Energy landscapes and catalysis in nitric-oxide synthase.
Energy landscapes and catalysis in nitric-oxide synthase.
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一氧化物合酶中的能量景观和催化。
DOI：
10.1074/jbc.m114.548834
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发表时间：
2014-04-25
期刊：
影响因子：
--
通讯作者：
Scrutton NS
中科院分区：
文献类型：
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作者：
Sobolewska-Stawiarz A;Leferink NGH;Fisher K;Heyes DJ;Hay S;Rigby SEJ;Scrutton NS
背景：蛋白结构域动力学和钙调素结合参与了NO合酶中电子流的调节。结果：展示了一个对酶催化重要的动态构象景观。结论：NO的合成需要一个复杂的构象景观，钙调素作为一个关键的化学驱动因素，通过调节动态景观。意义：详细了解构象景观为发现针对动态界面的抑制剂提供了新的机会。一氧化氮（NO）在哺乳动物生理中起着多种作用。它参与血压调节、神经传递和免疫反应，是由NO合成酶（NOS）催化的复杂电子转移反应产生的。在神经元NOS （nNOS）中，蛋白质结构域动力学和钙调素结合参与调节NADPH通过FAD和FMN辅助因子向血红素加氧酶结构域（NO生成位点）的电子流。基于nNOS还原酶晶体结构的简单模型表明，fmn结合结构域在fad结合结构域与血红素加氧酶结构域之间穿梭电子的大规模运动中起着重要作用。然而，在酶催化过程中，NOS酶的动态结构转变的分子水平的洞察力是缺乏的。我们利用脉冲电子-电子双共振谱法推导了nNOS多种构象态的域间距离关系。通过电子转移和周转的变压动力学研究，这些距离关系与酶活性相关。NADPH和钙调素的结合显示影响域间距离关系以及反应化学。钙调素结合的一个重要作用是抑制nNOS向分子氧的非定式电子转移，从而防止活性氧的积累。除了由nNOS还原酶的静态晶体结构推导的简单模型外，还需要一个复杂的构象景观来催化nNOS。对这一景观的详细了解有助于我们对nNOS催化/电子转移的理解，并可能为发现在这一多维能量景观的动态蛋白质界面结合的小分子抑制剂提供新的机会。
Background: Protein domain dynamics and calmodulin binding are implicated in regulating electron flow in NO synthase. Results: A dynamic conformational landscape important for enzyme catalysis is demonstrated. Conclusion: NO synthesis requires a complex landscape of conformations, with calmodulin as a key driver of chemistry through modulation of the dynamic landscape. Significance: Detailed understanding of conformational landscapes provides new opportunities for inhibitor discovery targeted at the dynamic interfaces. Nitric oxide (NO) plays diverse roles in mammalian physiology. It is involved in blood pressure regulation, neurotransmission, and immune response, and is generated through complex electron transfer reactions catalyzed by NO synthases (NOS). In neuronal NOS (nNOS), protein domain dynamics and calmodulin binding are implicated in regulating electron flow from NADPH, through the FAD and FMN cofactors, to the heme oxygenase domain, the site of NO generation. Simple models based on crystal structures of nNOS reductase have invoked a role for large scale motions of the FMN-binding domain in shuttling electrons from the FAD-binding domain to the heme oxygenase domain. However, molecular level insight of the dynamic structural transitions in NOS enzymes during enzyme catalysis is lacking. We use pulsed electron-electron double resonance spectroscopy to derive inter-domain distance relationships in multiple conformational states of nNOS. These distance relationships are correlated with enzymatic activity through variable pressure kinetic studies of electron transfer and turnover. The binding of NADPH and calmodulin are shown to influence interdomain distance relationships as well as reaction chemistry. An important effect of calmodulin binding is to suppress adventitious electron transfer from nNOS to molecular oxygen and thereby preventing accumulation of reactive oxygen species. A complex landscape of conformations is required for nNOS catalysis beyond the simple models derived from static crystal structures of nNOS reductase. Detailed understanding of this landscape advances our understanding of nNOS catalysis/electron transfer, and could provide new opportunities for the discovery of small molecule inhibitors that bind at dynamic protein interfaces of this multidimensional energy landscape.