SPECTROSCOPIC STUDIES OF NONHEME IRON ENZYMES

单核非血红素铁活性位点存在于广泛的 参与多种重要生物功能的酶 需要分子氧。这些包括脂氧合酶（脂肪酸 氢过氧化）、博来霉素（DNA中涉及抗癌药物 裂解）、二醇内和二醇外双加氧酶（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 状态涉及这类不同酶的催化， 并且观察到底物和氧结合的中间体。多 相对于血红素，人们对这些酶中的活性位点知之甚少 作为非血红素铁中心的系统在光谱上 容易接近这项研究计划的总体目标是 发展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，以获得对催化机制的分子水平洞察 并了解活性部位几何形状的差异， 电子结构与O2和底物的差异有关 反应性这些研究也将有助于 阐明非血红素和血红素的异同 铁网站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 电子结构的非血红素亚铁和铁的网站，并确定 非血红素亚铁的NO络合物的不寻常的电子结构 位置及其与可能的氧中间体的关系。具体 本提案的目标是：l）完成VTVH MCD的开发 作为非血红素亚铁活性位点的强有力探针; 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的活性位点; 3)扩展对脂氧合酶的研究，使其与两种相互冲突的 晶体结构和哺乳动物的酶与系统 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） 继续对博来霉素进行研究，以了解博来霉素的每个步骤。 催化机制，确定铁网站的影响，由于 与DNA的相互作用，并确定博来霉素与血红素的关系， 其他非血红素铁系统; 5）extradiol的相关结果 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， 与激活的差异有关，并可能影响 环裂解; 6）定义铁位点的相互作用， 苯丙氨酸羟化酶与蝶呤辅因子，探索关键步骤 的催化机制，并确定如何突变，影响 酶的反应性改变了活性位点及其与 辅因子和底物; 7）理解亚铁离子的相互作用 具有α-酮戊二酸辅因子的棒胺酸合酶的位点， 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， 导致羟基化相对于闭环化学。

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.