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如何在代谢关键转化中激活双氧。非血红素二铁酶具有多种与二氧有关的基本功能，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hypusine羟化酶）。一般来说，双氧活化的共同机制涉及双铁(III)-过氧中间体和由此衍生的高价氧化铁。该项目的目标将通过结合仿生学和光谱方法来实现。基于过去在模拟这些位点的结构和光谱特性方面的成就，我们建议合成三足配体的前体配合物，使它们与O2或过氧化物反应，并表征由此产生的亚稳中间体。我们对二铁（II）配合物(无论是铁(II) -铁（III)-超氧或二铁(III)-过氧）的O2加合物，以及具有Fe(III) - Fe（IV）和Fe(IV) - Fe（IV）氧化态的中间体非常感兴趣。这些配合物将尽可能通过x射线晶体学和各种技术进行表征，如NMR， EPR, UV-vis-NIR，拉曼，M ' ssbauer，电喷雾质谱，电化学和EXAFS。与这些努力平行，我们的光谱专业知识将应用于阐明甲烷单加氧酶中间体和人类脱氧hypusine羟化酶的二铁位点结构。在这个竞争性修订中，GM-38767增加了一个新的特异性靶点，因为最近发现寄生虫沙眼衣原体的核糖核苷酸还原酶（RNR）用作启动Fe(III)-O-Mn（IV）中心核糖核苷酸还原所需的氧化剂，而不是大肠杆菌和哺乳动物RNR中所特有的二铁(III)/酪氨酸自由基组合。这表明，这种金属取代使这种寄生细菌能够绕过哺乳动物细胞典型免疫反应中产生的双铁RNR对NO的敏感性。对于新的特定目标，建议使用我们正在进行的合成双铁中间体的工作中开发的方法来获得和表征相应的FeMn类似物，即Fe(III)Mn(III)-过氧，Fe(III)-O-Mn（IV）和Fe(IV)-O-Mn（IV）物种。这些新型FeMn复合物的性质将与它们的双铁复合物的性质进行比较，以评估自然界在这种关键酶中选择金属中心的化学基础。

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.