Redox properties of heme-oxygenase in NO synthases

NO合酶中血红素加氧酶的氧化还原特性

• 批准号: 6668857
• 负责人: MEKKI BAYACHOU
• 金额: $ 14.4万
• 依托单位: CLEVELAND STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-06 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6668857
• 关键词: arginine; biological signal transduction; chemical kinetics; computer simulation; electrochemistry; electron transport; enzyme activity; enzyme mechanism; enzyme substrate; heme oxygenase; isozymes; molecular dynamics; molecular film; nitric oxide; nitric oxide synthase; oxidation reduction reaction; tetrahydrobiopterin; thermodynamics

DESCRIPTION (provided by applicant): Nitric oxide (NO) is undeniably one of the most important biological signaling species discovered in the recent two decades. Age-old as it seems, nitric oxide only recently emerged as a key player involved in control mechanisms of cardiovascular activities, in neurotransmission, and as potent weapon in antibacterial and antiviral action of macrophages. NO is synthesized in vivo from the amino acid L-arginine by a class of enzymes known as Nitric Oxide Synthases (NOSs) requiring a P450-heme, a biopterin cofactor and molecular oxygen in its oxygenase domain. Although the biochemistry of these enzymes has been the focus of vigorous investigations in the last decade, a lot is yet to be learned about their molecular functioning, and especially their post-translational regulation; understanding the details of these processes may open avenues for potential NOS targeted therapies. In this regard, we propose a direct-electrochemical study to investigate mechanisms of electron transfer to, and oxygen activation by nitric oxide synthases. We aim to develop fast protocols to study the effect of the cofactor tetrahydrobiopterin on the electronic properties of the heme as well as on oxygen activation in the NOS catalysis. Similarly, we want to use direct-electrochemistry to measure the effect of substrate arginine and NOS-inhibitors, especially endogenous methylarginines, on redox properties of the heme-oxygenase in NOS. To this end, we use immobilized NOS-oxygenase domain (NOSoxy) in thin films on electrodes to perform fast and direct electrochemistry (i.e. without mediators). We synergistically use computational methodologies to complement and guide our experimental endeavors. Specific aims of our proposed study are: 1) Measure thermodynamic redox potentials and kinetics of charge transfer to iron-heme in NOSoxys by direct electrochemistry and quantify the effects of binding of substrate L-arginine, cofactor biopterin, and NOS-inhibitors (such as endogenous methylarginines). 2) Compare direct electrochemical behavior of different isoforms (i.e. neuronal NOS: nNOS, inducible NOS: iNOS, etc.), as well as wild-type NOSoxys versus mutants, and correlate experimental results on oxygen activation and catalysis to computational findings.

描述（由申请人提供）：一氧化氮（NO）无疑是近二十年来发现的最重要的生物信号物质之一。一氧化氮看似历史悠久，但直到最近才成为心血管活动控制机制、神经传递以及巨噬细胞抗菌和抗病毒作用的有力武器。 NO 在体内由氨基酸 L-精氨酸通过一类称为一氧化氮合酶 (NOS) 的酶合成，需要 P450 血红素、生物蝶呤辅因子和加氧酶结构域中的分子氧。尽管这些酶的生物化学在过去十年中一直是大力研究的焦点，但关于它们的分子功能，特别是它们的翻译后调节，还有很多东西有待了解。了解这些过程的细节可能会为潜在的 NOS 靶向治疗开辟道路。在这方面，我们提出了一项直接电化学研究，以研究电子转移到一氧化氮合酶以及一氧化氮合酶活化氧的机制。我们的目标是开发快速方案来研究辅因子四氢生物蝶呤对血红素电子特性以及 NOS 催化中氧活化的影响。同样，我们希望使用直接电化学来测量底物精氨酸和 NOS 抑制剂，尤其是内源性甲基精氨酸，对 NOS 中血红素加氧酶的氧化还原特性的影响。为此，我们在电极薄膜中使用固定化的一氧化氮合酶加氧酶结构域（NOSoxy）来进行快速、直接的电化学（即没有介体）。我们协同使用计算方法来补充和指导我们的实验工作。我们提出的研究的具体目标是：1) 通过直接电化学测量 NOSoxys 中电荷转移到铁血红素的热力学氧化还原电位和动力学，并量化底物 L-精氨酸、辅因子生物蝶呤和 NOS 抑制剂（例如内源性甲基精氨酸）。 2) 比较不同异构体（即神经元 NOS：nNOS、诱导型 NOS：iNOS 等）以及野生型 NOSoxys 与突变体的直接电化学行为，并将氧活化和催化的实验结果与计算结果相关联。