SPECTROSCOPIC STUDIES OF NONHEME IRON ENZYMES

非血红素铁酶的光谱研究

• 批准号: 2734602
• 负责人: EDWARD I SOLOMON
• 金额: $ 26.29万
• 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 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2734602
• 关键词: active sites; alpha ketoglutarate; atomic absorption spectrometry; biomagnetism measurement; bleomycin; circular dichroism; electron spin resonance spectroscopy; enzyme substrate complex; gene mutation; hydroxylation; intermolecular interaction; lipoxygenase; oxidoreductase inhibitor; oxygenases; phenylalanine; protein structure function; pteridines

Mononuclear non-heme iron active sites are present in a wide range of enzymes involved in a variety of important biological functions requiring dioxygen. These include the lipoxygenases (fatty acid hydroperoxidation), bleomycin (anticancer drug involved in DNA cleavage), intra- and extradiol dioxygenases (degradation of aromatic rings), tetrahydropterin dependent hydroxylases (phenylalanine metabolism), and the alpha-ketoglutarate-dependent enzymes (substrate hydroxylation and ring closure). Both the ferrous and ferric oxidation states are involved in catalysis for different enzymes in this class, and substrate and oxygen bound intermediates have been observed. Much less is known about the active sites in these enzymes relative to heme systems as the non-heme iron centers are less spectroscopically accessible. The general goals of this research program have been to develop new spectroscopic methods for the investigation of non-heme iron active sites and to apply these methods to the study of the above enzymes to obtain molecular level insight into the catalytic mechanisms and to understand the differences in the active site geometric and electronic structure which relate to differences in O2 and substrate reactivity. These studies should also contribute significantly toward elucidating the similarities and differences between non-heme and heme iron sites. Studies thus far have emphasized variable-temperature variable-field magnetic circular dichroism (VTVH MCD) combined with other excited state spectroscopic methods to probe the geometric and electronic structure of non-heme ferrous and ferric sites and to define the unusual electronic structure of the NO complex of non-heme ferrous sites and its relation to possible oxygen intermediates. The specific aims of this proposal are to: l) Complete the development of VTVH MCD as a powerful probe of non-heme ferrous active sites; 2) Develop Fe L- edge X-ray absorption spectroscopy as a new probe of non-heme iron active sites, particularly for ferric centers and oxygen intermediates; 3) Extend studies on lipoxygenases to correlate to the two conflicting crystal structures and to the mammalian enzymes with systematic mutations which influence reactivity, define active site intermediates, and probe the interaction of the active site with inhibitors: 4) Continue studies on bleomycin to understand each of the steps of the catalytic mechanism, determine the effect on the iron site due to the interaction with DNA, and define the relation of bleomycin to heme and other non-heme iron systems; 5) Correlate results on the extradiol dioxygenases with parallel data on the intradiol dioxygenases to determine differences in substrate-iron active site interactions which relate to differences in activation and could influence the position of ring cleavage; 6) Define the interaction of the ferrous site of phenylalanine hydroxylase with the pterin cofactor, probe the key steps of the catalytic mechanism, and determine how mutations which affect the enzyme's reactivity change the active site and its interactions with cofactor and substrate; 7) Understand the interaction of the ferrous site of clavaminate synthase with the alpha-ketoglutarate cofactor, define the interaction of this binary complex with O2 and analogs, and determine the interactions of this site with different substrates which lead to hydroxylation versus ring closure chemistry.

单核非血红素铁活性位点广泛存在于 酶参与多种重要的生物功能 需要双氧。这些包括脂氧合酶（脂肪酸 氢过氧化）、博莱霉素（参与 DNA 的抗癌药物 裂解），二醇内和二醇双加氧酶（芳香族化合物的降解） 环），四氢蝶呤依赖性羟化酶（苯丙氨酸 代谢）和α-酮戊二酸依赖性酶（底物 羟基化和闭环）。亚铁和三价铁的氧化 状态参与此类不同酶的催化， 并观察到底物和氧结合中间体。很多 关于这些酶相对于血红素的活性位点知之甚少 系统，因为非血红素铁中心的光谱较少 可以访问。该研究计划的总体目标是 开发新的光谱方法来研究非血红素铁 活性位点并将这些方法应用于上述研究 酶以获得分子水平的催化机制洞察 并了解活性位点几何和 与 O2 和底物差异有关的电子结构 反应性。这些研究也应该对以下方面做出重大贡献： 阐明非血红素和血红素之间的异同 铁矿点。迄今为止的研究都强调变温 变场磁圆二色性 (VTVH MCD) 结合 其他激发态光谱方法来探测几何和 非血红素亚铁和三价铁位点的电子结构并定义 非血红素亚铁NO复合物的不寻常电子结构 位点及其与可能的氧中间体的关系。具体的 该提案的目标是： l) 完成 VTVH MCD 的开发 作为非血红素亚铁活性位点的强大探针； 2) 开发Fe L- 边缘X射线吸收光谱作为非血红素铁的新探针 活性位点，特别是铁中心和氧中间体； 3) 扩展对脂氧合酶的研究，将这两种相互冲突的酶联系起来 晶体结构和哺乳动物酶的系统研究 影响反应性的突变，定义活性位点中间体， 并探测活性位点与抑制剂的相互作用：4) 继续对博莱霉素进行研究，了解其每个步骤 催化机制，确定由于 与 DNA 的相互作用，并定义博莱霉素与血红素和 其他非血红素铁系统； 5) 关联额外二醇的结果 双加氧酶与内二醇双加氧酶的平行数据 确定底物-铁活性位点相互作用的差异 与激活差异有关，并可能影响 环裂； 6) 定义亚铁位点的相互作用 苯丙氨酸羟化酶与蝶呤辅因子，探究关键步骤 催化机制，并确定突变如何影响 酶的反应性改变活性位点及其与 辅因子和底物； 7）了解铁的相互作用 具有α-酮戊二酸辅因子的棒棒糖合酶位点， 定义该二元复合物与 O2 和类似物的相互作用，以及 确定该位点与不同底物的相互作用 导致羟基化与闭环化学反应。