SPECTROSCOPIC STUDIES OF NON-HEME IRON ENZYMES

非血红素铁酶的光谱研究

• 批准号: 3297866
• 负责人: EDWARD I SOLOMON
• 金额: $ 17.08万
• 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 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3297866
• 关键词: Raman spectrometry; active sites; analog; bleomycin; catalyst; circular dichroism; circular magnetic dichroism; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate complex; ethylenediaminetetraacetate; intermolecular interaction; ligands; metalloenzyme; nitric oxide; oxidoreductase; oxygenases; phenylalanine 4 monooxygenase; protein structure function; superoxide dismutase

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 metabolism and regulation of prostaglandin synthesis), extra and intradiol dioxygenases (tyrosine and tryptophan metabolism and degradation of aromatic rings), bleomycin (anticancer drug involved in DNA cleavage), tetrahydroprotein dependent hydroxylases (phenylalanine metabolism related to mental disorders), alpha-ketoglutarate dependent dioxygenases (collagen synthesis), monooxygenases (hydrocarbon and fatty acid hydroxylation), isopenicillin N synthase and superoxide dismutase. Much less is known about the active sites in these enzymes relative to heme systems as these are far less spectroscopically accessible, particularly the high spin ferrous sites which are often not detectable by EPR and do not exhibit intense charge transfer transitions in the visible spectrum. Our research is directed toward developing an excited state spectroscopic approach for the study of non-heme iron enzymes. Absorption, CD, low temperature Magnetic Circular Dichroism (MCD) and resonance Raman spectroscopies are used to study the excited states and variable temperature variable field MCD of an excited state is used to probe the ground state sublevel splittings even in high spin ferrous sites which do not exhibit EPR signals. The data is analyzed in terms of ligand field theory which defines the splitting of the d orbitals as a probe of the electronic and geometric structure of the iron center, and Xalpha-scattered wave molecular orbital calculations of the ligand-to-metal charge transfer transitions which probe the electronic structure associated with substrate and small molecule binding to the iron center. Spectral studies are directed toward the non-heme ferrous and ferric sites, catalytic intermediates and NO complexes of the ferrous sites which serve as stable analogues of reactive small molecule binding. Initial studies have now demonstrated that this excited state spectral approach can provide important information on the active sites in each of these forms of the enzymes. The goals of these studies are to define the geometric and electronic structure of the iron center, its interaction with small molecules and substrate, and the effects of substrate on small molecule binding in that for many non-heme iron enzymes substrate binding enhances dioxygen reactivity. These spectral studies on non-heme iron enzymes should provide molecular level insight into their catalytic mechanisms, define geometric and electronic structure differences which relate to differences in reactivity over the different enzymes in this class, and allow a correlation to heme which in some cases appear to have parallel reactivity.

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（脂肪酸代谢和 前列腺素合成的调节）、额外和内二醇双加氧酶 （酪氨酸和色氨酸的代谢和芳香环的降解）， 博莱霉素（参与 DNA 切割的抗癌药物）、四氢蛋白 依赖性羟化酶（与精神相关的苯丙氨酸代谢） 疾病），α-酮戊二酸依赖性双加氧酶（胶原蛋白 合成），单加氧酶（烃和脂肪酸羟基化）， 异青霉素 N 合酶和超氧化物歧化酶。 知之甚少 关于这些酶中相对于血红素系统的活性位点，因为这些 光谱上的可观测性要差得多，尤其是高自旋 含铁位点通常无法通过 EPR 检测到，并且不会表现出 可见光谱中强烈的电荷转移跃迁。 我们的研究旨在开发激发态光谱 非血红素铁酶的研究方法。 吸收，CD，低 温度 磁圆二色性 (MCD) 和共振拉曼 光谱用于研究激发态和变量 激发态的温度可变场 MCD 用于探测 即使在高自旋含铁位点也会发生基态亚能级分裂 不显示 EPR 信号。 数据根据配体场进行分析 该理论将 d 轨道的分裂定义为 铁中心的电子和几何结构以及 Xalpha 散射 配体到金属电荷转移的波分子轨道计算 探测与基底相关的电子结构的跃迁 和与铁中心结合的小分子。 光谱研究是 针对非血红素亚铁和三价铁位点，催化 亚铁位点的中间体和 NO 络合物作为稳定 反应性小分子结合的类似物。 初步研究现已 证明这种激发态光谱方法可以提供 有关每种形式的活性位点的重要信息 酶。 这些研究的目标是定义几何和 铁中心的电子结构及其与小分子的相互作用 分子和底物，以及底物对小分子的影响 对于许多非血红素铁酶来说，底物结合增强 双氧反应性。 这些关于非血红素铁酶的光谱研究 应提供对其催化机制的分子水平洞察， 定义与以下相关的几何和电子结构差异 此类中不同酶的反应性差异，以及 允许与血红素相关，在某些情况下似乎具有平行关系 反应性。