Regulation of Nitric Oxide Synthase through Formation of the Output State

描述(申请人提供)：关于一氧化氮合酶(NOS)如何在分子水平上严格调控一氧化氮(NO)的生物合成，仍有许多未知之处。这是值得注意的，因为体内NO产生的偏差与越来越多的严重疾病有关，这些疾病缺乏有效的治疗，包括中风、感染性休克和癌症。与诱导型一氧化氮合酶不同，内皮和神经元型一氧化氮合酶亚型(eNOS和nNOS)是由钙调素(CaM)通过促进催化显著的域间电子转移(IET)过程控制的。有人认为，CaM通过黄素单核苷酸(FMN)结构域从屏蔽的电子接受(输入)状态到新的电子给予(输出)状态的构象变化来激活eNOS和nNOS中NO的合成。一氧化氮合酶输出状态中的FMN-血红素IET对于催化血红素合成NO是必不可少的。然而，一氧化氮合酶输出状态的形成机制尚不清楚，这是从分子水平上理解一氧化氮合酶催化调控的关键障碍。本研究的重点是探讨人全长eNOS和nNOS酶中CaM激活的产出态形成的分子机制。我们假设，生产性的FMN/血红素相互作用，CaM与NOS的特异性结合，以及FMN结构域中独特的自抑制插入，协同控制NO产生的输出态的形成。这一假设将通过三个互补和协同的目标对酶中离散的FMN-血红素IET步骤的动力学进行量化来验证。我们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，作为形成NOS输出态的直接测量。实验设计将把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)技术与定点突变结合起来。这项研究将在分子水平上显著提高对一氧化氮合酶调控的基本理解，并将为如何选择性地调节一氧化氮合酶以达到治疗目的提供新的重要见解。长期以来，由于缺乏可靠的技术来确定FMN-血红素IET动力学，一氧化氮合酶输出态的形成机制一直被研究得不够。我们创新的激光闪光光解方法为通过形成输出态详细研究一氧化氮合酶的调控奠定了基础。鉴于这一实验项目的新颖性和探索性，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.