CHARACTERIZATION OF THE FAD BINDING DOMAIN OF CYTOCHROME-P450 REDUCTASE

The redox potentials of FAD and FMN of Cytochrome P450 reductase (redu ctase) are equivalent in solution but differ by 138 mV when bound to r eductase. The interaction of each flavin with its flavin binding domai n confers the unique electron transferring abilities to each flavin. I n order to determine flavin binding properties and activity of the FAD binding domain, we have expressed in pTrcHis three fragments (1161, 1 244, and 1556 bp) of rat liver reductase cDNA encompassing the propose d FAD and NADPH binding domain. The FAD binding fragments from cells h arboring the 1161- and 1556-bp-containing vectors were stable and boun d 0.66 and 0.71 mol FAD/mol enzyme, respectively. Both fragments reduc e ferricyanide (54 and 104% of FMN-less reductase/mol bound flavin, re spectively) and participate in the transhydrogenation reaction of 3-Ac Py-ADP (41 and 65% of FMN-less reductase/mol bound flavin, respectivel y). FAD-less fragments were purified and reconstituted with 8-amino-FA D and 8-chloro-FAD to determine binding efficiencies. (C) 1996 Academi c Press, Inc.