Synthetic Models and Spectroscopy of Metal-Oxo Proteins

描述(由申请人提供)：本提案的总体目标是了解生物双铁中心如何激活氧气以进行代谢关键的转化。非血红素二铁酶具有多种氧依赖的基本功能，包括DNA的生物合成(核糖核苷酸还原酶)、铁储存(铁蛋白)和有机底物的羟基化(甲烷单加氧酶、芳烃羟基酶、脱氧亚硫氨酸羟基酶)。一般来说，氧的活化被认为需要一个共同的机制，涉及二铁(III)-过氧基中间体。重要的项目目标是了解二铁(III)-过氧基中间体如何转化为相应的高价铁氧物种，这些物种很可能作为底物转化的关键氧化剂，并描述高价中间体的结构、电子和反应性能。这些目标将通过生化和仿生方法来实现。生化工作将主要集中在人脱氧亚硫氨酸羟基酶上，这是一种将真核细胞启动因子5A上的脱氧亚硫氨酸残基羟化以产生成熟形式的酶，这是真核细胞增殖所必需的，并与HIV-1转录启动有关。这种酶具有双铁活性部位，并以一种异常稳定的双铁(III)-过氧基形式分离出来，但仍能底物羟化。将结合生化和光谱技术对双铁活性部位进行研究，以深入了解其作用模式。仿生的努力将集中在产生和捕获与非血红素双铁酶的氧化还原循环中观察到的二铁(III)-过氧基和高价铁中间体有关的亚稳物种。特别令人感兴趣的是具有不寻常的Fe(III)-O-Fe(IV)和Fe(IV)-O-Fe(IV)单元的络合物，这些基序与酶催化过程中形成的氧化物种有关。这些新的配合物将通过各种光谱技术进行表征，以确定它们的结构和电子性质。相应的具有Fe(III)-O-Mn(IV)和Fe(IV)-O-Mn(IV)单元的配合物也将被合成，以模拟与最近从寄生虫沙眼衣原体中发现的具有FeMn活性中心(而不是二铁中心)的核糖核苷酸还原酶(RNR)相关的高价中间体。了解高价FeFe和FeMn络合物反应性的差异可能有助于开发更好的方法来治疗此类人类病原体的感染。非血红素双铁酶执行需要氧气的各种代谢关键功能。例如，核糖核苷酸还原酶是控制DNA生物合成的关键酶，而脱氧亚硫氨酸羟化酶是形成成熟的真核细胞起始因子5a所必需的，而真核细胞起始因子5a是细胞增殖所必需的。了解这些酶是如何工作的，可以导致开发治疗癌症、艾滋病和衣原体等人类病原体感染的新药策略。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to understand how dioxygen is activated by biological diiron centers to carry out metabolically critical transformations. Nonheme diiron enzymes perform a variety of dioxygen-dependent essential functions, including the biosynthesis of DNA (ribonucleotide reductase), iron storage (ferritin), and the hydroxylation of organic substrates (methane monooxygenase, arene hydroxylases, deoxyhypusine hydroxylase). In general, dioxygen activation is proposed to entail a common mechanism involving diiron(III)- peroxo intermediates. Important project goals are to understand how the diiron(III)-peroxo intermediates are converted to corresponding high-valent iron-oxo species that very likely act as the key oxidants for substrate transformation and to describe the structural, electronic, and reactivity properties of the high-valent intermediates. These goals will be accomplished by both biochemical and biomimetic approaches. The biochemical effort will primarily focus on human deoxyhypusine hydroxylase, an enzyme that hydroxylates a deoxyhypusine residue on eukaryotic initiation factor 5A to generate a mature form that is required for eukaryotic cell proliferation and implicated in HIV-1 transcription initiation. This enzyme has a diiron active site and is isolated in an unusually stable diiron(III)-peroxo form that is nevertheless capable of substrate hydroxylation. The diiron active site will be investigated by a combination of biochemical and spectroscopic techniques to gain insight into its mode of action. The biomimetic effort will focus on generating and trapping metastable species that relate to diiron(III)-peroxo and high-valent iron intermediates observed in the redox cycles of the nonheme diiron enzymes. Of particular interest are complexes with unusual Fe(III)-O-Fe(IV) and Fe(IV)-O-Fe(IV) units, motifs associated with the oxidizing species formed during enzyme catalysis. These novel complexes will be characterized by a variety of spectroscopic techniques to determine their structures and electronic properties. Corresponding complexes with Fe(III)-O-Mn(IV) and Fe(IV)-O-Mn(IV) units will also be synthesized to model high-valent intermediates associated with the recently discovered ribonucleotide reductase (RNR) with a FeMn active site (instead of a diiron site) from the parasite Chlamydia trachomatis. Understanding the difference in the reactivity properties of high-valent FeFe and FeMn complexes may contribute to the development of better methods for treating infections from such human pathogens. Nonheme diiron enzymes perform a variety of metabolically critical functions that require O2. For example, ribonucleotide reductase is a key enzyme that controls DNA biosynthesis, while deoxyhypusine hydroxylase is required for the formation of mature eukaryotic initiation factor 5a that is essential for cell proliferation. Understanding how these enzymes work can lead to the development of new drug strategies for treating cancer, AIDS, and infections of human pathogens like chlamydiae.