The Hinge Segment of Human NADPH-Cytochrome P450 Reductase in Conformational Switching: The Critical Role of Ionic Strength.
The Hinge Segment of Human NADPH-Cytochrome P450 Reductase in Conformational Switching: The Critical Role of Ionic Strength.
复制标题
DOI：
10.3389/fphar.2017.00755
复制
发表时间：
2017
影响因子：
5.6
通讯作者：
Kranendonk M
中科院分区：
文献类型：
--
作者：
Campelo D;Lautier T;Urban P;Esteves F;Bozonnet S;Truan G;Kranendonk M
NADPH-细胞色素P450还原酶(CPR)是微粒体细胞色素P450的氧化还原伙伴，是双黄素还原酶家族的原型。CPR包含3个不同的功能结构域：FMN结合域(受体还原)、连接区(铰链)和连接/FAD结构域(NADPH氧化)。已经证明，CPR的机制显示出一个重要的步骤，它从紧凑的闭合构象(锁定状态)转换到开放构象的集合(非锁定状态)，后者使电子转移到氧化还原伙伴。锁定和非锁定状态之间的构象平衡高度依赖于离子强度，这支持了在FMN和连接的FAD结构域之间的界面存在临界盐相互作用的假设。在这里，我们证明了铰链片段的特定残基在控制CPR的构象平衡中起着重要作用。我们构建了6个人CPR单突变体和2个双突变体，分别针对铰链片段的G240、S243、I245和R246残基，目的是改变铰链片段的灵活性或潜在的离子相互作用。我们测量了这8个突变体在不同盐浓度下细胞色素c的还原，无论是可溶性的还是膜结合的人CPR。所有突变体都能还原细胞色素c，但效率不同，最大细胞色素c还原速率向低盐浓度移动。特别是，残基R246似乎在铰链和连接结构域的界面上存在的盐桥网络中起着关键作用。有趣的是，突变的影响虽然相似，但在可溶性或膜结合的环境中表现出特定的差异。我们的结果表明，铰链链段的静电和柔性特性对于电子从CPR到其氧化还原伙伴的转移是至关重要的。
NADPH-cytochrome P450 reductase (CPR) is a redox partner of microsomal cytochromes P450 and is a prototype of the diflavin reductase family. CPR contains 3 distinct functional domains: a FMN-binding domain (acceptor reduction), a linker (hinge), and a connecting/FAD domain (NADPH oxidation). It has been demonstrated that the mechanism of CPR exhibits an important step in which it switches from a compact, closed conformation (locked state) to an ensemble of open conformations (unlocked state), the latter enabling electron transfer to redox partners. The conformational equilibrium between the locked and unlocked states has been shown to be highly dependent on ionic strength, reinforcing the hypothesis of the presence of critical salt interactions at the interface between the FMN and connecting FAD domains. Here we show that specific residues of the hinge segment are important in the control of the conformational equilibrium of CPR. We constructed six single mutants and two double mutants of the human CPR, targeting residues G240, S243, I245 and R246 of the hinge segment, with the aim of modifying the flexibility or the potential ionic interactions of the hinge segment. We measured the reduction of cytochrome c at various salt concentrations of these 8 mutants, either in the soluble or membrane-bound form of human CPR. All mutants were found capable of reducing cytochrome c yet with different efficiency and their maximal rates of cytochrome c reduction were shifted to lower salt concentration. In particular, residue R246 seems to play a key role in a salt bridge network present at the interface of the hinge and the connecting domain. Interestingly, the effects of mutations, although similar, demonstrated specific differences when present in the soluble or membrane-bound context. Our results demonstrate that the electrostatic and flexibility properties of the hinge segment are critical for electron transfer from CPR to its redox partners.
登录
查看更多内容
影响因子：
2.9
作者：
Davydov, DR;Kariakin, AA;Peterson, JA
通讯作者：
Peterson, JA
影响因子：
3.4
作者：
Frances, Oriane;Fatemi, Fataneh;Truan, Gilles
通讯作者：
Truan, Gilles
影响因子：
2.9
作者：
Grunau, A;Paine, MJ;Gutierrez, A
通讯作者：
Gutierrez, A
影响因子：
4.8
作者：
Bridges, A;Gruenke, L;Waskell, L
通讯作者：
Waskell, L
影响因子：
3.8
作者：
Murataliev, MB;Ariño, A;Feyereisen, R
通讯作者：
Feyereisen, R