Transient catalytic oxygen species in iron enzymes

项目描述（申请人提供）：本项目主要研究铁酶催化循环中瞬态氧中间体的结构。具体的重点是氧金属配体的原子振动及其质子化状态的分辨率使用连续流共振拉曼光谱。使用由PI设计和实施的独特的，最先进的实验装置，我们将检查反应开始后短时间内形成的物种的同位素差异拉曼光谱。我们的方法的主要优点是能够在低至-70°C的温度下在液体样品中进行反应，并且死体积很小。这使我们能够将化学反应的速度放慢几个数量级，并允许在不需要大量生物样品的情况下对反应的早期阶段进行仔细检查。我们将研究两类铁酶。首先，我们将研究我们最近在单核非血红素铁和？-酮戊二酸依赖双加氧酶我们将利用紫外/可见光谱的激发来表征这些中间体在不同温度下的特征，以便分离它们的光谱特征。这些物种将使用同位素标记的底物和培养基进一步研究。将进行一系列综合建模、计算和比较研究，以明确确定新物种的结构。第二组包括两种关键的细菌二铁酶。甲烷单加氧酶是高等生物中细胞色素P450的强大类似物，它能够将甲烷氧化成甲醇。其高度氧化的中间Q含有前所未有的FeIVFeIV核心。核糖核苷酸还原酶在DNA复制中起着关键作用，是哺乳动物相应酶的良好模型。其高度氧化的中间体X负责通过产生蛋白质自由基来激活酶。在过去的二十年里，人们对X和Q的结构进行了广泛的研究，但仍然存在争议。利用我们的新方法，我们试图确定X和Q中的氧配体的数量、类型和质子化状态，这对于解决它们特定的催化机制至关重要。

DESCRIPTION (provided by applicant): This project focuses on the structures of transient oxygen intermediates occurring in the catalytic cycles of iron enzymes. Specific focus is on the resolution of atomic vibrations of oxygenic metal ligands and their protonation states using continuous flow resonance Raman spectroscopy. Using a unique, state of the art experimental setup, designed and implemented by the PI, we will examine isotope-difference Raman spectra of species that are formed for a short time following the start of the reactions. The major advantage of our approach is the ability to carry out reactions in liquid samples at temperatures as low as -70¿C with minuscule dead volume. This allows us to slow down the rates of chemical reactions by orders of magnitude and permits close examination of the early phases of the reaction without the need for prohibitive amounts of biological samples. We will examine two classes of iron enzymes. First, we will study transient intermediates that we recently identified in the mono-nuclear non-heme iron and ?-ketoglutarate dependent dioxygenase, TauD. We will characterize these intermediates at various temperatures using excitations across the UV/visible spectrum in order to isolate their spectral signatures. These species will be further investigated using isotope-labeled substrates and medium. A range of synthetic modeling, computational and comparative studies will be carried out that will allow unambiguous identification of the structures of the new species. The second group includes two key bacterial di-iron enzymes. Methane monooxygenase is a powerful analog of cytochrome P450 in higher organisms, which is capable of oxidizing methane to methanol. Its highly oxidized intermediate Q contains an unprecedented FeIVFeIV core. Ribonucleotide reductase plays a key role in DNA replication and is a good model for the corresponding mammalian enzyme. Its highly oxidized intermediate X is responsible for activation of the enzyme by generating a protein radical. Structures of both X and Q have been studied extensively over the last two decades, but remain controversial. Using our novel approach we seek to identify the number, type, and protonation states of oxygenic ligands in X and Q, which is critical for resolving their specific catalytic mechanisms. PUBLIC HEALTH RELEVANCE: Short-lived highly oxidized oxygen-metal complexes are the catalytic centerpieces of many enzymes exhibiting a diverse range of critical physiological functions. Using a unique experimental setup, which us allows to dramatically slow down chemical reactions, we seek to establish the detailed structures of such intermediates. We will investigate three representative enzymes: ribonucleotide reductase is involved in DNA replication and two bacterial oxygenases provide important models of analogous enzymes in mammals.