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

描述（由申请人提供）：包括人类在内的所有需氧生物的生命都依赖于金属对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的氧化能力。Mn和非血红素Fe双加氧酶催化底物芳香环的裂解，同时加入来自O2的两个氧原子。这个反应是大自然回收大量封存在芳香化合物中的碳的关键一步。色氨酸2,3-双加氧酶（TDO）催化l -色氨酸（L-Trp）的双氧插入和吲哚环的氧化裂解，将其转化为n -甲酰基犬尿氨酸（NFK）。犬尿氨酸途径是NAD从头生物合成的主要途径，NAD是所有生命系统中必需的氧化还原辅助因子之一。该途径中间代谢物的改变可导致许多生理和病理状况，包括：白内障形成、脑性疟疾、阿尔茨海默病、艾滋病毒感染、亨廷顿病和缺血性脑损伤。TDO负责氧化细胞内和细胞外池中99%以上的游离l -色氨酸。此外，TDO调节的色氨酸水平可以影响血清素（一种已知的神经递质）的合成。这两类酶代表了自然界如何进化出有效的底物氧化策略的两种基本差异。锰和非血红素铁双加氧酶的活性金属位点对底物和氧都有可用的金属配位位点。相比之下，含有TDO的血红素只有一个可用的金属配位位点，它与O2结合，底物L-Trp与蛋白质在一个靠近但远离Fe的口袋中结合。仿生模型复合物的研究将增强我们解释生物分子复杂电子特性的能力，并帮助我们理解催化中重要的金属酶的结构和化学方面。我们的目标是通过研究金属活性位点在酶翻转天然底物时发生的原子水平变化来深入了解这些酶是如何发挥其功能的。预计这样的研究将提供更好的理解自然如何构建酶活性位点来执行选择性和有效的底物氧化。我们在EPR方法上的进步使这项研究成为可能。我们已经创建了EPR光谱解释软件，它允许前所未有的能力定量表征几乎所有顺磁性金属位点在蛋白质和酶。

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.