SPECTROSCOPIC STUDIES OF MONONUCLEAR NONHEME IRON ENZYME

单核非血红素铁酶的光谱研究

• 批准号: 6385733
• 负责人: EDWARD I SOLOMON
• 金额: $ 32.26万
• 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/6385733
• 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参与需要O2多种生物学功能的一系列酶中。 其中包括脂氧酶（LOS，脂肪酸氢过氧化），博伐霉素（BLM，DNA裂解），内部和性内部二加氧酶（芳族环的降解），翼龙依赖性羟基酶（氨基酸代谢）和α-酮酸盐（Alpha-ketoglutaratarate） ）依赖性酶（羟基化和环闭合）。 FEIII的底物激活和FEII位点激活的底物激活，并且已经观察到关键的中间体。 关于这些酶相对于血红素系统，知之甚少，因为非血红素铁中心在光谱上较少可访问。 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，以获取对其催化机制的分子水平洞察力，并定义了与O2和底物反应性差异有关的活性位点几何和电子结构的差异。 这些研究还应为阐明非血红素与铁位点的关系以及在非血红素环境中的O2激活的性质做出重大贡献。 研究强调了磁圆二色性，并结合了其他激发态光谱方法，以探测非血红素FEII和FEIII位点的几何和电子结构以及可能的氧中间体的类似物。目前的特定目的是：1）开发研究方法，用于研究高旋转FEIII位点，从而提供D轨道的配体场分裂和关键配体金属键的共价； 2）发展L边缘光谱法作为非血红素铁位相对于血红素部位的差异轨道共价探针； 3）确定配体变异对LO的FEII和FEIII位点的影响及其对反应性的贡献； 4）确定FEII位点在链蛋白依赖性酶中耦合羟基化中的作用； 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位点与非依赖α-KG依赖性的相关FEII酶的相关性如何； 7）确定底物的结合如何激活o2反应性的二加氧酶中的FeII位点； 8）定义二加氧酶内FeIII位点底物激活的性质； 9）确定DNA结合对FEIIBLM的影响，以及其催化胜任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，并评估它们相对于血红素位点的反应性的可能贡献。