flavin-adenine-dinucleotide and 7-ethoxycoumarin

It has been proposed that negatively charged amino acids on the surface of reductase and positively charged amino acids on the surface of P450 mediate the binding of both proteins through electrostatic interactions. In this study, we used a site-directed mutagenesis approach to determine a role for two lysine residues (Lys271 and Lys279) of cytochrome P4501A1 in the interaction of P4501A1 with reductase. We prepared two mutants P4501A1Ile271 and P4501A1Ile279 with a mutation of the lysine at positions 271 and 279, respectively. We observed a strong inhibition (>80%) of the 7-ethoxycoumarin and ethoxyresorufin deethylation activity in the reductase-supported system for both mutants. In the cumene hydroperoxide-supported system, P4501A1Ile279 exhibited wild-type activity, but the P4501A1Ile271 mutant activity remained low. The CD spectrum and substrate-binding assay indicated that the secondary structure of P4501A1Ile271 is perturbed. To evaluate further the involvement of these P4501A1 lysine residues in reductase binding, we measured the KM of reductase for wild type and mutants. Both wild type and P4501A1Ile271 reached saturation in the range of reductase concentrations tested with KM values 5.1 and 11.2 pM, respectively. The calculated KM value for P4501A1Ile279 increased 9-fold, 44.4 pM, suggesting that the mutation affected binding of reductase to P4501A1. Stopped-flow spectroscopy was employed to evaluate the effect of mutations on electron transfer from reductase to heme iron. Both wild type and P450Ile279 showed biphasic kinetics with a approximately 40% participation of the fast step in the total activity. On the other hand, only single-phase kinetics for iron reduction was observed for P450Ile271, suggesting that the low activity of this mutant can be attributed not only to major structural changes but also to a disturbance in the electron transport.

Topics: Amino Acid Substitution; Benzene Derivatives; Binding Sites; Circular Dichroism; Coumarins; Cytochrome P-450 CYP1A1; Electron Transport; Flavin-Adenine Dinucleotide; Heme; Iron; Kinetics; Lysine; Mutation; NADPH-Ferrihemoprotein Reductase; Oxazines; Protein Binding; Protein Structure, Secondary

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 (less than 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., Müller, F., & Visser, A. J. W. G. [(1989) Biochemistry 28, 8416-8425] from time-resolved fluorescence anisotropy measurements.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Binding Sites; Coumarins; Cytochrome P-450 Enzyme System; Energy Metabolism; Energy Transfer; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Fluorescence; Heme; Lipids; Microsomes, Liver; NADPH-Ferrihemoprotein Reductase; Protein Conformation; Rats; Water