EPR and Mossbauer Characterization of Mn and Fe Proteins, Models, Intermediates

Mn 和 Fe 蛋白质、模型、中间体的 EPR 和穆斯堡尔表征

• 批准号: 8462633
• 负责人: MICHAEL P HENDRICH
• 金额: $ 27.51万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8462633
• 关键词: Active Sites; Aerobic; Affect; Alzheimer' s Disease; Anabolism; Aromatic Compounds; Binding; Biochemical; Biological; Biomimetics; Carbon; Catalysis; Cataract; Catechols; Cerebral Malaria; Chemicals; Chemistry; Complex; Computer software; Data; Dioxygen; Dioxygenases; Electronics; Enzymes; Freezing; Goals; HIV Infections; Heme; Human; Huntington Disease; Hydrogen Bonding; Indoles; Ischemic Brain Injury; Kinetics; Kynurenine; Lead; Life; Ligands; Measures; Metals; Methodology; Modeling; N' -formylkynurenine; Nature; Neurotransmitters; Organism; Oxidation-Reduction; Oxygen; Pathway interactions; Physiological; Property; Proteins; Reaction; Recovery; Research; Route; Series; Serotonin; Site; Spectrum Analysis; Superoxides; System; Techniques; Tryptophan; Tryptophanase; cofactor; electronic structure; extracellular; heme a; insight; metalloenzyme; novel; oxidation

DESCRIPTION (provided by applicant): Life for all aerobic organisms, including humans, depends on the activation of O2 by metals to provide selective and rapid oxidation of biological molecules. Metalloenzymes have evolved a variety of chemical pathways to efficiently utilize the oxidizing power of abundant O2. Mn and non-heme Fe dioxygenases catalyze the cleavage of the aromatic ring of the substrate with incorporation of both oxygen atoms from O2. This reaction is a key step in the ability of Nature to reclaim large quantities of carbon sequestered in aromatic compounds. Tryptophan 2,3-dioxygenase (TDO) catalyzes the insertion of dioxygen and oxidative cleavage of the indole ring of L-tryptophan (L-Trp), converting it to N-formylkynurenine (NFK). The kynurenine pathway constitutes the major route of de novo biosynthesis of NAD, one of the essential redox cofactors in all living systems. The alteration of intermediate metabolites of this pathway can lead to numerous physiological and pathological conditions, including: cataract formation, cerebral malaria, Alzheimer's disease, HIV infection, Huntington's disease and ischemic brain injury. TDO is responsible for oxidizing over 99% of the free L-Trp in intracellular and extracellular pools. In addition, the levels of tryptophan regulated by TDO can affect the synthesis of serotonin, a known neurotransmitter. These two classes of enzymes represent two such fundamental differences in how Nature has evolved strategies for efficient substrate oxidation. The active metal site of the Mn and non-heme Fe dioxygenases has available metal coordination sites for both the substrate and O2. In contrast, the heme containing TDO has only one available metal coordination site, which binds O2, and the substrate L-Trp binds to the protein in a pocket close, but away from the Fe. Biomimetic model complexes will be studied to both enhance our ability to interpret complicated electronic properties of biomolecules and to aid our understanding of the structural and chemical aspect of metalloenzymes that are important in catalysis. Our goal in this proposal is to provide insight into how these enzymes perform their function by studying the atomic level changes that occur in the metal active site as the enzymes turn over their natural substrate. It is anticipated that such studies will provide a better understanding of how Nature constructs enzymatic active sites to perform selective and efficient oxidation of substrates. This research is made possible by our advances in EPR methodology. We have created software for the interpretation of EPR spectra which allows an unprecedented ability to quantitatively characterize virtually all paramagnetic metal sites in proteins and enzymes.

描述（申请人提供）：包括人类在内的所有有氧生物的寿命取决于金属对O2的激活，以提供生物分子的选择性和快速氧化。金属酶已经进化了多种化学途径，以有效利用丰富的O2的氧化能力。 Mn和非血红素Fe二加氧酶通过掺入O2的两个氧原子来催化底物的芳族环的裂解。该反应是自然界回收大量碳含量的碳化合物能力的关键步骤。 色氨酸2,3-二加氧酶（TDO）催化了二氧化物的插入和L-色氨酸（L-TRP）的吲哚环的氧化裂解，从而将其转化为N-Formylylkynurenarine（NFK）。 Kynurenine途径构成了NAD的新生生物合成的主要途径，NAD是所有生活系统中必不可少的氧化还原辅助因子之一。该途径中间代谢物的改变可能导致许多生理和病理状况，包括：白内障形成，脑疟疾，阿尔茨海默氏病，HIV感染，亨廷顿氏病和缺血性脑损伤。 TDO负责在细胞内和细胞外池中氧化超过99％的游离L-TRP。另外，由TDO调节的色氨酸水平会影响5-羟色胺（一种已知的神经递质）的合成。 这两类酶在自然如何发展有效底物氧化的策略方面表示了两个这种基本差异。 Mn和非血红素Fe二氧酶的活性金属位点可用于基板和O2的金属配位位点。相比之下，包含TDO的血红素只有一个可用的金属配位位点，该位点结合O2，底物L-TRP在靠近的口袋中与蛋白质结合，但远离FE。 将研究仿生模型复合物，以增强我们解释生物分子的复杂电子特性的能力，并帮助我们理解在催化中很重要的金属酶的结构和化学方面。 我们在该提案中的目标是通过研究金属活性位点发生的原子水平变化如何在酶转向其自然底物中的原子水平变化，从而提供有关这些酶如何执行其功能的见解。可以预料，这样的研究将更好地理解自然如何构建酶促活性位点以对底物进行选择性和有效的氧化。 我们在EPR方法方面的进步使这项研究成为可能。我们创建了用于解释EPR光谱的软件，该软件允许定量表征蛋白质和酶中所有顺磁性金属位点的前所未有的能力。