A Well-Balanced Preexisting Equilibrium Governs Electron Flux Efficiency of a Multidomain Diflavin Reductase
A Well-Balanced Preexisting Equilibrium Governs Electron Flux Efficiency of a Multidomain Diflavin Reductase
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DOI：
10.1016/j.bpj.2015.01.032
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发表时间：
2015-03-24
影响因子：
3.4
通讯作者：
Truan, Gilles
中科院分区：
文献类型：
--
作者：
Frances, Oriane;Fatemi, Fataneh;Truan, Gilles
二黄素还原酶是双结构域电子转移蛋白，其结构重定向是必要的，以解释各种分子内和分子间的电子转移步骤。利用小角度x射线散射和核磁共振数据，我们描述了nadph -细胞色素P450还原酶（CPR）在不同实验条件下的构象自由能图，这是一种典型的由两个共价结合的黄素结构域组成的双结构域氧化还原酶。CPR酶存在于先前描述的刚性、锁定状态和新特征的高度柔性、解锁状态之间的盐和ph依赖的快速平衡中。我们进一步证明，在静息条件下，最大电子通量是由锁态域界面的可调稳定性决定的。这是合理的动力学方案耦合快速构象采样和缓慢的化学反应速率。因此，据我们所知，在催化循环中与快速随机区域接触相关的调节区域界面稳定性为提高我们对多区域酶功能的理解提供了一个新的范例。
Diflavin reductases are bidomain electron transfer proteins in which structural reorientation is necessary to account for the various intramolecular and intermolecular electron transfer steps. Using small-angle x-ray scattering and nuclear magnetic resonance data, we describe the conformational free-energy landscape of the NADPH-cytochrome P450 reductase (CPR), a typical bidomain redox enzyme composed of two covalently-bound flavin domains, under various experimental conditions. The CPR enzyme exists in a salt- and pH-dependent rapid equilibrium between a previously described rigid, locked state and a newly characterized, highly flexible, unlocked state. We further establish that maximal electron flux through CPR is conditioned by adjustable stability of the locked-state domain interface under resting conditions. This is rationalized by a kinetic scheme coupling rapid conformational sampling and slow chemical reaction rates. Regulated domain interface stability associated with fast stochastic domain contacts during the catalytic cycle thus provides, to our knowledge, a new paradigm for improving our understanding of multidomain enzyme function.