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双氧以进行代谢关键转化。非血红素二铁酶具有多种依赖二氧的基本功能，包括DNA的生物合成（核糖核苷酸还原酶）、铁的储存（铁蛋白）和有机底物的羟基化（甲烷单加氧酶、芳烃羟化酶、脱氧氢碱基羟化酶）。一般来说，双氧活化被认为需要一个涉及双铁(III)-过氧中间体的共同机制。重要的项目目标是了解二铁(III)-过氧中间体如何转化为相应的高价氧化铁，这些氧化铁很可能是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。这些目标将通过生化和仿生两种方法来实现。生物化学方面的努力将主要集中在人类脱氧hypusine羟化酶上，这种酶将真核起始因子5A上的脱氧hypusine残基羟化，从而产生真核细胞增殖所需的成熟形式，并与HIV-1转录起始有关。该酶具有二铁活性位点，并且以异常稳定的二铁(III)-过氧形式分离，但仍能进行底物羟基化。双铁活性位点将通过生物化学和光谱技术的结合来研究，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二铁酶氧化还原循环中观察到的二铁(III)-过氧和高价铁中间体相关的亚稳物种。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）单元的配合物，这些基序与酶催化过程中形成的氧化物质相关。这些新型配合物将通过各种光谱技术来确定其结构和电子性质。还将合成Fe(III)-O-Mn（IV）和Fe(IV)-O-Mn（IV）单元的相应配合物，以模拟最近发现的与沙眼衣原体中具有FeMn活性位点（而不是二铁位点）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复合物的反应性差异可能有助于开发更好的方法来治疗这些人类病原体的感染。非血红素二铁酶执行各种代谢关键功能，需要氧气。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hypusine羟化酶是形成成熟的真核起始因子5a所必需的，而真核起始因子5a是细胞增殖所必需的。了解这些酶是如何工作的，将有助于开发治疗癌症、艾滋病和人类病原体（如衣原体）感染的新药物策略。

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.