Redox properties of heme-oxygenase in NO synthases

说明(申请人提供)：一氧化氮(NO)是近二十年来发现的最重要的生物信号物质之一。尽管一氧化氮似乎由来已久，但直到最近才成为参与心血管活动控制机制、神经传递以及巨噬细胞抗细菌和抗病毒作用的有力武器。NO是由氨基酸L-精氨酸在体内由一类称为一氧化氮合酶(NOSS)的酶合成的，需要一个P450-血红素、一个生物蝶呤辅因子和其加氧酶区域的分子氧。尽管在过去的十年里，这些酶的生物化学一直是积极研究的焦点，但关于它们的分子功能，特别是它们的翻译后调控，还需要了解很多；了解这些过程的细节可能会为潜在的一氧化氮合酶靶向治疗开辟道路。在这方面，我们建议用直接电化学研究来研究一氧化氮合酶的电子转移和氧激活的机制。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性质的影响以及在一氧化氮合酶催化中对氧激活的影响。同样，我们想用直接电化学来测量底物精氨酸和一氧化氮合酶抑制剂，特别是内源性甲基精氨酸对一氧化氮合酶中血红素加氧酶氧化还原特性的影响。为此，我们在电极上的薄膜中使用固定化的一氧化氮合酶结构域(NOSoxy)来进行快速、直接的电化学(即不需要介体)。我们协同使用计算方法来补充和指导我们的实验工作。1)用直接电化学法测定NOSoxys中铁-血红素的热力学氧化还原电位和电荷转移动力学，定量底物L-精氨酸、辅因子生物蝶呤和一氧化氮合酶抑制剂(如内源性甲基精氨酸)的结合效应。2)比较不同亚型(神经型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进行关联。

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.