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

计划摘要 一氧化氮合酶(NOS)如何产生一氧化氮(NO)仍是一个未知数。 受到严格监管。这是值得注意的，因为体内一氧化氮合酶不受调控地产生NO是一个关键 问题是越来越多的疾病缺乏有效的治疗，包括癌症和 心血管疾病。在合理设计有效的预防和治疗策略之前 针对不受调控的NO产生，必须清楚地了解NOS的控制机制 催化作用。一氧化氮合酶功能的一个重要组成部分是结构域间的调节 NO合成所需的电子转移(IET)过程。这个项目的长期目标是 从分子水平上研究一氧化氮合酶中关键的IET过程的机制，以期 确定用于控制NOS功能的键序列。有观点认为，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需要一种 黄素单核苷酸(FMN)结构域的构象变化 接受(输入)状态变为新的给电子(输出)状态。假定的输出状态为 被设想为FMN结合和加氧酶结构域之间的复合体，从而促进高效 在加氧酶区域，FMN和催化亚铁血红素之间的IET。体内的FMN-HEME 一氧化氮合酶的输出状态对一氧化氮的合成是必不可少的。然而，输出状态的机制 队形尚不清楚，这构成了理解凸轮的关键障碍 更全面地掌握了一氧化氮合酶的催化机理。这项研究的重点是调查 分子水平上CaM激活的输出态形成机制。我们假设 特异的CaM结合和FMN/HO相互作用是两个关键的结构决定因素 用于形成一氧化氮合酶的输出状态。这一假说将通过量化FMN-血红素来检验 在经过验证的一氧化氮合酶输出状态模型中，通过两个互补的和 协同目标。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。实验设计将整合我们的 激光闪光光解方法和定点突变生物物理技术，以 确定特定氨基酸在钙调素控制的输出态形成中的机械作用 ENOS和nNOS。拟议的研究将极大地提高对 一氧化氮合酶的调控，并将为如何选择性地调节一氧化氮合酶提供重要的新见解 用于治疗目的。

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.