Synthetic Models and Spectroscopy of Metal-Oxo Proteins

金属氧化蛋白的合成模型和光谱学

• 批准号: 6616418
• 负责人: LAWRENCE QUE
• 金额: $ 32.72万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6616418
• 关键词: Mossbauer spectrometry; Raman spectrometry; X ray crystallography; active sites; adduct; biomimetics; chemical models; electron spin resonance spectroscopy; electrospray ionization mass spectrometry; enzyme activity; enzyme induction /repression; hydroxylation; infrared spectrometry; iron compounds; iron sulfur protein; metalloproteins; nuclear magnetic resonance spectroscopy; oxidation; oxygen; peptide chemical synthesis; peroxides; ultraviolet spectrometry

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.

描述（由申请人提供）：二龙氧蛋白具有活性位点，该位点由羧酸酯支撑的Oxo或Hydroxo基团桥接的金属中心组成。现在，这种不断扩展的金属蛋白包括在生物学中执行多种功能的蛋白质：二氧化物传输（hemerythrin），核糖核苷酸向脱氧核糖核苷酸（核糖核苷酸还原酶），铁储存（铁蛋白），铁蛋白），磷酸酯酯化酶（纯酸磷酸磷酸酶）的转化（核糖核苷酸还原酶）的转化（核糖核苷酸还原酶）。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。许多可溶性酶的序列基序表示富含羧酸盐的二烯位点，而新兴的膜结合的亚类似乎具有富含组氨酸的二烷存位。 该提案的重点是使用仿生和生物物理方法的组合来理解二铁中心的氧气激活。提出了二烷活性位点的氧气激活，以需要一种涉及二龙（III） - 过敏中间体和从中得出的高价值铁氧物种的共同机制。基于对此类部位的结构和光谱特性进行建模的成就，它提议合成与O2或过氧化物反应的前体配合物，以提供亚稳态中间体并表征这些中间体的光谱和反应性。引人注目的是中间体，例如铁（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）Fe（IV）Fe（IV）Fe（iv）的物种。这些复合物将尽可能以X射线晶体学为特征，以及各种光谱技术，例如NMR，EPR，UV-VIS-NIR，Raman，Mossbauer，Electrospray质谱和EXAFS。停止流量和常规动力学方法都将用于表征形成和分解的机理。这些瞬态复合物朝向一系列底物的氧化反应率将进行研究，并将其与酶活性位点的氧化反应能力进行比较。同时，将对非血红素二氮酶中间体的EXAF和共振拉曼研究本身进行，以深入了解其核心结构。特定感兴趣的是甲烷单加氧酶周期中的stearoyl-ACP Delta-9-尿素酶和人H链和大肠杆菌铁蛋白以及中间体O，P，P，Q和T的过氧中间体。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非常吻合。