Coupled motions direct electrons along human microsomal P450 Chains.
Coupled motions direct electrons along human microsomal P450 Chains.
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DOI：
10.1371/journal.pbio.1001222
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发表时间：
2011-12
期刊：
影响因子：
9.8
通讯作者：
Scrutton NS
中科院分区：
文献类型：
--
作者：
Pudney CR;Khara B;Johannissen LO;Scrutton NS
通过生物氧化还原链的定向电子转移可以通过将反应化学与单个氧化还原酶的构象变化相耦合来实现。蛋白质结构域的运动通常与生物的电子转移有关，但运动的一般意义尚不清楚。运动与电子从人细胞色素P450还原酶(CPR)到所有微粒体细胞色素P450(Cyps)的转移有关。我们的假设是，运动与酶化学的紧密耦合可以发出信号，指示电子从CPR向下游Cyps的转移，并支持复杂的氧化还原链上的矢量电子转移。我们开发了一种新的方法来研究催化过程中动态变化的时间依赖性，该方法报告了CPR构象状态的变化。FRET与CPR中电子传递的停流研究有关，CPR中的电子传递包含酶表面的供体-受体荧光团。CPR的开启和关闭状态与催化循环中的关键步骤相关，这表明氧化还原化学和NADPH结合是如何驱动酶的连续开启和关闭的。具体地说，我们提供的证据表明，CPR中黄素部分的减少导致CPR开放，而配体结合导致CPR关闭。建立了一个动态反应循环，其中CPR通过采用闭合状态来优化黄素辅因子之间的内部电子传递，并通过采用更开放的状态来发出信号来与CYP酶配对。这种复杂的、时间上的酶运动控制被用来催化NADPH→FAD→FMN→血红素的定向电子转移，从而促进所有微粒体P450催化的反应。对于CPR更广泛的生物学功能至关重要的运动与人类NADPH-CPR-CYP氧化还原链中的酶化学紧密相连。光是氧化还原化学就足以驱动功能上必要的大规模构象变化。我们的研究不依赖于随机构象采样，而是强调了运动与酶化学的紧密耦合的必要性，以便沿着复杂的氧化还原链进行矢量电子转移。酶是一种蛋白质，它催化大量的化学反应，通常与其他酶合作。我们详细地了解了其中许多反应的化学机制；然而，酶在催化(或蛋白质动力学)过程中的物理运动的重要性正变得越来越明显。在本研究中，我们在一种名为细胞色素P450还原酶(CPR)的酶上放置了荧光标记，以探索随着反应化学的进行蛋白质物理构象的动态变化。CPR催化来自小分子供体(称为NADPH)的电子转移，最终将它们传递给它们的伙伴酶，称为Cyps。我们能够将CPR中特定的构象变化与不同的化学步骤联系起来。我们发现，化学转化本身诱导酶采用其与Cyps有效相互作用所需的构象。这些发现使我们建立了CPR活性的模型，在该模型中，从NADPH到CPR再到CYP的路径上的电子转移与酶的物理构象控制紧密结合。
Directional electron transfer through biological redox chains can be achieved by coupling reaction chemistry to conformational changes in individual redox enzymes. Protein domain motion is often implicated in biological electron transfer, but the general significance of motion is not clear. Motion has been implicated in the transfer of electrons from human cytochrome P450 reductase (CPR) to all microsomal cytochrome P450s (CYPs). Our hypothesis is that tight coupling of motion with enzyme chemistry can signal “ready and waiting” states for electron transfer from CPR to downstream CYPs and support vectorial electron transfer across complex redox chains. We developed a novel approach to study the time-dependence of dynamical change during catalysis that reports on the changing conformational states of CPR. FRET was linked to stopped-flow studies of electron transfer in CPR that contains donor-acceptor fluorophores on the enzyme surface. Open and closed states of CPR were correlated with key steps in the catalytic cycle which demonstrated how redox chemistry and NADPH binding drive successive opening and closing of the enzyme. Specifically, we provide evidence that reduction of the flavin moieties in CPR induces CPR opening, whereas ligand binding induces CPR closing. A dynamic reaction cycle was created in which CPR optimizes internal electron transfer between flavin cofactors by adopting closed states and signals “ready and waiting” conformations to partner CYP enzymes by adopting more open states. This complex, temporal control of enzyme motion is used to catalyze directional electron transfer from NADPH→FAD→FMN→heme, thereby facilitating all microsomal P450-catalysed reactions. Motions critical to the broader biological functions of CPR are tightly coupled to enzyme chemistry in the human NADPH-CPR-CYP redox chain. That redox chemistry alone is sufficient to drive functionally necessary, large-scale conformational change is remarkable. Rather than relying on stochastic conformational sampling, our study highlights a need for tight coupling of motion to enzyme chemistry to give vectorial electron transfer along complex redox chains. Enzymes are proteins that catalyze a large array of chemical reactions, often in partnership with other enzymes. We understand in detail the chemical mechanisms of many of these reactions; however, the importance of the physical movements of enzymes during catalysis (or protein dynamics) is, increasingly, becoming apparent. In this study, we have placed fluorescent markers on an enzyme called cytochrome P450 reductase (CPR) to probe the dynamic changes in the physical conformation of the protein as the reaction chemistry proceeds. CPR catalyses the transfer of electrons from a small molecule donor (called NADPH), ultimately passing them to their partner enzymes called CYPs. We were able to correlate specific conformational changes with distinct chemical steps in CPR. We found that the chemical transformation itself induces the enzyme to adopt conformations that are required for its efficient interaction with CYPs. These findings have allowed us to develop a model of CPR activity in which electron transfer along the pathway from NADPH through CPR to CYP is tightly integrated with physical conformational control of the enzyme.
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DOI：
10.1126/science.1198542
发表时间：
2011-04-08
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Bhabha G;Lee J;Ekiert DC;Gam J;Wilson IA;Dyson HJ;Benkovic SJ;Wright PE
通讯作者：
Wright PE
影响因子：
3.2
作者：
Pudney, Christopher R.;McGrory, Tom;Scrutton, Nigel S.
通讯作者：
Scrutton, Nigel S.
影响因子：
4.8
作者：
Garcin, ED;Bruns, CM;Getzoff, ED
通讯作者：
Getzoff, ED
影响因子：
2.9
作者：
Grunau, A;Paine, MJ;Gutierrez, A
通讯作者：
Gutierrez, A
影响因子：
2.9
作者：
Gutierrez, A;Lian, LY;Roberts, GCK
通讯作者：
Roberts, GCK