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

项目摘要 铁的活性部位在生物体内的氧活化中起着关键作用。这些都存在于单核非血红素铁中 (MNHFe)、双核非血红素铁(BNHFe)和血红素酶，进一步分为不同的亚类 (见下文)。我们开发了一系列光谱方法，使详细研究几何图形成为可能 FeII活性中心及其氧中间体的电子结构，电子的耦合 这些光谱数据的结构计算和实验支持的计算的使用 评估反应坐标。根据我们的最新进展，硫磺中FeIO2·-中间体的形成 氧化/加氧酶(SOX)是由底物硫配位稳定的，而对于Rieske 这种中间体的双加氧酶(RDO)的产生是艰难的，但其亲电攻击是由随后的 里斯克中心的激子质子耦合电子转移(PCET)。外源双加氧酶(EDO) 还利用FeIO2·-反应中间体对配位儿茶酸盐底物进行亲电攻击。我们 现在建议完成EDO的反应坐标，并确定为什么这些酶是选择性的 在外向插入和切割中，具有铁活性部位的吲哚双加氧酶(IDO)是 选择性地进行传统的卵裂。对于RDO和EDO，也可以形成FeIII(Ooh)，但相对 不具反应性，而在许多BNHFe酶中，形成的过氧化中间体在 亲电化学。我们在这里的重点是了解第二个铁在双核中的额外贡献 激活过氧化氢反应性的铁中心。α-酮戊二酸(AKG)和蝶呤依赖的羟基酶 (PDH)，生成FeIV=O中间体，并继续与衬底反应。我们在这里的研究重点是 FeII/O2反应生成这些FeIV=O物种的机理及其如何使选择性 羟基化、脱饱和度和亲电芳香取代(EAS)。BNHFe酶中可溶甲烷 单加氧酶(SMMO)，过氧二铁中间体，继续形成高价Fe2IVO2中间体Q 氢原子从甲烷的强C-H键中抽出(HAA)。我们这里的重点是确定为什么 只有这种双核过氧基继续形成高价中间体，定义了几何和电子结构 Q的结构和对该Fe2IVO2中间体相对于MNHFeIV=O的增强反应活性的理解 中间体。在含和不含卟啉的血红素酶中也形成FeIV=O中间体 氧化，在P450的情况下，也比在MNHFe位置上的FeIV=O物种更活跃。我们在这里的学习 是针对光谱测定的影响，它的氧化和变异的 FeIV=O键上的跨轴配体；到目前为止，所有的主题都是通过计算来解决的。我们的研究 对理解氧的活化和反应的机制有重要的贡献 不同亚类铁位置的坐标和总体上对理解氧在 生物和化学中的超氧、过氧和氧合水平。

Project Summary Iron active sites play key roles in O2 activation in biology. These are present in mononuclear non-heme Fe (MNHFe), binuclear non-heme Fe (BNHFe) and heme enzymes, which further divide into different subclasses (vide infra). We have developed a range of spectroscopic methods that enable the detailed study of the geometric and electronic structures of the FeII active sites and their oxygen intermediates, the coupling of electronic structure calculations to these spectroscopic data and the use of calculations supported by experiment in evaluating reaction coordinates. From our recent progress, formation of an FeIIIO2•- intermediate in the sulfur oxidizing/oxygenating enzymes (SOx) is stabilized by the substrate sulfur coordination, while for the Rieske dioxygenases (RDO) generation of this intermediate is uphill but its electrophilic attack is driven by a subsequent exergonic proton coupled electron transfer (PCET) from the Rieske center. The extradiol dioxygenases (EDO) also utilize an FeIIIO2•- reactive intermediate in electrophilic attack on a coordinated catecholate substrate. We now propose to complete the reaction coordinate for the EDOs and determine why these enzymes are selective in extradiol insertion and cleavage while the intradiol dioxygenases (IDO), which have a ferric active site, are selective for intradiol cleavage. For both the RDOs and EDOs, an FeIII(OOH) can also be formed but is relatively unreactive, while in a number of the BNHFe enzymes, peroxide intermediates are formed that are reactive in electrophilic chemistry. Our focus here is to understand the additional contributions of the second Fe in binuclear ferric centers that activate peroxide reactivity. For the a-ketoglutarate (aKG) and pterin dependent hydroxylases (PDH), an FeIV=O intermediate is formed and proceeds to react with substrate. Our research here focuses on the FeII/O2 reaction mechanisms for the generation of these FeIV=O species and how they enable selective hydroxylation, desaturation and electrophilic aromatic substitution (EAS). In the BNHFe enzyme soluble methane monooxygenase (sMMO), the peroxo biferric intermediate goes on to form a high valent Fe2IVO2 intermediate Q that H-atom abstracts (HAA) from the strong C-H bond of methane. Our focuses here are on determining why only this binuclear peroxo goes on to form a high valent intermediate, defining the geometric and electronic structure of Q and understanding the enhanced reactivity of this Fe2IVO2 intermediate relative to the MNHFeIV=O intermediates. An FeIV=O intermediate is also formed in the heme enzymes both with and without porphyrin oxidation and is also more reactive in the case of P450 than FeIV=O species in MNHFe sites. Our studies here are directed toward spectroscopically determining the effects of the porphyrin, its oxidation and of variation of the trans axial ligand on the FeIV=O bond; all topics thus far mostly addressed through calculations. Our studies should significantly contribute toward understanding the mechanisms of O2 activation and the reaction coordinates for the different subclasses of iron sites and generally toward understanding O2 activation at the superoxo, peroxo and oxo levels in biology and chemistry.