Synthetic Models and Spectroscopy of Metal-Oxo Proteins

描述（由申请人提供）：二铁氧蛋白具有活性位点，由氧桥接的金属中心或羧酸桥支撑的羟基组成。这一不断扩大的金属蛋白类别现在包括在生物学中执行各种功能的蛋白质：双氧运输（甲氰菊酯），核糖核苷酸转化为脱氧核糖核苷酸（核糖核苷酸还原酶），铁储存（铁蛋白），磷酸酯水解（紫酸磷酸酶），以及通过氧激活有机底物的氧化（甲烷单加氧酶，脂肪酸去饱和酶，烷烃和芳烃羟化酶）。已知可溶和膜结合两种形式。许多可溶性酶具有指示富含羧酸二铁位点的序列基序，而新兴的膜结合亚类似乎具有富含组氨酸的二铁位点。本研究的重点是利用仿生和生物物理相结合的方法来了解双铁中心的氧活化。氧在双铁活性位点的活化被认为是一个涉及双铁(III)-过氧中间体和由此衍生的高价氧化铁的共同机制。基于过去在这些位点的结构和光谱特性建模方面取得的成就，我们建议合成前体配合物，与O2或过氧化物反应产生亚稳中间体，并表征这些中间体的光谱和反应性特性。我们非常感兴趣的是中间产物，如二铁（II）配合物的O2加合物，铁（III）的过氧衍生物，以及具有Fe(III)Fe（IV）和Fe(IV)Fe（IV）形式氧化态的物质。这些配合物将尽可能通过x射线晶体学和各种光谱技术进行表征，如NMR， EPR, UV-vis-NIR，拉曼，穆斯堡尔，电喷雾质谱和EXAFS。堵流和常规动力学方法都将用于表征形成和分解的机制。这些瞬态复合物对一系列底物的氧化反应性将被研究，并与酶活性位点的氧化反应性进行比较。同时，将对非血红素二铁酶中间体本身进行EXAFS和共振拉曼研究，以深入了解其核心结构。特别感兴趣的是硬脂酰acp δ 9-去饱和酶和人H链和大肠杆菌铁蛋白的过氧化物中间体，以及甲烷单加氧酶循环中的O， P， Q和T中间体。这些生物物理实验与生成这些酶中间体的合成模型的努力相吻合。

DESCRIPTION (provided by applicant): The diiron-oxo proteins have active sites consisting of metal centers bridged by oxo or hydroxo groups supported by carboxylate bridges. This expanding class of metalloproteins now includes proteins that perform a variety of functions in biology: dioxygen transport (hemerythrin), the conversion of ribonucleotides to deoxyribonucleotides (ribonucleotide reductase), iron storage (ferritin), phosphate ester hydrolysis (purple acid phosphatases), and oxidations of organic substrates via oxygen activation (methane monooxygenase, fatty acid desaturases, alkane and arene hydroxylases). Both soluble and membrane-bound forms are known. Many of the soluble enzymes have a sequence motif indicative of a carboxylate-rich diiron site, while the emerging membrane-bound subclass appears to have a histidine-rich diiron site. The focus of this proposal is to understand oxygen activation by diiron centers using a combination of biomimetic and biophysical approaches. Oxygen activation at a diiron active site is proposed to entail a common mechanism involving diiron(III)-peroxo intermediates and high-valent iron-oxo species derived therefrom. Building on past accomplishments in modeling structural and spectroscopic properties of such sites, it is proposed to synthesize precursor complexes that react with O2 or peroxides to afford metastable intermediates and characterize the spectroscopic and reactivity properties of these intermediates. Of great interest are intermediates such as O2 adducts of diiron(II) complexes, peroxo derivatives of iron(III), and species with Fe(III)Fe(IV) and Fe(IV)Fe(IV) formal oxidation states. These complexes will be characterized by x-ray crystallography whenever possible and by a variety of spectroscopic techniques such as NMR, EPR, UV-vis-NIR, Raman, Mossbauer, electrospray mass spectrometry, and EXAFS. Both stopped-flow and conventional kinetic methods will be used to characterize the mechanisms of formation and decomposition. The oxidative reactivities of these transient complexes towards a range of substrates will be investigated and compared with those of enzyme active sites. In parallel, EXAFS and resonance Raman studies of nonheme diiron enzyme intermediates themselves will be carried out to gain insight into their core structures. Of specific interest are the peroxo intermediates of stearoyl-ACP delta-9-desaturase and human H chain and E. coli ferritins as well as intermediates O, P, Q, and T in the methane monooxygenase cycle. These biophysical experiments dovetail well with the efforts to generate synthetic models for these enzyme intermediates.