Spectroscopic Definition of Electronic Structure and Contributions to Reactivity of Binuclear Non-Heme Iron Enzymes

双核非血红素亚铁活性位点参与了与二氧的广泛反应，包括O2结合（血红蛋白）、羟基化（甲烷单加氧酶，MMO）、去饱和（去饱和酶）、酪氨酸自由基形成（核糖核苷酸还原酶，RR，通过质子偶联电子转移（PCET）过程）、双电子还原（铁蛋白的铁氧化酶位点和红红蛋白的相关过氧化物酶位点）及其四电子还原为水(红红蛋白：O2氧化还原酶、NO还原酶和植物中的替代氧化酶)。这些蛋白的结构存在于至少一种氧化态，fe2iii -过氧化物和高价铁-氧中间体已被捕获，相关的氧-非血红素二铁模型配合物和从头设计的蛋白质类似物已被合成。采用变温变场磁圆二色性（VTVH MCD）的光谱学方法来探测双联活性位点，并根据每个铁和桥接配体的几何和电子结构分析激发态和基态相互作用。VTVH MCD结合其他光谱（共振拉曼激发谱，x射线吸收，EPR等）已被进一步应用于定义蛋白质和模型中许多氧中间体的几何和电子结构及其与反应性的相关性。将这些数据与电子结构计算（DFT）相结合，提供了对几何和电子结构/功能相关性的重要见解。目前的研究重点是利用这种光谱/计算方法从结构上确定RR和MMO反应坐标中的中间体。这些研究将定义与激活O2反应性位点相关的双联静息状态的结构变化，确定导致底物反应性差异的几何和电子结构差异（去饱和、羟基化和PCET），并大致评估上述列出的蛋白质和酶（以及从头设计的类似物）的几何和电子结构差异，这些差异有助于它们在O2生物化学中的不同作用。铁酶和铁蛋白在许多重要的生物功能中起着关键作用。本研究的重点是了解双核非血红素铁酶的结构和功能，为反应过程和催化提供分子水平的见解。所研究的酶在催化过程、生物技术、环境调节和DNA生物合成中具有重要意义。作为本研究的一部分，所开发的科学方法已应用于生物学和催化中各种重要类别的金属位点。参与这项研究的博士后、研究生和本科生都受过广泛的光谱方法、量子化学计算和酶学方面的训练，并已成为学术界和工业界的重要贡献者。这些实验室中用于研究双核活性位点的仪器和理论资源已通过一系列合作提供给生物无机界，这些合作也利用这些资源加强对该领域的影响。PI还试图通过组织专题讨论会、书籍、综述文章和一般性讲座和系列讲座来提高人们对生物无机化学中不同光谱方法的重要性和信息内容的普遍认识。也有很多休假的访客花时间在斯坦福大学PI的实验室里，以进一步了解他们在这一领域的知识，从知名的生物无机/生物物理化学家，到当地的高中化学老师，以及对加强研究感兴趣的本科学院的少数民族教授。该项目由分子和细胞生物科学部的分子生物物理学项目和化学部的无机、生物无机和有机金属项目共同资助。

Binuclear non-heme ferrous active sites are involved in a wide-range of reactions with dioxygen including O2 binding (hemerythrin), activation for hydroxylation (methane monooxygenase, MMO), desaturation (desaturase), tyrosine radical formation (ribonucleotide reductase, RR, by a proton coupled electron transfer (PCET) process), reduction by two-electrons (ferroxidase site of ferritin, and the related peroxidase site of rubrerythrin), and its four-electron reduction to water (rubredoxin: O2 oxidoreductase, NO reductase, and the alternative oxidase in plants). Structures exist for many of these proteins in at least one oxidation state, Fe2III-peroxide and high valent iron-oxo intermediates have been trapped, and relevant oxygen-non-heme diiron model complexes and de novo designed protein analogues have been synthesized. Spectroscopic methodology emphasizing variable-temperature variable-field magnetic circular dichroism (VTVH MCD) has been developed to probe the biferrous active sites and analyze the excited and ground state interactions in terms of the geometric and electronic structure of each iron and the bridging ligands. VTVH MCD combined with other spectroscopies (resonance Raman excitation profiles, X-ray absorption, EPR, etc.) have further been applied to define the geometric and electronic structures of a number of the oxygen intermediates in the proteins and models and their relevance to reactivity. Combining these data with electronic structure calculations (DFT) has provided significant insight into geometric and electronic structure/function correlations. Research now focuses on using this spectroscopic/computational methodology to structurally define the intermediates in the reaction coordinates of RR and MMO. These studies will define the structural changes of the biferrous resting state associated with activating the site for O2 reactivity, determine geometric and electronic structural differences that contribute to differences in substrate reactivity (desaturation, hydroxylation and PCET), and generally evaluate geometric and electronic structure differences over the range of proteins and enzymes listed above (and de novo designed analogues) which contribute to their different roles in O2 biochemistry. Iron enzymes and proteins play key roles in many important biological functions. The focus of this research is to understand the structure and function of binuclear non-heme iron enzymes, providing molecular level insight into reaction processes and catalysis. The enzymes studied are important in catalytic processes, biotechnology, environmental regulation, and DNA biosynthesis. The scientific methodologies developed as part of this study have applications to a variety of important classes of metal sites in biology and catalysis. Postdoctoral associates, graduate students and undergraduates involved in this research are well trained in a wide range of spectroscopic methods, quantum chemistry calculations and enzymology, and have gone on to become significant contributors in academia and industry. The instrumental and theoretical resources available in these labs for the study of binuclear active sites have been made accessible to the bioinorganic community through a range of collaborations which also leverage these resources to enhance impact on the field. The PI has also attempted to enhance the general awareness of the importance and information content of the different spectroscopic methods in bioinorganic chemistry through organizing symposia, books, overview articles and presentations of general lectures and lecture series on this topic. There have also been a wide-range of sabbatical visitors spending time in the PI's labs at Stanford to further their knowledge in this area ranging from well established bioinorganic/biophysical chemists, to a local high school chemistry teacher and an under-represented minority Professor from an undergraduate college interested in enhancing research. This project is jointly funded by the Molecular Biophysics Program in the Division of Molecular and Cellular Biosciences and the Inorganic, Bioinorganic and Organometallic Program in the Chemistry Division.