The mechanisms of nitric oxyde synthesis by nitric oxide synthases

一氧化氮合酶（NOS）合成信号和氧化应激分子一氧化氮（NO）。该研究计划的长期目标是确定NOS活性位点的结构，功能和动力学特性，以了解分子水平上的催化循环和酶活性调节机制。由NOS合成的NO在血管和神经系统中用作信号分子。它也是一种有毒分子，被免疫系统用于宿主防御病原体和癌细胞。NO参与人类生理学和疾病的许多方面，包括动脉粥样硬化、阿尔茨海默病和帕金森病，意味着需要亚型特异性抑制剂来调节神经元NOS（nNOS）、内皮NOS（eNOS）和诱导型NOS（iNOS）。我们期望获得对NOS的催化机制的透彻理解，包括提供血红素-配体底物相互作用的详细描述和识别关键的动力学中间体，将提高我们对NOS化学的理解，并且从长远来看，将有助于开发有效的、异构体特异性的人类NOS抑制剂。因此，该提案的具体目标是：A-通过共振拉曼光谱表征使用非天然底物的NOS催化循环期间形成的第一氧化中间体，并表征活性位点突变体的氧化复合物。这些研究将有助于确定在催化循环的重要性，底物和血红素结合的双氧在活性位点之间的极性和空间相互作用，也将有助于破译近端半胱氨酸连接到血红素所发挥的作用。B-表征NOS突变体的氧化形式，以确定催化循环中蛋白质动力学的参与。C-表征与过氧化物反应形成的催化中间体。后两项研究旨在更好地了解NOS合成NOS的机制。D-表征血红素结合配体（O2、NO和CO）和NOS抑制剂与所有三种哺乳动物NOS之间的相互作用，以解释抑制剂结合和亚型特异性的结构基础。总之，这些研究将提供在分子水平上的NO合成机制的理解，确定重要的因素调节酶活性和确定异构体特异性的NOS的结构决定因素。

Nitric oxide synthases (NOSs) synthesize the signaling and oxidative stress molecule nitric oxide (NO). The long-term objectives of this research program are to determine the structural, functional and dynamic properties of the active site of NOSs to understand the catalytic cycle and mechanisms of regulation of the enzymatic activity at the molecular level. The NO synthesized by NOSs is used as a signaling molecule in the vascular and neural systems. It is also a toxic molecule used by the immune system in host defenses against pathogens and cancer cells. The involvement of NO in many aspects of human physiology and diseases, including atherosclerosis, Alzheimer's and Parkinson's, means that isoform-specific inhibitors are needed to regulate neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). We expect that gaining a thorough understanding of the catalytic mechanisms of NOSs, including providing a detailed picture of heme-ligand substrate interactions and identifying key kinetic intermediates, will improve our comprehension of the chemistry of NOSs and, in the long term, will help in the development of potent, isoform-specific inhibitors of human NOSs. The specific objectives of this proposal thus are: A-Characterize by resonance Raman spectroscopy the first oxygenated intermediate formed during the catalytic cycle of NOSs using non-natural substrates and characterize the oxygenated complex of active site mutants. These studies will help determine the importance in the catalytic cycle of polar and steric interactions between substrates and the heme-bound dioxygen at the active site and will also help decipher the role played by the proximal cysteine ligated to the heme. B- Characterize the oxygenated form of NOS mutants to determine the involvement of protein dynamics in the catalytic cycle. C-Characterize catalytic intermediates formed by the reaction with peroxide. The latter two studies aim to get a better understanding of the mechanisms of NOS synthesis by NOSs. D-Characterize the interactions between heme-bound ligands (O2, NO and CO) and NOS inhibitors with all three mammalian NOSs to decipher the structural basis for inhibitor binding and isoform specificity. Together, these studies will provide an understanding at the molecular level of the mechanism of NO synthesis, identify important factors modulating the enzymatic activity and identify structural determinants for isoform-specificity of NOSs.