Electron Transfer in Iron and Copper Oxygenases and Oxidases

项目摘要/摘要 加氧酶和氧化酶必须协调四个质子和四个电子的传递 O2，以防止形成有害的、部分还原的活性氧物种。带来的风险 活性中间体是如此之大，以至于好氧生物需要保护氧气的机制- 当初级电子/质子转移机制被破坏时，从失活中利用酶。 从受挫的反应中间体传递强氧化孔的自由基转移途径 与细胞内抗氧化剂反应的蛋白质表面的酶活性部位可以提供这样的 保护。这些自由基转移途径很可能是由色氨酸、酪氨酸、半胱氨酸和 可能是甲硫氨酸残留物。本研究计划将重点放在细胞色素P450(P450)，2-氧代-2-氧代-天冬氨酸 依赖于戊二酸的非血红素铁加氧酶(20G-Fe)和多铜氧化酶(MCOs)。 细胞色素P450是与异种生物有关的血红素加氧酶超家族的成员 代谢和生物合成途径。在哺乳动物中，这些功能包括药物新陈代谢、转化 将亲脂分子转化为更极性的产物，以增强消除、类固醇生物合成和 二十烷类化合物的合成和降解。细胞色素P450也负责66%的酶 致癌物质的激活。阐明P450在存在时避免失活的机制 不同底物的组合有助于确定治疗药物的有效性并降低 药物与药物的不良相互作用。用于癌症治疗和预防的药物被设计成针对 P450通过竞争性抑制和基于机制的不可逆抑制。要了解 P450对这些化合物的生物反应，有必要描述其作用机制。 酶用来保护自己免受降解。 来自20G-Fe超家族的酶使用2-氧基戊二酸作为2电子供体共底物， Fe2+作为辅因子，O2影响有机底物的羟基化。60-70岁的人2OG-Fe 酶表现出广泛的生物学功能，包括胶原生物合成、赖氨酸羟化 RNA剪接蛋白，DNA修复，RNA修饰，染色质调节，表皮生长因子- 如结构域修饰、低氧感应和脂肪酸代谢。酶的周转率也是 在某些情况下伴随着活性氧物种的产生。关于ROS产生的解释 这些途径将提供对这些酶的功能和错误功能的更深层次的了解。 血液和肠道中的铜蓝蛋白和肝磷脂是与铁有关的MCOs。 氧化。氧还原发生在三核铜中心(TNC)，并提供第四电子 通过一个遥远的类型1铜中心。与活动站点相邻的Trp或Trp/Tyr可以瞬时地提供 第四个电子。拟议的研究将阐明TNC近端Trp/Tyr残基的作用。

PROJECT SUMMARY/ABSTRACT Oxygenase and oxidase enzymes must coordinate the delivery of four protons and four electrons to O2 in order to prevent the formation of harmful, partially reduced reactive oxygen species. The risks posed by reactive intermediates are so great that aerobic organisms need mechanisms to protect oxygen- utilizing enzymes from inactivation when primary electron/proton transfer mechanisms are disrupted. Radical transfer pathways that deliver strongly oxidizing holes from frustrated reactive intermediates in enzyme active sites to the protein surface for reaction with intracellular antioxidants can provide such protection. These radical transfer pathways are likely constructed from chains of Trp, Tyr, Cys, and possibly Met residues. This research program will focus on the cytochromes P450 (P450), the 2-oxo- glutarate dependent nonheme iron oxygenases (2OG-Fe), and the multicopper oxidases (MCOs). The cytochromes P450 are members of a superfamily of heme oxygenases involved in xenobiotic metabolic and biosynthetic pathways. In mammals these functions include drug metabolism, conversion of lipophilic molecules to more polar products for enhanced elimination, steroid biosynthesis, and eicosanoid synthesis and degradation. Cytochromes P450 also are responsible for 66% of enzymatic activation of carcinogens. Elucidating the mechanisms by which P450s avoid inactivation in the presence of diverse substrates can contribute to defining therapeutic drug efficacies and mitigating the risks of adverse drug-drug interactions. Drugs for cancer treatment and prevention have been designed to target P450s through competitive inhibition and mechanism based irreversible inhibition. To understand the biological response of P450s to these compounds, it is essential to delineate the mechanisms that the enzymes use to protect themselves against degradation. Enzymes from the 2OG-Fe superfamily use 2-oxoglutarate as a 2-electron donating co-substrate, Fe2+ as a cofactor, and O2 to effect the hydroxylation of organic substrates. The 60-70 human 2OG-Fe enzymes exhibit a wide array of biological functions including collagen biosynthesis, lysyl hydroxylation of RNA splicing proteins, DNA repair, RNA modification, chromatin regulation, epidermal growth factor- like domain modification, hypoxia sensing, and fatty acid metabolism. Enzymatic turnover also is accompanied in some cases by generation of reactive oxygen species. Elucidation of ROS generating pathways will provide deeper insight into the functioning and mis-functioning of these enzymes. The blood and intestinal human enzymes ceruloplasmin and hephaestin are MCOs involved in iron oxidation. Oxygen reduction occurs at a trinuclear copper center (TNC) and a fourth electron is provided by a distant type 1 copper center. A Trp or Trp/Tyr adjacent to the active site may transiently provide the fourth electron. The proposed studies will elucidate the role of the TNC proximal Trp/Tyr residues.