LOCATION OF FUNCTIONAL CENTERS IN THE MICROSOMAL CYTOCHROME-P450 SYSTEM

Fluorescence quenching and energy-transfer studies have been carried out to determine the position of FAD and FMN groups of NADPH-cytochrome P450 reductase and of the heme and substrate groups of cytochrome P450 with respect to the lipid/water interphase. Quenching by iodine of the fluorescence of the flavins of the reductase shows a biphasic pattern, due to the different accessibility of FAD and FMN to the solvent with Stern-Volmer constants of 7.9 X 10(-4) and 2.7 x 10(-3) mM-1, respectively. Both prosthetic groups appear to be buried within the three-dimensional structure of the native reductase, FAD more deeply embedded than FMN and with a relative contribution to the total fluorescence of flavins of 84% (FAD) and 16% (FMN). The lack of significant energy transfer (<5%) from FAD + FMN to the rhodamine group of the N-labeled phosphatidylethanolamine incorporated in membranes reconstituted with NADPH-cytochrome P450 reductase and phosphatidylcholine points out that both groups are located at a distance greater than 5 nm from the lipid/water interphase. Steady-state fluorescence intensity and anisotropy data obtained with native and FMN-depleted NADPH-cytochrome P450 reductase show that energy transfer between both prosthetic groups occurs in the native reductase with an efficiency of ca. 31%, consistent with a separation between these groups of 2 nm as suggested earlier by Bastiaens, P. I. H., Bonants, P. J. M., Muller, F., & Visser, A. J. W. G. [(1989) Biochemistry 28, 8416-8425] from time-resolved fluorescence anisotropy measurements. The efficiency of energy transfer between the donor/acceptor pairs DPH/heme or 4-heptadecyl-7-hydroxycoumarin/heme allows us to estimate that the heme group is approximately 5 nm from the lipid/water interphase. The binding site of the substrate 7-ethoxycoumarin in cytochrome P450 is located at less than 5 nm from the heme group, and not at the lipid surface.