SPECTROSCOPIC STUDIES OF NONHEME IRON ENZYMES

单核非血红素铁的活性部位广泛存在于 参与多种重要生物功能的酶 需要氧气。其中包括脂肪氧合酶(脂肪酸 氢过氧化)、博莱霉素(DNA参与的抗癌药物 裂解)、醇内和醇外双加氧酶(芳香族的降解 环)，四氢蝶呤依赖羟基酶(苯丙氨酸 代谢)，以及依赖于α-酮戊二酸的酶(底物 羟基化和环关闭)。亚铁和铁氧化 在这一类中，状态参与了不同酶的催化作用， 观察到了底物和氧结合中间体。大有可为 关于这些酶中相对于亚铁血红素的活性部位，我们知之甚少。 作为非血红素铁中心的系统在光谱上较少 无障碍。这项研究计划的总体目标是 开发研究非血红素铁的新光谱方法 并将这些方法应用于上述研究 酶可在分子水平上深入了解催化机理 并了解活动站点的几何形状和 与氧气和衬底的差异有关的电子结构 反应性。这些研究还应对以下方面做出重大贡献 浅谈非血红素与血红素的异同 铁质遗址。到目前为止，研究都强调了可变温度 变场磁圆二向色性(VTVH MCD)与 其他激发态光谱方法探测几何和 非血红素铁和铁中心的电子结构和定义 非亚铁血红素NO络合物的反常电子结构 位置及其与可能的氧中间体的关系。具体的 这项建议的目的是：L)完成VTVH MCD的开发 作为非血红素铁活性中心的有力探索者；2)开发铁L- 边缘X射线吸收光谱作为非血红素铁的新探针 活性中心，特别是铁中心和氧中间体； 3)扩大对脂氧合酶的研究，使其与这两个相互冲突的因素相关联 晶体结构和对哺乳动物酶的系统研究 影响反应性、定义活性中心中间体的突变， 并探讨活性部位与抑制剂的相互作用：4) 继续对博莱霉素的研究，以了解 催化机理，确定对铁中心的影响 与DNA的相互作用，并确定博莱霉素与血红素和 其他非血红素铁系统；5)外部醇相关结果 双加氧酶与传统双加氧酶的平行数据 确定底物-铁活性部位相互作用的差异 与激活的差异有关，并可能影响 环解理；6)定义亚铁位置的相互作用 苯丙氨酸羟基酶与蝶呤辅酶，探索关键步骤 催化机制，并确定突变是如何影响 酶活性改变活性部位及其与酶的相互作用 辅因子和底物；7)了解黑素的相互作用 克拉维酸合成酶与α-酮戊二酸辅因子的结合部位， 定义该二元络合物与O2和类似物的相互作用，以及 确定该位置与不同底物的相互作用 导致羟基化和环合化学。

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.