Biochemistry of Nitric Oxide Synthesis

描述（由申请人提供）：三种NO合成酶（iNOS， nNOS和eNOS）在人类健康和疾病中广泛发挥作用。我们的目标是明确NO合成的机制和NOS催化的结构-功能方面，这应该有助于制定临床策略来控制NO的可用性。所有NOS都含有血红素和黄素，并催化l -精氨酸的两步氧化生成NO。nos在黄素酶中是独特的，因为它们含有6r -四氢生物蝶呤（HUB）作为结合的辅因子。此外，它们的H4B辅因子在催化过程中经历了新的单电子跃迁。我们的前期研究揭示了H4B自由基形成与三种NOS中氧活化和催化步骤的关系，并探讨了H4B氧化还原功能的一些结构功能方面的问题。我们目前的目标是描述生物化学、动力学、分子生物学和生物物理学的研究，通过进一步定义H4B功能的机制、调节和影响，提高我们对NOS催化的理解。目的1。研究调节H4B自由基形成的结构、热力学和NOS同工酶特异性基础。在NO合成的两个反应（精氨酸羟基化和n -羟基氧化）中，H4B将一个电子转移到血红素-二氧基中间体上。我们假设H4B自由基形成的动力学和程度是由周围的蛋白质残基和翼蛋白环结构本身调节的。我们将测试特定的NOS残基和蝶呤结构类似物的功能，并研究NOS中H4B自由基形成的热力学控制。研究不依赖氧化还原的翼图素对NOS中fei02中间体的还原、稳定性和随后的反应性的影响。我们将利用无氧化还原活性的翼图素来研究结合的翼图素如何影响NOS黄蛋白向血红素的电子转移、feio2中间体的稳定性以及NOS血红素氧的进一步反应活性，从而产生Arg和NONA的产物。这项工作将提供一个全面的观点，如何影响羽蝶呤NOS催化。目标3。研究H4B自由基的还原转变及其如何被调节。在两个NO合成反应中形成的H4B自由基必须还原回H4B，酶才能继续催化。这是如何发生的尚不清楚。我们假设在NO合成的每个反应中，H4B自由基通过不同的机制被减少。我们已经开发了监测H4B自由基在单催化转化反应中的减少的方法，并将研究其机制和调控方面。目标4。启动NOS中结合的H4B的13C、15N和HSQC NMR研究。我们假设NOS蛋白为H4B创造了一个有利于其单电子氧化还原转变的电子环境。我们将对NOS或其突变体中结合的富含13C-和15n的H4B进行核磁共振研究，以验证从我们的蛋白质晶体结构中得出的特定假设。相关性：通过阐明一氧化氮的产生和调控方式，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 NO synthases (iNOS, nNOS, and eNOS) function broadly in human health and disease. Our goal is to define the mechanism of NO synthesis and the structure-function aspects of NOS catalysis, which should help to develop clinical strategies to control NO availability. All NOS contain heme and flavins and catalyze a two-step oxidation of L-arginine to make NO. NOSs are unique among flavoheme enzymes because they contain 6R-tetrahydrobiopterin (HUB) as a bound cofactor. Moreover, their H4B cofactor undergoes novel one- electron transitions during catalysis. Our previous studies showed how H4B radical formation relates to steps in oxygen activation and catalysis in the three NOS, and probed some of the structure-function aspects of H4B redox function. Our current Aims describe biochemical, kinetic, molecular biological, and biophysical studies that will advance our understanding of NOS catalysis by further defining the mechanisms, regulation, and impact of H4B functions. Aim 1. Investigate the structural, thermodynamic, and NOS isozyme-specific basis for regulating H4B radical formation. H4B transfers an electron to a heme-dioxy intermediate in both reactions of NO synthesis (Arg hydroxylation and N-hydroxyArg oxidation). We hypothesize that the kinetics and extent of H4B radical formation are tuned by surrounding protein residues and by the pterin ring structure itself. We will test the function of specific NOS residues and pterin structural analogs, and investigate thermodynamic control of H4B radical formation in NOS. Aim 2. Investigate redox-independent effects of pterins on the reduction, stability, and subsequent reactivity of the FeII02 intermediate in NOS. We will utilize redox-inactive pterins to investigate how bound pterins impact NOS flavoprotein electron transfer to the heme, the stability of the FeIIO2 intermediate, and the further reactivity of NOS heme-oxy species to generate products from Arg and NONA. This work will provide a comprehensive view of how pterin influences NOS catalysis. Aim 3. Investigate the reductive transitions of the H4B radical and how they are regulated. The H4B radical formed in both reactions of NO synthesis must be reduced back to H4B before the enzyme can continue catalysis. How this occurs is unclear. We hypothesize that the H4B radical is reduced by distinct mechanisms in each reaction of NO synthesis. We have developed methods to monitor H4B radical reduction in single catalytic turnover reactions and will examine the mechanistic and regulatory aspects. Aim 4. Initiate 13C, 15N, and HSQC NMR studies of bound H4B in NOS. We hypothesize that the NOS protein creates an electronic environment for H4B that favors its one-electron redox transitions. We will perform NMR studies on 13C- and 15N-enriched H4B bound in NOS or its mutants to test specific hypotheses derived from our protein crystal structures. Relevance: By clarifying how nitric oxide production occurs and is regulated, our work may help to develop treatments for human diseases that involve making too much or too little nitric oxide.