Reconstitution of the Enzymatic Activities of Cytochrome P450s Using Recombinant Flavocytochromes Containing Rat Cytochrome b5 Fused to NADPH–Cytochrome P450 Reductase with Various Membrane-Binding Segments

The role of the hydrophobic membrane-binding segments of NADPH–cytochrome P450 reductase (CPR) and cytochrome b5 remain undefined. We have expressed four different recombinant flavocytochromes containing b5 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 paper. Of interest is the presence of internal “membrane-binding” hydrophobic segments in these flavocytochromes. This paper describes the ability of these flavocytochromes to reconstitute in vitro two P450 activities that have been reported to be stimulated by the addition of b5 (the 17,20-lyase activity of CYP17A and the 6β hydroxylation of testosterone catalyzed by CYP3A4) and two P450 reactions that do not respond to the presence of b5 (the 17α-hydroxylation of progesterone catalyzed by CYP17A and the omega hydroxylation of lauric acid catalyzed by CYP4A1). The present study shows that a hydrophobic “membrane-binding” segment must be present in the artificial flavocytochromes in order to successfully reconstitute in vitro hydroxylation 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 in the purified recombinant flavocytochromes. The hypothesis is proposed that differences in the surface topology of a P450 may dictate differences in their docking with the CPR or b5 component of a fusion protein, resulting in differences in the rates of electron transfer to the P450.