Control Mechanisms of the Nitric Oxide Synthases

描述（由申请人提供）：三种一氧化氮合酶（NOS）进化到在人类健康和疾病中起作用。我们希望确定调节NOS催化的机制和蛋白质结构特征。NOS黄蛋白结构域将NADPH电子转移到加氧酶结构域的血红素中，从而使NO合成成为可能。催化调节是复杂的，因为NO在离开酶之前与NOS血红素结合。我们开发了一个全球动力学模型，其中包含了催化的这些和其他方面。全局模型认为，三种NOS的独特催化特性是由于三个动力学参数的差异：铁血红素还原（kr）、铁血红素- no解离（kd）和铁血红素- no络合物的氧化（ox）。控制NOS电子转移和设置kr、kd和kox值的机制和结构基础尚不清楚。我们的目标是通过生物化学、动力学、分子生物学和生物物理学的研究来解决这些问题，从而从分子水平上理解NOS的控制机制。目的1。是什么调节NOS中的电子转移和血红素还原（kr） ？FMN模块的电子转移是NOS催化的核心特征。我们将研究伴侣子结构域、FMN氧化还原状态和钙调蛋白如何调节FMN模块的构象平衡及其与电子受体的相互作用，结构域间表面电荷相互作用如何调节FMN电子转移和NOS催化，以及特定的FMN-蛋白质相互作用和两个连接铰链元件的组成如何控制NOS中FMN模块的电子转移。是什么调节亚铁血红素- no络合物与O2 （ox）的反应？cox反应是关键，因为它决定了NOS的催化行为，并产生不同于NO的n -氧化物产物。我们将研究cox如何受NOS血红素中点电位、控制O2进入血红素的蛋白质结构特征以及FMN和NOSoxy亚结构域之间的相互作用的调控，并将研究几种相关血红素硫酸酶的cox机制和调控。目标3。NOS酶如何调节NO释放（kd）？控制铁血红素-NO解离（kd）的因子对NOS特别重要，因为其新生成的NO分子在离开酶之前与铁血红素协调。我们将研究NOS二聚体结构、底物结合和结构以及血红素口袋开口的大小如何控制NO kd。目标4。通过蛋白质工程生成具有新型催化行为的NOS。在目标1-3的背景下，我们将创建具有kr， kox和kd的非本地组合的NOS变体。这些研究将测试NOS的全局模型和我们对NOS动力学控制机制的理解，揭示NOS酶的功能是否会在动力学参数超过其自然范围时发生变化，并可能为NO递送治疗产生有用的超级NOS变体。相关性：通过澄清一氧化氮的产生是如何在酶水平上被调节的，我们的工作可能有助于开发涉及产生过多或过少一氧化氮的人类疾病的治疗方法。公共卫生相关性：三种一氧化氮合酶在人类健康和疾病中发挥着广泛的作用。我们希望确定这些酶的蛋白质成分如何调节其一氧化氮的产生。通过澄清一氧化氮的产生是如何在酶水平上被调节的，我们的工作可能有助于开发涉及产生过多或过少一氧化氮的人类疾病的治疗方法。

DESCRIPTION (provided by applicant): Three nitric oxide synthase enzymes (NOS) evolved to function in human health and disease. We wish to define the mechanisms and the protein structural features that regulate NOS catalysis. The NOS flavoprotein domain transfers NADPH electrons to a heme in the oxygenase domain and this enables NO synthesis. Catalytic regulation is complex because NO binds to the NOS heme before it exits the enzyme. We developed a global kinetic model that incorporates these and other facets of catalysis. The global model posits that the unique catalytic profiles of the three NOS are due to differences in three kinetic parameters: Ferric heme reduction (kr), ferric heme-NO dissociation (kd), and oxidation of the ferrous heme-NO complex (kox). The mechanisms and structural basis for controlling NOS electron transfer and for setting the kr, kd, and kox values are still unclear. Our Aims propose biochemical, kinetic, molecular biological, and biophysical studies to tackle these issues, in order to provide a molecular-level understanding of NOS control mechanisms. Aim 1. What regulates electron transfer and heme reduction (kr) in NOS? Electron transfer by the FMN module is a central feature of NOS catalysis. We will investigate how partner subdomains, FMN redox status, & calmodulin regulate the conformational equilibrium of the FMN module and its interactions with electron acceptors, how inter-domain surface charge interactions regulate FMN electron transfer and NOS catalysis, and how a specific FMN-protein interaction and the composition of two connecting hinge elements govern FMN module electron transfer in NOS. Aim 2. What regulates reaction of the ferrous heme-NO complex with O2 (kox)? The kox reaction is key because it determines NOS catalytic behavior and generates an N-oxide product that is distinct from NO. We will investigate how kox may be regulated by the NOS heme midpoint potential, protein structural features that control O2 access to the heme, and interactions between the FMN and NOSoxy subdomains, and will study the kox mechanism and regulation in several related heme-thiolate enzymes. Aim 3. How do NOS enzymes regulate their NO release (kd)? Factors that control ferric heme-NO dissociation (kd) are particularly important for NOS because its newly-generated NO molecules coordinate to the ferric heme before leaving the enzyme. We will investigate how the NO kd is controlled by NOS dimeric structure, substrate binding & structure, and the size of the heme pocket opening. Aim 4. Generate NOS with novel catalytic behaviors through protein engineering. Within the context of Aims 1-3 we will create NOS variants that possess non- native combinations of kr, kox and kd. These will test the global model and our understanding of kinetic control mechanisms in NOS, will reveal if NOS enzyme function can change when the kinetic parameters exceed their natural ranges, and may generate useful super-NOS variants for NO delivery therapy. Relevance: By clarifying how nitric oxide production is regulated at the enzyme level, our work may help to develop treatments for human diseases that involve making too much or too little nitric oxide. PUBLIC HEALTH RELEVANCE: Three nitric oxide synthase enzymes function broadly in human health and disease. We wish to determine how the protein components of these enzymes regulate their nitric oxide production. By clarifying how nitric oxide production is regulated at the enzyme level, our work may help to develop treatments for human diseases that involve making too much or too little nitric oxide.