Mechanisms of Electron Transfer in Nitric Oxide Synthases: the Output State in Ni

描述(申请人提供)：计划摘要关于一氧化氮合酶(NOS)如何产生一氧化氮(NO)如何受到严格调控，仍有许多未知之处。这是值得注意的，因为体内一氧化氮合酶不受控制地产生NO是越来越多缺乏有效治疗的疾病的关键问题，包括癌症和心血管疾病。在合理设计针对无调控NO产生的有效预防和治疗策略之前，必须清楚地了解NOS催化的控制机制。一氧化氮合酶功能的一个重要组成部分是调节NO合成所需的域间电子转移(IET)过程。该项目的长期目标是在分子水平上研究一氧化氮合酶中关键的IET过程的机制，以确定控制一氧化氮合酶功能的关键序列。有人认为，钙调素(CaM)激活内皮细胞和神经元型一氧化氮合酶(eNOS和nNOS)合成NO需要黄素单核苷酸(FMN)结构域从原来的电子接受(输入)状态转变为新的电子给予(输出)状态。假设的输出状态被设想为FMN结合结构域和加氧酶结构域之间的复合体，从而促进FMN和加氧酶结构域中催化血红素之间的有效IET。一氧化氮合酶输出状态中的FMN-heme IET对NO的合成是必不可少的。然而，其产出态的形成机制尚不清楚，这为更全面地理解CaM控制的NOS催化机制提供了关键的障碍。本研究的重点是在分子水平上研究CaM激活的输出态的形成机制。我们假设，特定的CaM结合和生产性的FMN/heme相互作用是形成NOS输出态的两个关键结构决定因素。这一假设将通过量化FMN-血红素IET动力学来验证，该模型通过两个互补和协同的目标在一个得到良好验证的NOS输出状态模型中进行。我们开发了创新的激光闪光光解方法来直接测定一氧化氮合酶输出状态下的FMN-HEME IET。实验设计将结合我们的激光闪光光解方法和生物物理技术与定点突变，以确定特定氨基酸在CaM控制的eNOS和nNOS输出态形成中的机制作用。拟议的研究将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重要的新见解。一氧化氮合酶(NOS)产生的一氧化氮(NO)在生理条件下受到严格调控，在狭窄的范围内既提供正常的信号通路，又保护机体免受过量NO的损害。因此，一氧化氮合酶家族是开发治疗癌症、中风和高血压等多种疾病的新药的关键靶点；然而，由于对一氧化氮合酶调节的分子机制了解不深，尚未开发出可用于临床治疗的一氧化氮合酶抑制剂/激动剂。拟议的研究将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。

DESCRIPTION (provided by applicant): Program Summary There is still much unknown about how nitric oxide (NO) production by nitric oxide synthase (NOS) is tightly regulated. This is remarkable because unregulated NO production by NOS in vivo is a critical problem in an increasing number of diseases lacking effective treatments, including cancer and cardiovascular diseases. Before logically designing effective preventive and therapeutic strategies targeting unregulated NO production, one must clearly understand the control mechanisms of NOS catalysis. An important component of the function of the NOS enzyme is the regulation of interdomain electron transfer (IET) processes required for NO synthesis. The long-term goal of this project is to investigate the mechanisms of the crucial IET processes in NOS at the molecular level, in order to determine the key sequences for controlling the NOS function. It is proposed that the calmodulin (CaM) activation of NO synthesis in endothelial and neuronal NOS (eNOS and nNOS) requires a conformational change of the flavin mononucleotide (FMN) domain from its original electron- accepting (input) state to a new electron-donating (output) state. The putative output state is envisioned as a complex between the FMN binding and oxygenase domains, thus facilitating efficient IET between the FMN and the catalytic heme in the oxygenase domain. The FMN-heme IET within the NOS output state is essential for NO synthesis. However, the mechanism of the output state formation remains unclear, which thus constitutes a critical barrier for understanding the CaM controlled NOS catalytic mechanisms more completely. The focus of this study is to investigate the mechanisms of CaM-activated output state formation at the molecular level. We hypothesize that specific CaM binding and productive FMN/heme interactions are two critical structural determinants for formation of the NOS output state. This hypothesis will be tested by quantitating the FMN-heme IET kinetics in a well-validated model of the NOS output state through two complementary and synergistic Aims. We have developed innovative laser flash photolysis approaches to directly determine the FMN-heme IET within the NOS output state. The experimental design will integrate our laser flash photolysis approach and biophysical techniques with site-directed mutagenesis, in order to determine mechanistic roles of specific amino acids in CaM-controlled formation of the output state in eNOS and nNOS. The proposed studies will significantly improve the fundamental understanding of NOS regulation, and will provide important new insight as to how NOS might be selectively modulated for therapeutic purposes. Nitric oxide (NO) production by nitric oxide synthase (NOS) is tightly regulated under physiological conditions within narrow ranges to both provide normal signaling pathways yet protect against damages from excess NO. The NOS family is thus a key target for development of new pharmaceuticals for a wide range of diseases such as cancer, stroke and hypertension; however, due to a poor understanding of the molecular mechanisms of NOS regulation, no NOS inhibitors/activators have yet been developed for clinical treatments. The proposed studies will significantly improve the fundamental understanding of NOS regulation, and will provide new important insight as to how NOS might be selectively modulated for therapeutic purposes.