present in the a rtificial flavocytochromes in order to successfully reconstitute in vitro h ydroxylation activities with P450s. Differences in the effectiveness of the different flavocytochromes to reconstitute enzymatic activities depends on the P450 tested and the nature of the hydrophobic linker segment present i n the purified recombinant flavocytochromes, The hypothesis is proposed tha t differences in the surface topology of a P450 may dictate differences in their docking with the CPR or b(5) component of a fusion protein, resulting in differences in the rates of electron transfer to the P450. (C) 2001 Aca demic Press.