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

双核非血红素活性中心参与了与氧气的广泛反应，包括O2结合(高氯氰菊酯)，羟化活化(甲烷单加氧酶，MMO)，去饱和酶(去饱和酶)，酪氨酸自由基形成(核糖核苷酸还原酶，RR，通过质子耦合电子转移(PCET)过程)，双电子还原(铁蛋白的铁氧化物酶位置，以及相关的红花红蛋白的过氧化物酶位置)，以及它的四电子还原为水(Rubredoin：O2氧化还原酶，NO还原酶，和植物中的交替氧化酶)。这些蛋白质中的许多结构至少以一种氧化态存在，Fe2III-过氧化铁和高价铁氧中间体被捕获，并合成了相应的氧-非血红素二铁模型络合物和从头设计的蛋白质类似物。利用变温变场磁圆二色谱(VTVH-MCD)研究了双铁的活性中心，并根据每个铁和桥联配体的几何结构和电子结构分析了激发态和基态相互作用。VTVH MCD与其他光谱(共振拉曼激发谱、X射线吸收、EPR等)相结合。进一步用于确定蛋白质和模型中一些氧中间体的几何和电子结构及其与反应活性的相关性。将这些数据与电子结构计算(DFT)相结合，为几何和电子结构/功能关联提供了重要的见解。现在的研究集中在使用这种光谱/计算方法来从结构上定义RR和MMO反应坐标中的中间体。这些研究将确定与激活O2反应位点相关的双亚铁休眠状态的结构变化，确定导致底物反应性差异(去饱和、羟化和PCET)的几何和电子结构差异，并总体评估上述蛋白质和酶(以及从头设计的类似物)在O2生物化学中起不同作用的几何和电子结构差异。铁酶和蛋白质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，提供对反应过程和催化的分子水平的洞察。所研究的酶在催化过程、生物技术、环境调节和DNA生物合成中都很重要。作为这项研究的一部分，开发的科学方法适用于生物和催化中各种重要的金属位置。参与这项研究的博士后助理、研究生和本科生在广泛的光谱方法、量子化学计算和酶学方面都受到了良好的培训，并成为学术界和工业界的重要贡献者。通过一系列的合作，生物无机界可以获得这些实验室中可用于研究双核活性中心的仪器和理论资源，这些合作也利用这些资源来加强对实地的影响。该协会还试图通过组织专题讨论会、书籍、概述文章以及关于这一主题的一般性讲座和系列讲座的介绍，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。也有各种各样的休假参观者在斯坦福国际的实验室里花费时间来加深他们在这一领域的知识，从著名的生物无机/生物物理化学家，到当地的高中化学教师和对加强研究感兴趣的本科学院的一名代表不足的少数族裔教授。该项目由分子和细胞生物科学部的分子生物物理学计划和化学部的无机、生物无机和有机金属计划共同资助。

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.