Transient catalytic oxygen species in iron enzymes

描述(申请人提供)：本项目重点研究铁酶催化循环中的瞬时氧中间体的结构。重点是利用连续流动共振拉曼光谱对含氧金属配体的原子振动及其质子化状态的分辨。使用由PI设计和实施的独特的、最先进的实验装置，我们将检查在反应开始后的短时间内形成的物种的同位素差拉曼光谱。我们方法的主要优势是能够在温度低至-70℃的液体样品中进行反应，死体积极小。这使我们能够将化学反应的速度减慢数量级，并允许密切检查反应的早期阶段，而不需要令人望而却步的生物样本。我们将检测两类铁酶。首先，我们将研究我们最近在单核非血红素铁和依赖于酮戊二酸的双加氧酶TauD中发现的瞬时中间产物。我们将利用紫外可见光谱中的激发来表征这些中间体在不同温度下的特性，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介质进行进一步研究。将进行一系列综合建模、计算和比较研究，以便明确识别新物种的结构。第二类包括两种关键的细菌双铁酶。甲烷单加氧酶是高等生物中一种功能强大的细胞色素P450类似物，能够将甲烷氧化为甲醇。其高度氧化的中间体Q包含一个前所未有的FeIVFeIV核心。核糖核苷酸还原酶在DNA复制中起着关键作用，是研究相应哺乳动物酶的良好模型。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，X和Q的结构都得到了广泛的研究，但仍然存在争议。使用我们的新方法，我们试图确定X和Q中含氧配体的数量、类型和质子化状态，这对于解决其特定的催化机理至关重要。 与公共健康相关：短命的高度氧化的氧-金属络合物是许多酶的催化中心，表现出一系列关键的生理功能。使用一种独特的实验装置，我们可以极大地减缓化学反应，我们寻求建立这种中间体的详细结构。我们将研究三种具有代表性的酶：参与DNA复制的核苷酸还原酶和两种细菌加氧酶，它们为哺乳动物提供了重要的类似酶模型。

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.