SPECTROSCOPIC STUDIES OF MONONUCLEAR NONHEME IRON ENZYME

单核细胞非血红素铁活性位点存在于一系列参与各种需要O2的生物功能的酶中。 这些酶包括脂氧合酶（LO，脂肪酸氢过氧化）、博来霉素（BLM，DNA裂解）、二醇内和二醇外双加氧酶（芳环降解）、蝶呤依赖性羟化酶（氨基酸代谢）和α-酮戊二酸（α-KG）依赖性酶（羟基化和闭环）。 底物激活Fe III和O2激活Fe II网站被发现为不同的酶，和关键的中间体已被观察到。 相对于血红素系统，对这些酶的了解要少得多，因为非血红素铁中心在光谱上不太容易接近。 研究目标是开发新的光谱方法，用于调查非血红素铁活性位点，以获得分子水平的洞察其催化机制，并定义活性位点的几何和电子结构的差异，这涉及到O2和底物反应性的差异。 这些研究也应该有助于阐明显着的非血红素血红素铁网站和O2激活的性质在非血红素环境中的关系。 研究强调磁性圆二色性结合其他激发态光谱方法来探测非血红素FeII和FeIII位点和可能的氧中间体的类似物的几何和电子结构。目前的具体目标是：1）开发用于研究高自旋和低自旋Fe III位点的方法，该方法提供d轨道的配位场分裂和关键配体-金属键的共价性; 2）开发L边缘光谱作为非血红素铁位点相对于血红素位点的差示轨道共价性的探针; 3）确定配体变化对LO的Fe II和Fe III位点的影响及其对反应性的贡献; 4）确定FeII位点在蝶呤依赖性酶中偶联羟基化中的作用; 5）确定α-KG-FeII键的性质以及该位点如何受到经历羟基化、氧化环闭合或去饱和的不同底物的影响; 6）确定α-KG依赖性酶中的FeII位点如何与非α-KG依赖性的相关FeII酶相关; 7）确定底物结合如何激活外二醇双加氧酶中的FeII位点以进行O2反应性; 8）确定内二醇双加氧酶中FeIII位点激活底物的性质; 9）确定DNA结合对FeIIBLM的影响，以及其催化活性过氧化物中间体的电子结构; 10）确定一系列过氧化物-FeIII络合物的电子结构，并评估它们对相对于血红素位点的反应性的可能贡献。

Mononuclear non-heme iron active sites are present in a range of enzymes involved in a variety of biological functions requiring O2. These include the lipoxygenases (LOs, fatty acid hydroperoxidation), bleomycin (BLM, DNA cleavage), intra- and extradiol dioxygenases (degradation of aromatic rings), pterin-dependent hydroxylases (amino acid metabolism), and alpha- ketoglutarate (alpha-KG)-dependent enzymes (hydroxylation and ring closure). Substrate activation by FeIII and O2 activation by FeII sites are found for different enzymes, and key intermediates have been observed. Much less is known about these enzymes relative to heme systems as the non-heme iron centers are less spectroscopically accessible. Research goals have been to develop new spectroscopic methods for the investigation of non-heme iron active sites to obtain molecular level insight into their catalytic mechanisms and define differences in active site geometric and electronic structure which relate to differences in O2 and substrate reactivity. These studies should also contribute significantly toward elucidating the relation of non-heme to heme iron sites and the nature of O2 activation in a non-heme environment. Studies have emphasized magnetic circular dichroism combined with other excited state spectroscopic methods to probe the geometric and electronic structure of non-heme FeII and FeIII sites and analogs of possible oxygen intermediates. Present specific aims are to: 1) develop methodology for the study of high-and low-spin FeIII sites which provides the ligand field splitting of the d-orbitals and the covalencies of key ligand-metal bonds; 2) develop L-edge spectroscopy as a probe of differential orbital covalency of non-heme iron sites in relation to heme sites; 3) determine the effects of ligand variation on the FeII and FeIII sites of LO and their contribution to reactivity; 4) determine the role of the FeII site in coupled hydroxylation in the pterin-dependent enzymes; 5) define the nature of the alpha-KG-FeII bond and how this site is affected by different substrates which undergo hydroxylation, oxidative ring closure, or desaturation; 6) determine how the FeII site in the alpha-KG-dependent enzymes correlates to related FeII enzymes which are not alpha-KG-dependent; 7) determine how substrate binding activates the FeII site in the extradiol dioxygenases for O2 reactivity; 8) define the nature of substrate activation by FeIII sites in the intradiol dioxygenases; 9) determine the effects of DNA binding on FeIIBLM, and the electronic structure of its catalytically competent peroxide intermediate; 10) determine the electronic structures of a series of peroxide-FeIII complexes and evaluate their possible contributions to reactivity relative to heme sites.