Synchrotron NRVS and Campus Spectroscopy of Multinuclear Fe Proteins: Nitrogenase, Hydrogenase, Methane Monoxygenase, and NO Sensor Proteins

 描述(申请人提供)：生物铁-S团簇是含有2-8个铁原子的纳米颗粒，主要通过桥联S原子结合在一起。含有铁-S簇的蛋白质在生命系统中起着广泛的基本任务，包括化学催化 反应、对化学环境的感知、DNA的信号和修复以及分子结构的维持。我们这项提案的主要目标围绕四个关键问题： 铁-S团簇如何与小分子反应？它们如何催化反应，它们如何作为一氧化氮和氧气传感器工作，以及它们分解时会发生什么？ 氢酶活性部位‘H-簇’的组装有哪些步骤？ 较大的蛋白质环境如何影响小分子进入铁-S位点？ 铁-S团簇的化学如何影响三级蛋白质结构及其与脱氧核糖核酸的相互作用？ 我们研究的预期结果包括： 关于固定氮(固氮酶)或产氢(氢酶)的酶的催化中间体的信息 有关铁-S簇蛋白与信号分子NO和O2反应时的反应中间产物的信息，以及在簇传感器传递信号以影响dna时发生的变化 有关气体处理酶中的侧链贡献和气体通道的信息，这些酶处理或被小分子如氮气、氧气、一氧化碳和一氧化氮抑制。 获取这些信息的方法是光谱学。 利用光解/FT-IR、NRVS和共振拉曼光谱，这项工作将表征固氮酶(N2ase)如何与抑制剂CO结合，以及它与N2和更多还原中间体的络合物的结构，阐明氢化物何时以及如何结合在氢酶(H2酶)的活性部位， 使用各种时间尺度上的时间分辨光谱，我们的目标是确定NO和O2与各种蛋白质中的[4Fe-4S]簇反应的中间产物和最终产物，包括引起结核病的结核分枝杆菌的‘WhiB’蛋白质。 我们将开发一种新技术。61Ni同步加速器穆斯堡尔谱学，它应该在镍酶和一般化学中有广泛的应用。

 DESCRIPTION (provided by applicant): Biological Fe-S clusters are nanoparticles containing 2-8 Fe atoms that are held together primarily by bridging S atoms. Proteins that contain Fe-S clusters serve a wide variety of essential tasks in living systems, including catalysis of chemical reactions, sensing the chemical environment, signaling to and repair of DNA, and the maintenance of molecular structure. Our main goals for this proposal center on four key questions: How do Fe-S clusters react with small molecules? How do they catalyze reactions, how do they work as nitric oxide and oxygen sensors, and what happens when they decompose? What are the steps in assembly of the hydrogenase active site `H-cluster'? How does the larger protein environment affect access of small molecules to Fe-S sites? How does chemistry at an Fe-S cluster affect tertiary protein structure and interactions with DNA? The expected outcomes from our research include: Information about the catalytic intermediates of enzymes that fix nitrogen (nitrogenase) or produce hydrogen (hydrogenase) Information about reaction intermediates when Fe-S cluster proteins react with signaling molecules NO and O2, and changes that occur as the cluster sensor transduces the signal to affect DNA Information about side chain contributions and gas channels in gas-processing enzymes that process or are inhibited by small molecules such as N2, O2, CO, and NO. The approach to gain this information is spectroscopy. Using photolysis/FT-IR, NRVS, and resonance Raman spectroscopy, this work will characterize how nitrogenase (N2ase) binds the inhibitor CO, as well as the structures of its complex with N2 and more reduced intermediates, elucidate when and how hydrides bound at the active sites of the hydrogenases (H2ases), Using time-resolved spectroscopies on a variety of time-scales, we aim to define intermediates and final products for reactions of NO and O2 with [4Fe-4S] clusters in various proteins, including the `WhiB' proteins from the tuberculosis-causing bacterium Mycobacterium tuberculosis. We will develop a new technique. 61Ni synchrotron Mössbauer spectroscopy, that should have broad applications to Ni enzymes and to chemistry in general.