Regulation of Nitric Oxide Synthase through Formation of the Output State

描述（由申请人提供）：关于NO合酶（NOS）如何在分子水平上严格调节一氧化氮（NO）生物合成，仍有很多未知之处。这是值得注意的，因为体内偏离的NO产生与越来越多的缺乏有效治疗的严重疾病有关，包括中风、败血性休克和癌症。与诱导型NOS不同，内皮型和神经型NOS亚型（eNOS和nNOS）通过促进催化显著的结构域间电子传递（IET）过程由钙调素（CaM）控制。有人提出，钙调素激活NO合成eNOS和nNOS通过构象变化的黄素单核苷酸（FMN）域从其屏蔽的电子接受（输入）状态到一个新的电子捐赠（输出）状态。在NOS输出状态内的FMN-heme IET对于催化血红素处的NO合成是必不可少的。然而，形成NOS输出状态的机制仍不清楚，这是理解NOS催化在分子水平上的调节的关键障碍。本研究的重点是探讨全长人eNOS和nNOS酶中CaM激活的输出状态形成的分子机制。我们假设，生产FMN/血红素相互作用，特异性结合的钙调素NOS，和独特的自抑制插入FMN域协同控制形成的输出状态NO生产。这一假设将通过三个互补和协同的目的，通过定量动力学的离散FMN-血红素IET步骤的酶进行测试。我们已经开发了创新的激光闪光光解方法来确定FMN-heme 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。这项研究将显着提高在分子水平上的NOS调节的基本理解，并将提供新的重要的见解，如何NOS可能被选择性地调制为治疗目的。由于缺乏确定FMN-heme IET动力学的可靠技术，NOS输出状态形成的机制长期以来一直未得到充分研究。我们创新的激光闪光光解方法为通过形成输出状态详细研究NOS调节奠定了基础。鉴于这项实验性研究的新颖性和探索性，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.