Association of cytochromes P450 with their reductases: opposite sign of the electrostatic interactions in P450BM-3 as compared with the microsomal 2B4 system.

The role of electrostatic interactions in the association of P450s with their nicotinamide adenine dinucleotide phosphate- (NADPH) dependent flavoprotein reductases was studied by fluorescence resonance energy transfer. The fluorescent probe 7-(ethylamino)-3-(4‘-maleimidylphenyl)-4-methylcoumarin maleimide (coumarylphenylmaleimide, CPM) was introduced into the flavoprotein molecule at a 1:1 molar ratio. The interaction of P450 2B4 and NADPH−P450 reductase (CPR) from rabbit liver microsomes was compared with that of the isolated heme domain (BMP) and the flavoprotein domain (BMR) of P450BM-3. The cross-pairs of the components were also studied. Increasing ionic strength (0.05−0.5 M) was shown to result in the dissociation of the CPR−P450 2B4 complex with the dissociation constant increasing from 0.01 to 0.09 μM. This behavior is consistent with the assumption that charge pairing between CPR and P450 2B4 is involved in their association. In contrast, the electrostatic component of the interaction of the par...