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: 6874923
• 负责人: LAWRENCE QUE
• 金额: $ 28.88万
• 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/6874923
• 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）：二铁-氧代蛋白具有由金属中心组成的活性位点，所述金属中心由羧酸酯桥支撑的氧代或羟基基团桥接。这一不断扩大的金属蛋白类别现在包括在生物学中执行多种功能的蛋白质：双氧转运（血红蛋白）、核糖核苷酸向脱氧核糖核苷酸的转化（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）和通过氧活化（甲烷单加氧酶、脂肪酸去饱和酶、烷烃和芳烃羟化酶）氧化有机底物。 可溶形式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盐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。 该提案的重点是结合仿生和生物物理方法来了解二铁中心的氧活化。提出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-氧物种的共同机制。基于过去在模拟此类位点的结构和光谱特性方面取得的成就，建议合成与 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特性。人们非常感兴趣的是中间体，例如二铁 (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。这些配合物将尽可能通过 X 射线晶体学以及各种光谱技术（例如 NMR、EPR、UV-vis-NIR、拉曼、穆斯堡尔、电喷雾质谱和 EXAFS）进行表征。停流和传统动力学方法都将用于表征形成和分解的机制。将研究这些瞬时复合物对一系列底物的氧化反应性，并与酶活性位点的氧化反应性进行比较。与此同时，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中间体，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非常吻合。