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 microM. 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 partners in P450BM-3 was shown to have an opposite sign. The isolated BMP and BMR domains have very low affinity for each other and the dissociation constant of their complex decreases from 8 to 3 microM with increasing ionic strength (0.05-0.5 M). Importantly, the BMP-CPR and P450 2B4-BMR "mixed", heterogeneous pairs behave similarly to the pairs of BMP and P450 2B4 with their native electron donors. Therefore, the observed difference in the interaction mechanisms between these two systems is determined mainly by the different structure of the heme proteins rather than their flavoprotein counterparts. P450BM-3 is extremely efficient and highly coupled, with the reductase and the P450 domains tethered to one another. Therefore, in contrast to P450 2B4-CPR binding, very tight binding between the P450BM-3 redox partners would be of no value in the synchronization of complex formation during catalytic turnover.
Davydov
D R
DR
Institute of Biomedical Chemistry, Russian Academy of Medical Science, Moscow.
Kariakin
A A
AA
Petushkova
N A
NA
Peterson
J A
JA
D000818:Animals; D001426:Bacterial Proteins; D001665:Binding Sites; D003374:Coumarins; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism*; D005456:Fluorescent Dyes; D007700:Kinetics; D008301:Maleimides; D008862:Microsomes, Liver / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D055672:Static Electricity
D001426:Bacterial Proteins; D003374:Coumarins; D005456:Fluorescent Dyes; D008301:Maleimides; D003577:Cytochrome P-450 Enzyme System; C074193:N-(4-(7-(diethylamino)-4-methylcoumarin-3-yl))maleimide; D006899:Mixed Function Oxygenases; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases