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

描述(申请人提供)：包括人类在内的所有需氧生物的生命依赖于金属对O2的激活，以提供生物分子的选择性和快速氧化。金属酶已经进化出多种化学途径来有效地利用丰富的氧气的氧化能力。锰和非血红素铁双加氧酶催化底物芳香环的裂解，结合了O2中的两个氧原子。这一反应是大自然回收大量滞留在芳香化合物中的碳的能力的关键一步。色氨酸2，3-双加氧酶(TDO)催化氧的插入和L-色氨酸(L-色氨酸)吲哚环的氧化裂解，将其转化为N-甲酰犬尿氨酸(NFK)。犬尿氨酸途径构成了NAD从头合成的主要途径，NAD是所有生命系统中必不可少的氧化还原辅因子之一。这一途径的中间代谢产物的改变可导致许多生理和病理情况，包括：白内障形成、脑疟疾、阿尔茨海默病、HIV感染、亨廷顿病和缺血性脑损伤。TDO负责氧化胞内池和胞外池中99%以上的游离L-色氨酸。此外，TDO调节的色氨酸水平会影响已知的神经递质5-羟色胺的合成。这两类酶代表了自然界如何进化出高效底物氧化策略的两个根本区别。锰和非血红素铁双加氧酶的活性金属中心对底物和O2都有有效的金属配位。相比之下，含有TDO的血红素只有一个可用的金属配位点，它与O2结合，底物L-色氨酸在口袋里靠近蛋白质与蛋白质结合，但远离铁。我们将研究仿生模型化合物，以提高我们解释生物分子复杂电子性质的能力，并帮助我们理解在催化中重要的金属酶的结构和化学方面。我们在这项建议中的目标是通过研究当酶翻转其自然底物时在金属活性中心发生的原子水平变化，从而深入了解这些酶是如何发挥其功能的。预计这样的研究将更好地了解大自然如何构建酶活性部位来执行选择性和高效的底物氧化。我们在EPR方法论方面的进步使这项研究成为可能。我们已经创建了用于解释EPR谱的软件，该软件允许对蛋白质和酶中几乎所有顺磁性金属位置进行定量表征，这是前所未有的能力。 与公共健康相关：包括人类在内的所有需氧生物的生命依赖于金属对O2的激活，以提供生物分子的选择性和快速氧化。色氨酸双加氧酶在犬尿氨酸途径中起重要作用，是NAD生物合成的主要途径，NAD是所有生命系统中必不可少的氧化还原辅因子之一。这一途径的中间代谢产物的改变可导致许多生理和病理情况，包括：白内障形成、脑疟疾、阿尔茨海默病、HIV感染、亨廷顿病和缺血性脑损伤。此外，色氨酸双加氧酶调节的色氨酸水平会影响已知的神经递质5-羟色胺的合成。儿茶酚双加氧酶在自然界碳的回收中起着重要的作用，它为氧的活化和芳环的裂解提供了一种替代的氧化机制。

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. PUBLIC HEALTH RELEVANCE: Life for all aerobic organisms, including humans, depends on the activation of O2 by metals to provide selective and rapid oxidation of biological molecules. Tryptophan dioxygenase is important in the kynurenine pathway, constituting the major route of 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. In addition, the levels of tryptophan regulated by tryptophan dioxygenase can affect the synthesis of serotonin, a known neurotransmitter. Catechol dioxygenases are important in the recovery of carbon in Nature and present an alternative oxidation mechanism for the activation of O2 and cleavage of aromatic rings.