Regulation of Nitric Oxide Synthase through Formation of the Output State

描述（由申请人提供）：一氧化氮合酶（NOS）的生物合成是如何在分子水平上受到严格调控的，目前还不清楚。这是值得注意的，因为体内一氧化氮产生的偏差与越来越多缺乏有效治疗的严重疾病有关，包括中风、感染性休克和癌症。与诱导型NOS不同，内皮和神经元NOS异构体（eNOS和nNOS）是由钙调素（CaM）通过促进具有催化意义的结构域间电子转移（IET）过程来控制的。CaM通过黄素单核苷酸（FMN）结构域的构象变化，使其从受屏蔽的电子接受（输入）态转变为新的电子提供（输出）态，从而激活eNOS和nNOS中NO的合成。NOS输出状态下的fmn -血红素IET对催化血红素合成NO至关重要。然而，NOS输出态的形成机制尚不清楚，这是在分子水平上理解NOS催化调控的关键障碍。本研究的重点是探讨cam激活的全长人eNOS和nNOS酶输出态形成的分子机制。我们假设FMN/血红素的相互作用、CaM与NOS的特异性结合以及FMN结构域中独特的自抑制插入物协同控制了NO产生的输出状态的形成。这一假设将通过定量动力学的离散fmn -血红素IET步骤在酶通过三个互补和协同的目的进行测试。我们已经开发了创新的激光闪光光解方法来确定fmn -血红素IET动力学，作为NOS输出状态形成的直接测量。实验设计将整合我们的激光闪光光解方法和最先进的脉冲电子顺磁共振（EPR）技术与定点诱变。本研究将显著提高对NOS分子水平调控的基本认识，并将为NOS如何选择性调节以达到治疗目的提供新的重要见解。由于缺乏确定fmn -血红素IET动力学的可靠技术，NOS输出状态的形成机制长期未得到充分研究。我们创新的激光闪光光解方法为通过形成输出状态来详细研究NOS调节奠定了基础。鉴于这项实验冒险的新颖性和探索性，这项研究非常适合获得R21奖。

DESCRIPTION (provided by applicant): There is still much unknown about how nitric oxide (NO) biosynthesis by NO synthase (NOS) is tightly regulated at the molecular level. This is remarkable because deviated NO production in vivo has been implicated in an increasing number of serious diseases lacking effective treatments, including stroke, septic shock and cancer. Unlike inducible NOS, endothelial and neuronal NOS isoforms (eNOS and nNOS) are controlled by calmodulin (CaM) through facilitating catalytically significant interdomain electron transfer (IET) processes. It is proposed that CaM activates NO synthesis in eNOS and nNOS through a conformational change of the flavin mononucleotide (FMN) domain from its shielded electron-accepting (input) state to a new electron-donating (output) state. The FMN-heme IET within the NOS output state is essential for NO synthesis at the catalytic heme. However, the mechanism for formation of the NOS output state remains unclear, and this stands as a critical barrier for understanding regulation of NOS catalysis at the molecular level. The focus of this study is to investigate the molecular mechanism of CaM-activated output state formation in full length human eNOS and nNOS enzymes. We hypothesize that productive FMN/heme interactions, specific binding of CaM to NOS, and unique autoinhibitory insert in the FMN domain synergistically control formation of the output state for NO production. This hypothesis will be tested by quantitating kinetics of the discrete FMN-heme IET step in the enzymes through three complementary and synergistic Aims. We have developed innovative laser flash photolysis approaches to determine the FMN-heme IET kinetics as a direct measure of formation of the NOS output state. The experimental design will integrate our laser flash photolysis methodology and state-of-art pulsed electron paramagnetic resonance (EPR) techniques with site-directed mutagenesis. This study will significantly improve the fundamental understanding of NOS regulation at the molecular level, and will provide new important insight as to how NOS might be selectively modulated for therapeutic purposes. The mechanism for formation of the NOS output state has long been understudied due to lack of reliable techniques for determining the FMN-heme IET kinetics. Our innovative laser flash photolysis methodology sets the stage for a detailed investigation of NOS regulation through formation of the output state. Given the novel and exploratory nature of this experimental venture, this study is well suited for an R21 award.