X-ray spectroscopy of Highly Reactive Catalytic Intermediates in Oxygen Activating Metallo-Enzymes

 描述(申请人提供)：氧激活酶是一类重要的氧化还原活性蛋白质，在细胞功能中具有关键作用，在生物分子、信号转导和处理氧化应激的关键步骤中起辅助作用。在这些酶中，含铁的血红素蛋白长期以来一直被认为是至关重要的，特别是在细胞呼吸酶中，如细胞色素C氧化酶。一氧化氮合酶(NOS)是一种铁血红素酶，催化合成一氧化氮(NO)，NO是一种小信号分子，其在炎症和血管扩张中的作用使其成为广受欢迎的药物靶点。为了实现我们的利益 了解一氧化氮合酶在人体内的功能，并将其作为控制疾病的工具，有必要对其功能背后的催化机制有深入的了解。关于这种酶的许多情况都已为人所知。为了从氨基酸L-精氨酸中释放出NO，一氧化氮合酶两次激活O2，该反应的许多中间产物已经用各种方法进行了表征。然而，反应的一些关键步骤仍然是假设的，要么缺乏直接的实验证据，要么受到模棱两可的解释的影响。氧化还原活性铁对光还原造成的辐射损伤的敏感性使得X射线光谱和结晶学研究的收集变得困难--这是研究这些未知催化中间体的关键方法。最近，可用的超短飞秒脉冲X射线自由电子激光(XFEL)用户设备正在消除研究人员在传统长脉冲同步加速器源上收集X射线光谱和衍射图时所面临的障碍。飞秒XFEL脉冲避免了辐射损伤，因为辐射损伤发生在皮秒时间尺度上，导致了一种被称为“破坏前的信号”的测量范式。因此，现在有可能在生理条件下收集具有高光子通量的金属酶的有意义的数据，而不是低温，从而允许对微弱的、信息丰富的光谱信号甚至动力学结晶学进行时间分辨的X射线研究。这项提议的主要目标是利用这一新的范式，并将其扩展到一氧化氮合酶，以便能够直接观察催化金属的电子态和周围环境。为了实现这一点，我提出了将一氧化氮合酶与L精氨酸快速混合溶液引入X射线的方法的发展。此外，还提出了数据分析方法，该方法利用XFEL独特的脉冲结构，允许快速收集X射线吸收和化学特定的共振发射，这是对金属的电子状态和配体环境的深入探测。

 DESCRIPTION (provided by applicant): Dioxygen activating enzymes are an import class of redox-active proteins that have critical roles in cellular function, aiding in key steps of the synthesis of biomolecules, signaling agents, and handling oxidative stress. Of these enzymes, Iron-containing Heme proteins have long been recognized as vital, notably in cellular respiration enzymes like cytochrome c oxidase. Nitric Oxide Synthase (NOS) is an Iron Heme enzyme, which catalyzes the synthesis of Nitric Oxide (NO), a small signaling molecule whose role in inflammation and vasodilation make it a popular drug target. In order to realize the benefit of our knowledge of NOS's function in the human body and to harness it as a tool for controlling disease it is necessary to have a deep understanding of the catalytic mechanism that underlies its function. Much about this enzyme is known. NOS activates O2 twice in order to release NO from the amino acid L-Arginine, and many of the intermediates to this reaction have been characterized by various methods. However, some key steps of the reaction remain hypothetical, either lacking direct experimental evidence or suffering from ambiguous interpretations. The sensitivity of the redox-active Fe to radiation damage by photoreduction has made difficult the collection of X-ray spectroscopic and crystallographic studies - key methods that could interrogate these unknown catalytic intermediates. Recently available ultra-short femtosecond pulse X-ray free electron laser (XFEL) user facilities are lifting the obstacles researchers have faced when trying to collect X-ray spectra and diffraction patterns at conventional long-pulse synchrotron sources. The femtosecond XFEL pulses avoid radiation damage, as the radiation damage happens on the picosecond time scale, resulting in a measurement paradigm known as "signal before destruction". As a consequence, it is now possible to collect meaningful data from metalloenzymes with high photon flux at physiological conditions, rather than cryogenic temperatures, allowing for time resolved X-ray studies of weak, information rich spectral signals and even kinetic crystallography. The key objective of this proposal is to take advantage of this new paradigm and extend it to NOS so that direct observations of the catalytic metal's electronic state and surrounding environment can be made. To accomplish this, I propose the development of methods to introduce rapidly mixed solutions of NOS with L-arginine into the X-ray beam. Further, data analytic methods are proposed which take advantage of the unique XFEL pulse structure, permitting rapid collection of X-ray absorption and chemically specific resonant emission, an incisive probe of both the metal's electronic state and ligand environment.