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。我们的目标是确定NO合成的机制和NOS催化的结构-功能方面，这将有助于制定临床策略来控制NO的可用性。所有的NOS都含有血红素和黄素，并催化L-精氨酸的两步氧化，使NO。NOS在黄血红素酶中是独特的，因为它们含有6 R-四氢生物蝶呤（HUB）作为结合辅因子。此外，它们的H4 B辅因子在催化过程中经历了新的单电子跃迁。我们以前的研究表明，H4 B自由基的形成涉及到的步骤，在三个NOS的氧活化和催化，并探讨了一些H4 B氧化还原功能的结构-功能方面。我们目前的目标描述了生物化学，动力学，分子生物学和生物物理学的研究，将通过进一步定义H4 B功能的机制，调节和影响来促进我们对NOS催化的理解。目标1。研究调节H4 B自由基形成的结构、热力学和NOS同工酶特异性基础。H4 B在NO合成的两个反应（Arg羟基化和N-hydroxyArg氧化）中将电子转移到血红素二氧基中间体。我们推测，H4 B自由基形成的动力学和程度是由周围的蛋白质残基和蝶呤环结构本身调节的。我们将测试特定NOS残基和蝶呤结构类似物的功能，并研究NOS中H4 B自由基形成的热力学控制。目标2.研究蝶呤对NOS中FeII 02中间体的还原、稳定性和后续反应性的氧化还原非依赖性作用。我们将利用氧化还原失活的蝶呤来研究如何绑定蝶呤影响NOS黄素蛋白电子转移到血红素，FeIIO 2中间体的稳定性，以及NOS血红素氧物种的进一步反应性，以产生从精氨酸和NONA的产品。这项工作将提供一个全面的看法如何蝶呤影响NOS催化。目标3.研究H4 B自由基的还原转换以及它们是如何被调节的。在NO合成的两个反应中形成的H4 B自由基必须在酶可以继续催化之前还原回H4 B。这是如何发生的尚不清楚。我们推测，H4 B自由基减少NO合成的每个反应中的不同机制。我们已经开发了方法来监测H4 B自由基还原在单一的催化周转反应，并将研究的机制和监管方面。目标4。启动NOS中结合H4 B的13 C、15 N和HSQC NMR研究。我们假设NOS蛋白为H4 B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13 C和15 N富集的H4 B进行NMR研究，以测试来自我们的蛋白质晶体结构的特定假设。相关性：通过澄清一氧化氮的产生和调节方式，我们的工作可能有助于开发治疗人类疾病的方法，这些疾病涉及产生过多或过少的一氧化氮。

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.