Association of cytochromes P450 with their reductases: Opposite sign of the electrostatic interactions in P450BM-3 as compared with the microsomal 2B4 system
Association of cytochromes P450 with their reductases: Opposite sign of the electrostatic interactions in P450BM-3 as compared with the microsomal 2B4 system
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DOI：
10.1021/bi992936u
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发表时间：
2000-05-30
期刊：
影响因子：
2.9
通讯作者：
Peterson, JA
中科院分区：
文献类型：
--
作者：
Davydov, DR;Kariakin, AA;Peterson, JA
利用荧光共振能量转移技术研究了静电相互作用在P450与其依赖于NADPH的黄素蛋白还原酶之间的相互作用。将荧光探针7-（乙氨基）-3-（4 '-马来酰亚胺基苯基）-4-甲基香豆素马来酰亚胺（coumarylphenylmaleimide，CPM）以1：1的摩尔比引入黄素蛋白分子中。比较了P450 2B 4与兔肝微粒体NADPH P450还原酶（CPR）、p450 BM-3的血红素结构域（BMP）和黄素蛋白结构域（BMR）的相互作用。还研究了组分的交叉对。增加离子强度（0.05-0.5 M）显示导致CPR-P450 2B 4复合物的解离，解离常数从0.01 μ M增加到0.09 μ M。这种行为与CPR和P450 2B 4之间的电荷配对参与其关联的假设一致。相反，在P450 BM-3的合作伙伴的相互作用的静电分量被证明有一个相反的符号。分离的BMP和BMR结构域彼此具有非常低的亲和力，并且它们的复合物的解离常数随着离子强度的增加（0.05-0.5 M）从8 μ M降低到3 μ M。重要的是，BMP-CPR和P450 2B 4-BMR“混合”的异质对与BMP和P450 2B 4与其天然电子供体的对的行为相似。因此，这两个系统之间的相互作用机制中观察到的差异主要是由血红素蛋白的不同结构决定的，而不是它们的黄素蛋白对应物。P450 BM-3是非常有效和高度耦合的，还原酶和P450结构域相互连接。因此，与P450 2B 4-CPR结合相反，P450 BM-3氧化还原配偶体之间的非常紧密的结合在催化周转期间复合物形成的同步中没有价值。
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 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 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 mu M 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.