Synthetic Models and Spectroscopy of Metal-Oxo Proteins

描述（申请人提供）：二铁氧蛋白具有由羧酸桥支持的氧代或羟基团桥连的金属中心组成的活性位点。这类不断扩大的金属蛋白现在包括在生物学中执行各种功能的蛋白质：双氧运输（血红蛋白），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（紫色酸性磷酸酶），以及通过氧活化氧化有机底物（甲烷单加氧酶，脂肪酸去饱和酶，烷烃和芳烃羟化酶）。 可溶性和膜结合形式都是已知的。许多可溶性酶具有指示富含羧酸的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。 这个建议的重点是了解二铁中心使用仿生和生物物理方法相结合的氧激活。提出在二铁活性位点处的氧活化需要涉及二铁（III）-过氧中间体和由此衍生的高价铁-氧代物种的共同机制。建立在过去的成就，这些网站的结构和光谱特性建模，建议合成前体配合物，与O2或过氧化物反应，提供亚稳中间体和表征这些中间体的光谱和反应性能。非常感兴趣的是中间体，如二铁（II）络合物的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。这些复合物将尽可能通过X射线晶体学和各种光谱技术（如NMR、EPR、UV-vis-NIR、拉曼、穆斯堡尔、电喷雾质谱和EXAFS）进行表征。停流和传统的动力学方法将用于表征的形成和分解的机制。这些瞬态复合物对一系列底物的氧化反应性将进行研究，并与酶活性位点进行比较。同时，EXAFS和共振拉曼研究nonheme二铁酶中间体本身将进行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链的过氧中间体和E。大肠杆菌铁蛋白以及甲烷单加氧酶循环中的中间体O、P、Q和T。这些生物物理实验与为这些酶中间体生成合成模型的努力吻合得很好。

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.