Nitroxyl adducts as structural probes of oxygenase/substrate interactions

描述（由申请人提供）：基于血红素的加氧酶（例如各种细胞色素P450（cyt P450）、一氧化氮合酶（NOS）和血红素加氧酶（HO））的结构已被长期研究，希望这些数据可用于预测产物和抑制剂特异性。但是，大多数结构数据已获得的非生产状态的酶，因此可能不相关的活性物种。在这个R21中，我们提出了不寻常的亚铁硝酰基加合物（HNO-FeII）的这些加氧酶的结构类似物的亚铁双氧加合物（O2-FeII），直接之前营业额的应用。HNO与O2是等电子的，并且显示出与O2载体如肌红蛋白、血红蛋白和豆血红蛋白紧密且不可逆地结合。HNO-FeII加合物与O2-FeII加合物一样具有抗磁性，但寿命更长。最重要的是，亚硝酰氢化物在活性口袋的中心提供了一个光谱手柄;在1H NMR中的共振发生在大约100 ° C。15 ppm，远离其他基于蛋白质的信号，因此可以用于表征口袋内的底物结合，其状态类似于进行转换。结合的HNO还在口袋内形成H-键合相互作用，已知其在确定加氧酶活性中是重要的。证明了两种血红素加氧酶的稳定HNO加合物，通过1H NMR观察到约15 ppm的特征峰。类似的加合物已经在P450 BM 3和P450 cam中产生，有和没有底物;并且提出了类似的方法来获得nNOS的这种加合物。一个关键的实验将是使用抗磁性氢化物共振通过2D NOE方法绘制出P450 BM 3中的衬底位置，因为以前的NMR和晶体学研究给出了广泛不同的距离。这一探索性建议的一个关键重点是开发这种加合物的合成方法，遵循形成的动力学和产率，评估寿命和可能的反应性，并确定关键特性，如H/D交换率，这在它们用作结构探针时非常重要。最终，这些加合物可能是用于评估口袋内的底物和抑制剂的流动性。我们在进行这些研究方面处于独特的地位：PI Farmer的研究小组首先确定了肌红蛋白的HNO加合物，并表征了其结构和反应性，以及发现了此类加合物的几种合成路线。CoPI Poulos长期以来对血红素蛋白结构以及配体和抑制剂结合的表征感兴趣;他在蛋白质表达和所有目标蛋白质的晶体学特征方面具有专业知识，（HO、P450和NOS）.Project Narrative基于血红素的加氧酶是药物代谢中的关键参与者，在生理效应物如一氧化氮和类固醇的合成中，以及血红素等毒素的营养化。它们是药物寿命和作用的关键决定因素，本身是治疗剂的重要靶标（例如，真菌感染、癌症）是导致广泛的药物毒性的原因。它们活性的一个重要方面是在特定位点结合和羟基化靶点的能力。预测P450底物和抑制剂特异性的能力是P450参与的所有这些领域的核心。

DESCRIPTION (provided by applicant): The structures of heme-based oxygenases (e.g. various cytochrome P450 (cyt P450), nitric oxide synthase (NOS) and heme oxygenase (HO)) have been long studied with the hope that such data can be used to predict product and inhibitor specificity. But most structural data has been obtained for non-productive states of the enzymes, and thus may not be relevant to the active species. In this R21, we propose the application of unusual ferrous nitroxyl adducts (HNO-FeII) of these oxygenases as structural analogues of the ferrous dioxygen adducts (O2-FeII) that directly precedes turnover. HNO is isoelectronic to O2, and is shown to bind tightly and irreversibly to O2 carriers like myoglobin, hemoglobin and leghemoglobin. The HNO-FeII adducts are diamagnetic, like the O2-FeII adducts, but much longer lived. Most importantly, the nitrosyl hydride provides a spectroscopic handle at the very center of the active pocket; the resonance in 1H NMR occurs ca. 15 ppm, well away from other protein-based signals, and therefore may be used to characterize substrate binding within the pocket in a state analogous to that proceeding turnover. The bound HNO also forms H-bonding interactions within the pocket, which are known to be important in determining oxygenase activity. Stable HNO adducts are demonstrated for two heme oxygenases, the characteristic peak ca 15 ppm seen by 1H NMR. Similar adducts have been generated in P450BM3 and P450cam with and without substrates; and similar methods are proposed to obtain such adducts of nNOS. A key proposed experiment will be to use the diamagnetic hydride resonance to map out the substrate position in P450BM3 by 2D NOE methods, as previous NMR and crystallographic studies give widely different distances. A key focus of this exploratory proposal is the development of synthetic methodologies to such adducts, following kinetics and yields of formation, assessing lifetime and possible reactivity, and determining key properties such as H/D exchange rates that would be important in their use as structural probes. Ultimately, these adducts may be of use in assessing mobility of substrate and inhibitors within the pocket. We are in a unique position to perform these studies: the PI Farmer's group first identified such an HNO adduct of myoglobin, and has characterized its structure and reactivity, as well uncovering several synthetic routes to such adducts. CoPI Poulos has longstanding interest in heme protein structure and in the characterizations of ligand and inhibitor binding; he has expertise in protein expression and crystallographic characteristics of all the target proteins (HO, P450 and NOS).Project Narrative Heme-based oxygenases are key players in the metabolism of drugs, in the synthesis of physiological effectors like nitric oxide and steroids, and in the nuetralization of toxins such as the hemes themselves. They are the key determinant of drug lifetime and action, are themselves important targets for therapeutic agents (e.g., fungal infections, cancer), are responsible for a broad range of drug toxicities. An important aspect of their activity is the ability to bind and hydroxylate targets at specific sites. The ability to predict P450 substrate and inhibitor specificity is central to all of these areas of P450 involvement.