Synthetic Models and Spectroscopy of Metal-Oxo Proteins

描述(申请人提供)：双铁-氧代蛋白具有活性中心，由羧酸桥支持的氧基或羟基连接的金属中心组成。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：氧气运输(氢氰菊酯)、将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)、磷酸酯水解酶(紫色酸性磷酸酶)以及通过氧激活的有机底物的氧化(甲烷单加氧酶、脂肪酸脱饱和酶、烷烃和芳烃羟基酶)。可溶的和膜结合的形式都是已知的。许多可溶性酶都有一个序列基序，表明有一个富含羧酸的双铁中心，而新出现的膜结合亚类似乎有一个富含组氨酸的双铁中心。这项建议的重点是通过结合仿生学和生物物理学的方法来了解双铁中心的氧活化。双铁活性中心的氧活化被认为需要涉及双铁(III)-过氧基中间体及其衍生的高价铁-氧物种的共同机制。在过去对这些中心的结构和光谱性质建模的基础上，建议合成与O2或过氧化物反应的前体络合物，以提供亚稳态中间体，并表征这些中间体的光谱和反应性性质。最感兴趣的是中间体，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)形式氧化态的物种。这些络合物将尽可能通过X射线结晶学和各种光谱技术进行表征，如核磁共振、EPR、UV-VIS-NIR、拉曼、穆斯堡尔谱、电喷雾质谱学和EXAFS。停流和传统的动力学方法将被用来表征形成和分解的机制。这些暂态络合物对一系列底物的氧化反应性将被研究，并与酶活性中心的氧化反应性进行比较。同时，还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。特别令人感兴趣的是硬脂酰-ACP增量-9-去饱和酶和人H链的过氧化中间体和大肠杆菌铁蛋白，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很好地吻合。

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.