Spectroscopic Characterization of Oxygen Intermediates in Non-heme and Heme Iron Enzymes

项目摘要 铁酶在生物学中的O2活化中起着重要作用。这些分为四类，基于 它们的活性位点结构反映了它们的O2活化模式：非辅因子依赖性 单核非血红素铁（MNHFe）酶、辅因子（α-酮戊二酸（α-KG）和蝶呤） 依赖性MNHFe酶、双核NHFe酶和O2/H2 O2活化血红素 内切酶晶体结构和氧反应中间体存在的金属酶在所有 四个班。多年来，我们开发了新的光谱方法， 详细研究了NHFeII活性中心及其O2的几何和电子结构 中间体，现在已经开发出一种方法来定量研究铁中心， 高度共价和发色血红素环境。在我们过去5年的成就中， 年是：1）确定FeIII-O2-物种是所有子类中的反应中间体 非辅因子依赖性MNHFe酶; 2）定义O2反应坐标以生成 α-KG和蝶呤依赖酶中的FeIV=O中间体; 3）对于α-KG 从属亚类，定义了它们的FeIV=O酶的几何和电子结构 中间体以及这些中间体如何直接卤化， 卤化酶中的羟基化; 4）表明与MNHFe酶相反， 氢过氧化物中间体在双核NHFe酶中具有活性，可直接与 底物; 5）对于甲烷单加氧酶，其中过氧-二铁中间体迅速 转化为高价的2FeIV-氧代中间体Q，我们确定了Q（a）的结构 当前辩论的主题），并提供了其与甲烷的高反应性的见解; 6）使用 光谱方法，我们现在已经开发了血红素环境中的铁，以确定 实验上计算有争议的氧合血红蛋白的电子结构; 7）和 将该方法扩展到分析具有强Fe-氧代键的活性中心。我们的研究现在 旨在完成四个类别及其子类的反应坐标， 理解在superoxo，peroxo和FeIV-oxo水平上的O2活化，确定 第二个铁在双核NHFe酶的增强反应性，并了解 高价铁-氧代中间体的活化和选择性的差异 单核NH、双核NH和血红素酶活性位点。

Project summary Iron enzymes play major roles in O2 activation in biology. These divide into four classes based on their active site structures that reflect their mode of O2 activation: the non-cofactor dependent mononuclear non-heme iron (MNHFe) enzymes, the cofactor (α-ketoglutarate (α-KG) and pterin) dependent MNHFe enzymes, the binuclear NHFe enzymes and the O2/H2O2 activating heme enzymes. Crystal structures and oxygen reaction intermediates exist for metalloenzymes in all four classes. Over the years, we have developed new spectroscopic methods enabling the detailed study of the NHFeII active sites, and the geometric and electronic structures of their O2 intermediates and have now developed a method to quantitatively study the iron center in the highly covalent and chromophoric heme environment. Among our accomplishments in the past 5 years are: 1) determined that FeIII-O2- species are the reactive intermediates in all the subclasses of non-cofactor dependent MNHFe enzymes; 2) defined the O2 reaction coordinates to generate the FeIV=O intermediates in both the α-KG and pterin dependent enzymes; 3) for the α-KG dependent subclass, defined the geometric and electronic structures of their FeIV=O enzyme intermediates and how these direct halogenation over the thermodynamically favored hydroxylation in the halogenases; 4) showed that in contrast to the MNHFe enzymes, hydroperoxide intermediates are active in the binuclear NHFe enzymes for direct reaction with substrates; 5) for methane monooxygenase, where the peroxo-biferric intermediate rapidly converts to a high-valent 2FeIV-oxo intermediate Q, we have determined the structure of Q (a topic of current debate) and provided insight into its high reactivity with methane; 6) used the spectroscopic method we have now developed for iron in heme environments to determine experimentally the computationally controversial electronic structure of oxyhemoglobin; 7) and extended this method to analyze active sites with strong Fe-oxo bonds. Our studies are now directed toward completing the reaction coordinates of the four classes and their subclasses, understanding O2 activation at the superoxo, peroxo and FeIV-oxo levels, determining the role of the second iron in the enhanced reactivity of the binuclear NHFe enzymes, and understanding the differences in the activation and selectivity of high-valent iron-oxo intermediates in mononuclear NH, binuclear NH and heme enzyme active sites.