SPECTROSCOPIC STUDIES OF MONONUCLEAR NONHEME IRON ENZYME

单核非血红素铁酶的光谱研究

• 批准号: 6519304
• 负责人: EDWARD I SOLOMON
• 金额: $ 33.02万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6519304
• 关键词: X ray spectrometry; active sites; alpha ketoglutarate; atomic absorption spectrometry; biomagnetism measurement; bleomycin; circular dichroism; enzyme substrate complex; intermolecular interaction; iron compounds; ligands; lipoxygenase; method development; oxygenases; protein structure function; pteridines

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.

单核非血红素铁活性位点存在于涉及多种需要 O2 的生物功能的一系列酶中。 这些包括脂氧合酶（LO，脂肪酸氢过氧化）、博莱霉素（BLM，DNA 裂解）、二醇内和二醇双加氧酶（芳香环降解）、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）。 对于不同的酶，发现了 FeIII 位点的底物激活和 FeII 位点的 O2 激活，并且观察到了关键中间体。 由于非血红素铁中心的光谱不易接近，因此对这些酶相对于血红素系统的了解要少得多。 研究目标是开发新的光谱方法来研究非血红素铁活性位点，以获得对其催化机制的分子水平洞察，并定义与 O2 和底物反应性差异相关的活性位点几何和电子结构的差异。 这些研究还应有助于阐明非血红素与血红素铁位点的关系以及非血红素环境中 O2 活化的性质。 研究强调磁圆二色性与其他激发态光谱方法相结合，以探测非血红素 FeII 和 FeIII 位点以及可能的氧中间体类似物的几何和电子结构。目前的具体目标是： 1) 开发研究高自旋和低自旋 FeIII 位点的方法，提供 d 轨道的配体场分裂和关键配体-金属键的共价键； 2）开发L边光谱作为非血红素铁位点相对于血红素位点的差分轨道共价性的探针； 3)确定配体变化对LO的FeII和FeIII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的偶联羟基化中的作用； 5) 定义α-KG-FeII键的性质以及该位点如何受到经历羟基化、氧化闭环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酶相关联； 7) 确定底物结合如何激活 Extradiol 双加氧酶中的 FeII 位点以实现 O2 反应性； 8) 定义内二醇双加氧酶中 FeIII 位点激活底物的性质； 9)确定DNA结合对FeIIBLM的影响，及其具有催化活性的过氧化物中间体的电子结构； 10) 确定一系列过氧化物-FeIII复合物的电子结构，并评估它们对血红素位点相关反应性的可能贡献。