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

描述（由申请人提供）：程序摘要一氧化氮合酶（NOS）如何严格调节一氧化氮（NO）的产生仍然是未知的。这是值得注意的，因为NOS在体内不受调节的NO产生是越来越多缺乏有效治疗的疾病（包括癌症和心血管疾病）中的关键问题。在逻辑上设计有效的预防和治疗策略，针对不受管制的NO生产，必须清楚地了解NOS催化的控制机制。NOS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。本项目的长期目标是在分子水平上研究NOS中关键的IET过程的机制，以确定控制NOS功能的关键序列。本文提出钙调素（CaM）激活内皮型和神经型NOS（eNOS和nNOS）中NO合成需要黄素单核苷酸（FMN）结构域从其原始的电子接受（输入）状态到新的电子给予（输出）状态的构象变化。假定的输出状态被设想为FMN结合和加氧酶结构域之间的复合物，从而促进FMN和加氧酶结构域中的催化血红素之间的有效IET。在NOS输出状态内的FMN-heme IET对于NO合成是必不可少的。然而，输出态的形成机制仍不清楚，这构成了一个关键的障碍，更全面地了解钙调素控制的NOS催化机制。本研究的重点是在分子水平上研究钙调素激活的输出态形成的机制。我们假设，具体的钙调素结合和生产FMN/血红素相互作用是两个关键的结构决定因素形成的NOS输出状态。通过两个互补和协同的目的，在一个经过充分验证的NOS输出状态模型中定量测定FMN-血红素IET动力学，将对这一假设进行检验。我们已经开发出创新的激光闪光光解方法来直接确定NOS输出状态内的FMN-heme IET。实验设计将我们的激光闪光光解方法和生物物理技术与定点诱变相结合，以确定特定氨基酸在eNOS和nNOS输出状态的CaM控制形成中的机制作用。拟议的研究将显着提高NOS调节的基本理解，并将提供重要的新见解，如何NOS可能被选择性地调制为治疗目的。一氧化氮合酶（NOS）产生的一氧化氮（NO）在生理条件下在狭窄范围内受到严格调节，以提供正常的信号传导途径，同时保护免受过量NO的损害。因此，NOS家族是开发用于多种疾病如癌症、中风和高血压的新药的关键靶标;然而，由于对NOS调节的分子机制了解不多，尚未开发出用于临床治疗的NOS抑制剂/激活剂。拟议中的研究将显着提高NOS调节的基本理解，并将提供新的重要见解，如何NOS可能被选择性地调制为治疗目的。

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.