Catalytic Mechanisms and Properties of Heme Proteins

描述(申请人提供)：本项目的目标是确定通过一氧化氮合酶(NOS)调节NO合成的关键因素。目前已发现一氧化氮合酶的三种主要亚型：来自内皮细胞的eNOS、来自神经元的nNOS和来自巨噬细胞的iNOS。一氧化氮合酶是一种由两个结构域组成的二聚体酶，一个是含血红素的加氧酶结构域，在那里精氨酸被氧化成瓜氨酸和一氧化氮，另一个是还原酶结构域，它将电子从NADPH转移到血红素活性中心。每个表达的加氧酶结构域都由一个血红素基团、一个四氢生物蝶呤辅助因子(H4B)和底物结合位点组成，将被用作本项目中全长酶的模型。在目前的授权期，我们发现H4B与NO结合蛋白的结合显著扭曲了血红素的大循环，从而改变了血红素的氧化还原电位，并且NO自动抑制了酶并破坏了二聚体的相互作用。此外，我们还发现，色氨酸和与血红素铁配位的近端半胱氨酸之间的氢键在调节铁-半胱氨酸键的电子性质方面非常重要，而铁-半胱氨酸键是合成一氧化氮的关键。我们还用共振拉曼光谱表征了一氧化氮合酶催化过程中的第一个氧中间体。为了进一步阐明一氧化氮合酶功能的机制细节，提出了四(4)个系列的实验。首先，我们将系统地研究血红素近端键的铁硫伸缩方式，以评价其对催化活性的影响。其次，我们将利用实验室开发的新型微流控混合器和快速冷冻冷却装置，以及几种机械策略来捕获和识别催化循环中的各种氧中间体。第三，三(3)个最先进的EPR技术将被用来表征在反应过程中形成的H4B自由基。最后，将进一步研究血红素扭曲对酶催化性能的作用以及NO诱导的二聚体相互作用的破坏，以构建NO介导的调控机制的完整图景。在这项工作中，将研究所有三(3)种一氧化氮合酶亚型，并将系统地评估亚型的特性。该项目所产生的信息将为我们更好地了解与一氧化氮合酶相关的各种疾病状态奠定基础，并将对促进异构体特异性抑制剂的合理设计非常有价值。

DESCRIPTION (provided by applicant): The goal of this project is to determine the key factors that regulate NO synthesis by nitric oxide synthase (NOS). Three major isoforms of NOS has been discovered: eNOS from endothelia, nNOS from neurons and iNOS from macrophages. NOS is a dimeric enzyme that is comprised of two domains, a heme-containing oxygenase domain, where arginine is oxidized to citrulline and NO, and a reductase domain, which transfers electrons from NADPH to the heme active center. Each expressed oxygenase domain, which consists of a heme group, a tetrahydrobiopterin cofactor (H4B) and the substrate binding site, will be used as models for the full length enzymes in this project. In the current grant period, we have discovered that binding of H4B to NO-bound protein significantly distorts the heme macrocycle thereby altering the heme redox potential, and that NO auto-inhibits the enzyme and disrupts the dimeric interactions. In addition, we found that the H-bond between a tryptophan and the proximal cysteine that coordinates to the heme iron is very important in modulating the electronic properties of the iron-cysteine bond, which is critical for NO synthesis. We also characterized the first oxygen intermediate during NOS catalysis with resonance Raman spectroscopy. To further elucidate the mechanistic details of NOS function, four (4) series of experiments are proposed. First, the iron-sulfur stretching mode of the heme proximal bond will be systematically studied to evaluate its impact on the catalytic activity. Second, a novel microfluidic mixer coupled with a rapid freeze quenching device that we have developed in our lab along with several mechanistic strategies will be employed to trap and identify the various oxygen intermediates in the catalytic cycle. Third, three (3) state-of-the-art EPR techniques will be used to characterize the H4B radicals formed during the reaction. Finally, the role of heme distortion on the catalytic properties of the enzyme and the NO-induced disruption of the dimeric interactions will be further studied to construct an integrated picture of the NO-mediated regulatory mechanisms. In this work, all three (3) NOS isoforms will be studied, and isoform specific properties will be systematically evaluated. The information generated from this project will serve as a foundation to improve our understanding of various disease states linked to NOS and will be very valuable in facilitating the rational design of isoform specific inhibitors.