Structure-Function and Reacation Mechanism of eNOS

本研究的总体目标是为四氢生物蝶呤（BH4）的功能作用和内皮型一氧化氮合酶（eNOS）的整体反应机制提供分子认识。要验证的机制假设是基于钙调素（CaM/Ca +2）介导的还原酶和P450氧化酶之间的3/2偶联模型。为了验证第一个假设，即BH4及其自由基中间体之间的有效氧化还原循环需要NO合酶而不是超氧化物合酶活性，我们将获得不同生物蝶素氧化还原物种包括其自由基中间体在eNOS单次和多次转换反应中的完整动力学数据。生物蝶呤自由基的动力学将与其他氧化还原中心的氧化还原变化以及底物的消失和产物的形成相关联，以解决加氧酶结构域的完整催化机制。为了解决蛋白质和血红素对生物蝶呤氧化还原行为的影响，将产生涉及BH4结合和血红素反应性的关键残基的位点特异性突变。几种BH4类似物将在Aim 1中描述的动力学实验中使用。生物蝶呤自由基的结构将通过不同的光谱方法和DFT计算来表征。为了验证最后一个假设，即黄素的氧化还原状态和CaM的结合对还原酶和加氧酶活性的总体周转率和偶联起决定性作用，我们将研究在CaM存在和不存在的情况下还原酶结构域的氧化和还原反应机制。我们希望找到限速步骤，与血红素还原偶联的半反应，定义内部电子转移和构象门控。区域通信将通过操纵自抑制序列来评估，并评估CaM对电子转移的影响。第一个目的是研究加氧酶结构域的反应机理，以及BH4作为加氧酶两步反应的电子供体，以及在引导NO生成而不是硝基（HNO）或超氧化物（O2-）生成中的作用。第二个目标将提供关于BH4和血红素之间的结构/功能关系以及调节BH4氧化还原行为的因素的关键信息。最后的目的是了解还原酶和CaM在调节氧化还原偶联与加氧酶结构域中的作用，从而阐明整个eNOS的反应机制。

The overall goal of this proposal is to provide a molecular understanding of the functional role of tetrahydrobiopterin (BH4) and the overall reaction mechanism of endothelial-type nitric oxide synthase (eNOS). The mechanistic hypothesis to be tested is based on a 3/2 coupling model between the reductase and the P450 oxidase mediated by calmodulin, CaM/Ca +2.To test the first hypothesis that efficient redox cycling between BH4 and its radical intermediate warrants a NO synthase rather than a superoxide synthase activity, we will obtain complete kinetic data of different biopterin redox species including its radical intermediate during single and multiple turnover reaction of eNOS. The kinetics of the biopterin radical will be correlated with redox change of other redox centers and the substrate disappearance and product formation to resolve the full catalytic mechanism of the oxygenase domain. To address the protein and heme effect on the biopterin redox behavior site-specific mutation of critical residues involved in BH4 binding and heme reactivity will be generated. Several BH4 analogs will be utilized in the kinetic experiments described in Aim 1. The structure of the biopterin radical will be characterized by different spectroscopic methods and DFT calculations. To test the last hypotheses that redox state of the flavins and the binding of CaM have deciding effect on the overall turnover rate and coupling of the reductase and oxygenase activities, we will look into the oxidative and reductive reaction mechanism of the reductase domain in the presence and absence of CaM. We wish to locate the rate-limiting step(s), the half-reaction that couples to heme reduction, defining the internal electron transfer and conformational gating. Domain communication will be assessed by manipulating the autoinhibitory sequence and to evaluate the effect of CaM on electron transfer. The first Aim is to address the reaction mechanism of the oxygenase domain and the function of BH4 as the electron donor for the two steps of oxygenase reaction, and in steering NO formation instead of nitroxyl (HNO) or superoxide (O2-) production. The second aim will provide critical information regarding the structure/function relationship between the BH4 and heme and the factors that regulate BH4 redox behaviors. The last Aim will provide understanding of the role of reductase and CaM in regulating the redox coupling with the oxygenase domain thus lead to an elucidation of the reaction of the reaction mechanism of the whole eNOS.