SPECTROSCOPIC STUDIES OF NON HEME IRON ENZYMES

非血红素铁酶的光谱研究

• 批准号: 2180304
• 负责人: EDWARD I SOLOMON
• 金额: $ 19.74万
• 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/2180304
• 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存在于广泛的范围 参与各种重要生物学功能的酶需要 二恶英。 这些包括脂氧酶（脂肪酸代谢和 前列腺素合成的调节），额外的和二醇内二氧酶的调节 （酪氨酸和色氨酸代谢和芳香环的降解）， 博来霉素（参与DNA裂解的抗癌药），四氢蛋白蛋白 依赖性羟化酶（苯丙氨酸代谢与心理有关 疾病），α-酮戊二酸二加氧酶（胶原蛋白 合成），单加氧酶（烃和脂肪酸羟基化）， 亚匹单抗N合酶和超氧化物歧化酶。 少得多 关于这些酶相对于血红素系统中的活性位点， 在光谱上访问少得多，尤其是高旋转 EPR通常无法检测到的亚特林地点 可见光谱中的强电荷转移转变。 我们的研究旨在开发激发态光谱 研究非血红素铁酶的方法。 吸收，CD，低 温度磁圆二色性（MCD）和共振拉曼 光谱学用于研究激发态和可变状态 激发态的温度变量场MCD用于探测 即使在高自旋的亚铁部位，地基状态分裂也 不显示EPR信号。 数据通过配体领域进行分析 将D轨道分裂为探针的理论 铁中心的电子和几何结构，以及Xalpha散落的 配体对金属电荷转移的波分子轨道计算 探测与底物相关的电子结构的过渡 小分子与铁中心结合。 光谱研究是 针对非血红素亚铁和铁位，催化 中间体，没有稳定的亚铁部位的复合物 反应性小分子结合的类似物。 最初的研究现在已经 证明这种激发的状态光谱方法可以提供 有关每种形式的活动地点的重要信息 酶。 这些研究的目标是定义几何和 铁中心的电子结构，与小的相互作用 分子和底物，以及底物对小分子的影响 在许多非血红素铁酶底物结合中结合起来增强 二氧化反应性。 这些关于非血红素铁酶的光谱研究 应该提供分子水平的洞察力，以了解其催化机制 定义与几何和电子结构差异有关 该类别的不同酶的反应性差异，以及 允许血红素有相关性，在某些情况下似乎具有平行 反应性。