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 the ir nicotinamide adenine dinucleotide phosphate- (NADPH) dependent flavoprot ein reductases was studied by fluorescence resonance energy transfer. The f luorescent probe 7-(ethylamino)-3-(4'-maleimidylphenyl)-4-methylcoumarin ma leimide (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. T he cross-pairs of the components were also studied. Increasing ionic streng th (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 mu M. This behavior is consistent with the assumption that charge pairing between CPR and P450 2B4 is involved in their association. In contrast, the electr ostatic component of the interaction of the partners in P450BM-3 was shown to have an opposite sign. The isolated BMP and BMR domains have very low af finity for each other and the dissociation constant of their complex decrea ses from 8 to 3 mu M with increasing ionic strength (0.05-0.5 M). Important ly, the BMP-CPR and P450 2B4-BMR "mixed", heterogeneous pairs behave simila rly to the pairs of BMP and P450 2B4 with their native electron donors. The refore, the observed difference in the interaction mechanisms between these two systems is determined mainly by the different structure of the heme pr oteins rather than their flavoprotein counterparts. P450BM-3 is extremely e fficient and highly coupled, with the reductase and the P450 domains tether ed to one another. Therefore, in contrast to P450 2B4-CPR binding, very tig ht binding between the P450BM-3 redox partners would be of no value in the synchronization of complex formation during catalytic turnover.