Mechanisms of electron transfer in nitric oxide synthases

 描述(申请人提供)：内皮和神经元型一氧化氮合酶(eNOS和nNOS)以钙/钙调蛋白(CaM)依赖的方式产生NO。CaM激活的NO合成需要一个很大的构象变化，黄素单核苷酸(FMN)结构域在NOS的接受电子输入状态和给电子输出状态之间穿梭，从而将电子传递到蛋白质的各个结构域。在输出状态下，FMN和血红素结构域形成对接络合物，从而使FMN和催化血红素中心之间的域间电子转移(IET)成为可能。最近的质谱学和冷冻电子显微镜研究提供了IET和NOS催化过程中构象变化和结构域位置的大致情况。然而，关于CaM如何在一氧化氮合酶输出状态下激活限速的FMN/heme IET仍有许多未知之处。我们实验室的研究表明：(I)CaM通过促进结构域间的FMN/heme相互作用来控制输出态的形成；(Ii)在nNOS FMN结构域中的CaM响应的自动调节插入稳定了输出态；(Iii)观察到的IET速率受到IET能力对接复合体相对较少形成的限制。尽管取得了重大进展，但结构域对接界面上的特定残基在决定FMN/血红素IET动力学&对接的FMN/血红素复合体的构象和群体中的作用仍很大程度上尚不清楚。我们的目标是通过结合激光闪光光解、脉冲电子顺磁共振(EPR)、分子动力学和分子生物学的方法来解决这一差距，以实现在分子水平上理解CaM对NOS输出状态的激活。根据最近的研究结果，总体假设是特定的NOS/CaM相互作用和内在的NOS控制元件通过促进FMN/Home域间的对接来协同调节NOS的功能。在目标1中，我们将通过重点研究NOS血红素结构域的突变和预测的CaM/血红素结构域界面上的CaM表面残基来识别特定的NOS和CaM相互作用位点。我们将使用IET动力学和脉冲EPR来衡量特定突变造成的变化。这将揭示特定残基在促进FMN血红素IE以及在血红素结构域、FMN结构域和CaM之间形成界面方面的作用。目的2将通过集中于中风干预潜在重要部位的eNOS磷酸化来确定CaM反应控制元件如何调节一氧化氮合酶功能。我们将分析拟磷突变体与体外磷酸化eNOS的偶联，以确定磷酸化在eNOS调控中的机制作用。这些研究将对CaM控制的NO生成的NOS输出态的形成提供重要的分子基础，并可能有助于合理开发针对与电子转移相关的结构域对接界面的新的选择性NOS调节剂。

 DESCRIPTION (provided by applicant): Endothelial & neuronal NO synthase enzymes (eNOS & nNOS) produce NO in a Ca2+/calmodulin (CaM) dependent manner. CaM-activated NO synthesis requires a large conformational change, in which the flavin mononucleotide (FMN) domain shuttles between NOS's electron-accepting input state and electron-donating output state to deliver electrons across the domains of the protein. In the output state, the FMN and heme domains form a docking complex, thus enabling interdomain electron transfer (IET) between the FMN and the catalytic heme center. Recent mass spectrometry and cryo-electron microscopy studies have provided a general view of the conformational changes and domain placement during the IET and NOS catalysis. Yet, there is still much unknown about how CaM activates the rate-limiting FMN/heme IET in the NOS output state. Research from our lab demonstrates that: (i) CaM controls formation of the output state by facilitating interdomain FMN/heme interactions; (ii) a CaM-responsive autoregulatory insert in the nNOS FMN domain stabilizes the output state; and (iii) the observed IET rate is limited by the relatively infrequen formation of the IET-competent docking complex. Despite significant progress, the roles of specific residues at the domain docking interfaces in determining the FMN/heme IET kinetics & conformation and population of the docked FMN/heme complex remain largely unclear. Our Aims address this gap through combined approaches of laser flash photolysis, pulsed electron paramagnetic resonance (EPR), molecular dynamics, and molecular biology, to achieve a molecular-level understanding of CaM-activation of the NOS output state. Based on recent results, the overall hypothesis is that specific NOS/CaM interactions and intrinsic NOS control elements synergistically regulate NOS function by facilitating the FMN/heme interdomain docking. In Aim 1, we will identify specific NOS and CaM interaction sites by focusing on mutations in the NOS heme domain and CaM surface residues at the predicted CaM/heme domain interface. We will use IET kinetics and pulsed EPR to measure changes resulting from specific mutations. This will reveal the roles of specific residues in facilitating the FMN heme IE and in forming the interfaces between the heme domain, FMN domain, and CaM. Aim 2 will determine how CaM-responsive control elements regulate NOS function by focusing on eNOS phosphorylations at the sites of potential importance for stroke intervention. We will analyze phosphomimetic mutants coupled with in vitro phosphorylated eNOS to define mechanistic roles of the phosphorylations in eNOS regulation. The proposed studies will provide significant insights into the molecular underpinnings of CaM-controlled formation of the NOS output state for NO production, and may help rational development of new selective NOS modulators targeting the domain docking interfaces related to the electron transfer.