Synthetic Models and Spectroscopy of Nonheme Diiron Enzymes

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在代谢关键转化中如何激活双氧。非血红素二铁酶执行涉及二氧的多种基本功能，包括DNA生物合成（核糖核苷酸还原酶）、铁储存（铁蛋白）和有机底物的氧化（甲烷单加氧酶、脂肪酸去饱和酶、烷烃和芳烃羟化酶、肌醇加氧酶、脱氧羟腐胺赖氨酸羟化酶）。一般来说，提出双氧活化需要涉及二铁（III）-过氧中间体和由其衍生的高价铁-氧代物质的共同机制。该项目的目标将使用仿生和光谱方法相结合来实现。建立在过去的成就，这些网站的结构和光谱特性建模，建议合成的三脚架配体的前体配合物，使它们与O2或过氧化物反应，并表征从中衍生的亚稳中间体。非常感兴趣的是中间体，例如二铁（II）络合物（铁（II）铁（III）-超氧或二铁（III）-过氧物种）的O2加合物，以及具有Fe（III）Fe（IV）和Fe（IV）Fe（IV）氧化态的物种。这些络合物将尽可能通过X射线晶体学和各种技术如NMR、EPR、UV-vis-NIR、拉曼、穆斯堡尔、电喷雾质谱、电化学和EXAFS表征。平行于这些努力，我们的光谱专业知识将被应用于阐明甲烷单加氧酶中间体和人脱氧羟腐胺赖氨酸羟化酶的二铁网站结构。在本竞争性修订版中，添加了GM-38767的新特定目标，因为最近发现寄生虫沙眼衣原体的核糖核苷酸还原酶（RNR）使用Fe（III）-O-Mn（IV）中心作为启动核糖核苷酸还原所需的氧化剂，而不是E.大肠杆菌和哺乳动物RNR。有人建议，这种金属取代允许这种寄生菌规避二铁RNR的敏感性，在哺乳动物细胞的典型免疫反应中产生的NO。对于新的具体目标，建议使用我们正在进行的合成二铁中间体的工作中开发的方法，以获得和表征相应的FeMn类似物，即Fe（III）Mn（III）-过氧，Fe（III）-O-Mn（IV），和Fe（IV）-O-Mn（IV）物种。这些新的铁锰复合物的性质将与他们的二铁同行进行比较，以评估自然界的选择在这个关键的酶的金属中心的化学基础。 公共卫生相关性：非血红素二铁酶执行各种代谢关键功能，需要双氧激活。了解这些酶如何工作可以导致治疗某些人类疾病的新药策略的开发。例如，核糖核苷酸还原酶是控制DNA生物合成的关键酶，而脱氧羟腐胺赖氨酸羟化酶是形成细胞增殖所必需的成熟真核延伸因子5a所必需的;因此这两种酶都可以作为抗肿瘤或抗HIV治疗的靶标。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to understand how dioxygen is activated by biological diiron centers in metabolically critical transformations. Nonheme diiron enzymes perform a variety of essential functions involving dioxygen, including DNA biosynthesis (ribonucleotide reductase), iron storage (ferritin), and oxidations of organic substrates (methane monooxygenase, fatty acid desaturases, alkane and arene hydroxylases, myo-inositol oxygenase, deoxyhypusine hydroxylase). In general, dioxygen activation is proposed to entail a common mechanism involving diiron(III)-peroxo intermediates and high-valent iron-oxo species derived therefrom. The project goals will be accomplished using a combination of biomimetic and spectroscopic approaches. Building on past accomplishments in modeling structural and spectroscopic properties of such sites, it is proposed to synthesize precursor complexes of tripodal ligands, to react them with O2 or peroxides, and to characterize the metastable intermediates derived therefrom. Of great interest are intermediates such as O2 adducts of diiron(II) complexes (either iron(II)iron(III)-superoxo or diiron(III)-peroxo species), and species with Fe(III)Fe(IV) and Fe(IV)Fe(IV) oxidation states. These complexes will be characterized by X-ray crystallography whenever possible and by a variety of techniques such as NMR, EPR, UV-vis-NIR, Raman, M"ssbauer, electrospray mass spectrometry, electrochemistry, and EXAFS. Parallel to these efforts, our spectroscopic expertise will be applied to elucidating the diiron site structures of methane monooxygenase intermediates and human deoxyhypusine hydroxylase. In this competitive revision, a new specific aim for GM-38767 is added because of the recent discovery that ribonucleotide reductase (RNR) of the parasite Chlamydia trachomatis uses as the oxidant needed to initiate ribonucleotide reduction a Fe(III)-O-Mn(IV) center, rather than the diiron(III)/tyrosyl radical combination characterized in E. coli and mammalian RNRs. It is suggested that this metal substitution allows this parasitic bacterium to circumvent the sensitivity of diiron RNR to NO produced in the typical immune response of mammalian cells. For the new specific aim, it is proposed to use methodologies developed in our ongoing work on synthetic diiron intermediates to obtain and characterize corresponding FeMn analogs, namely Fe(III)Mn(III)-peroxo, Fe(III)-O-Mn(IV), and Fe(IV)-O-Mn(IV) species. The properties of these novel FeMn complexes will be compared with those of their diiron counterparts to assess the chemical basis for Nature's choice of metal centers in this crucial enzyme. PUBLIC HEALTH RELEVANCE: Nonheme diiron enzymes perform a variety of metabolically critical functions that require dioxygen activation. Understanding how these enzymes work can lead to the development of new drug strategies for treating some human diseases. For example, ribonucleotide reductase is a key enzyme that controls DNA biosynthesis, while deoxyhypusine hydroxylase is required for the formation of mature eukaryotic elongation factor 5a that is essential for cell proliferation; thus both enzymes may serve as targets for anti-tumor or anti-HIV therapy.