Nonheme diiron enzymes: understanding oxygen activation in human deoxyhypusine hy

描述（由申请人提供）：非血红素二铁酶参与许多生物和工业相关化合物的合成，包括甲醇的形成，抗生素的生物合成以及RNA构建DNA模块的创建。这些酶的催化循环包括O2的结合和随后的活化，以进行各种各样的化学反应。重要的是，这些酶的机制被认为依赖于一个共同的（-1,2-过氧）差质中间体的形成。这种结构被认为是稳定的，需要激活成更活泼的中间体进行后续催化。尽管这种酶超家族已经被研究多年，但围绕酶如何激活常见的过氧化物中间体以维持如此多样化的反应性的几个关键机制细节尚不清楚。在不同的蛋白质折叠和二铁配位环境中发现了新的二铁酶，这促使人们进一步研究蛋白质和二铁位点在形成和控制过氧反应活性中的作用。特别是，Que实验室已经在人脱氧hypusine羟化酶（hDOHH）中发现了一个过氧中间体。hDOHH催化翻译后氨基酸hypusine的羟基化。这种氨基酸仅存在于真核生物翻译起始因子5A （eIF5A）中，是细胞增殖所必需的，这使得hDOHH成为癌症和HIV治疗的一个有吸引力的靶点。hDOHH过氧化物中间体（hDOHHperoxo）稳定数天，使其成为迄今为止发现的寿命最长的过氧化物物种。此外，二铁簇被安置在一个独特的蛋白质折叠和协调环境，从所有其他非血红素二铁酶。本项目拟利用一系列生化和光谱技术研究调节hDOHHperoxo活化的机制和结构细节。具体来说，本提案将研究底物结合、pH和不同的二铁配位环境如何促进hDOHHperoxo的形成和激活。hDOHHperoxo的形成速率作为底物浓度，pH和氘化的函数将使用紫外可见光谱测量。将四个活性位点Glu配体（Glu57, Glu90, Glu208, Glu241）单独突变为Asp和Gln，以评估静电和立体效应对hDOHHperoxo稳定性的贡献。电子和几何细节的活动场地结构

DESCRIPTION (provided by applicant): Nonheme diiron enzymes are involved in the synthesis of many biologically and industrially relevant compounds, including the formation of methanol, the biosynthesis of antibiotics, and the creation of DNA building blocks from RNA. The catalytic cycles of these enzymes involve the binding and subsequent activation of O2 to carry out a wide variety of chemistry. Importantly, the mechanism of these enzymes is thought to depend on the formation of a common (¿-1,2-peroxo)diferric intermediate. This structure is proposed to be stable, requiring activation to a more reactive intermediate for subsequent catalysis. Although this enzyme superfamily has been studied for many years, several key mechanistic details surrounding how enzymes activate the common peroxo intermediate to maintain such diverse reactivity are unknown. The identification of new diiron enzymes within distinct protein folds and diiron coordination environments has prompted further investigation into the role of both the protein and the diiron site on formation and control of peroxo reactivity In particular, the Que laboratory has identified a peroxo intermediate in human deoxyhypusine hydroxylase (hDOHH). hDOHH catalyzes the post-translational hydroxylation of the amino acid hypusine. This amino acid is found only in the eukaryotic translational initiation factor 5A (eIF5A and is required for cell proliferation, making hDOHH an appealing target for cancer and HIV treatments. The hDOHH peroxo intermediate (hDOHHperoxo) is stable for days, making it the longest-lived peroxo species identified to date. Furthemore, the diiron cluster is housed in a protein fold and coordination environment unique from all other nonheme diiron enzymes. This project proposes to investigate mechanistic and structural details that regulate hDOHHperoxo activation using a host of biochemical and spectroscopic techniques. Specifically, this proposal will investigate how substrate binding, pH, and the distinct diiron coordination environment contribute to the formation and activation of hDOHHperoxo. The rates of hDOHHperoxo formation as a function of substrate concentration, varied pH and deuteration will be measured using UV-visible spectroscopy. Individual mutagenesis of four active site Glu ligands (Glu57, Glu90, Glu208, Glu241) to Asp and Gln will be performed to assess the contribution of electrostatics and sterics on hDOHHperoxo stability. Electronic and geometric details of the active site structure as a function of the above modifications (substrate, pH, coordinating ligand) will be ascertained using resonance Raman, M¿ssbauer, and XAS spectroscopies. Importantly, these studies will be carried out for the first time using a peroxo species competent in carrying out the native chemistry of the enzyme. Information obtained from these experiments will add to our knowledge of features that affect the reactivity of peroxo intermediates, particularly those found in new protein folds and distinct diiron coordination environments, furthering our understanding of how O-O bond activation is accomplished and may lead to a better understanding of how to target hDOHH for cancer and HIV therapeutics.