Nitroxyl adducts as structural probes of oxygenase/substrate interactions

描述（由申请人提供）：血红素加氧酶(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））的结构已经研究了很长时间，希望这些数据可以用于预测产物和抑制剂的特异性。但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。在本R21中，我们提出应用这些加氧酶的不寻常的亚铁硝基加合物（HNO-FeII）作为直接发生转换的亚铁二氧加合物（O2-FeII）的结构类似物。HNO与O2是等电子的，与O2载体如肌红蛋白、血红蛋白和血红蛋白紧密不可逆地结合。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，亚硝基氢化物在活性口袋的正中央提供了一个光谱手柄；1H NMR中的共振发生在15ppm左右，远离其他基于蛋白质的信号，因此可以用来表征在类似于进行翻转的口袋内的底物结合状态。结合的HNO也在口袋内形成氢键相互作用，这是确定加氧酶活性的重要因素。两种血红素加氧酶具有稳定的HNO加合物，1H NMR表征峰约为15ppm。P450BM3和P450cam在有底物和没有底物的情况下也产生了类似的加合物；并提出了类似的方法来获得nNOS的此类加合物。由于之前的核磁共振和晶体学研究给出了很大不同的距离，因此一个关键的实验将是使用抗磁性氢化物共振通过二维NOE方法绘制P450BM3中的底物位置。这一探索性建议的重点是开发这些加合物的合成方法，遵循地层动力学和产率，评估寿命和可能的反应性，并确定关键性质，如H/D交换率，这对它们作为结构探针的使用很重要。最终，这些加合物可用于评估底物和抑制剂在囊内的迁移性。我们处于一个独特的位置来进行这些研究：PI Farmer的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，以及发现了几种合成这种加合物的途径。CoPI Poulos长期以来对血红素蛋白结构和配体与抑制剂结合的表征感兴趣；他在所有靶蛋白（HO， P450和NOS）的蛋白质表达和晶体学特征方面具有专长。血红素基加氧酶在药物代谢、一氧化氮和类固醇等生理效应物的合成以及血红素等毒素的中和中发挥着关键作用。它们是药物寿命和作用的关键决定因素，本身也是治疗剂（例如，真菌感染、癌症）的重要靶点，是造成广泛药物毒性的原因。它们活性的一个重要方面是在特定位点结合和羟基化靶标的能力。预测P450底物和抑制剂特异性的能力对于P450参与的所有这些区域至关重要。

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.