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Isolation and characterization of reactive heme-oxygen intermediates in cytochrome P450 catalysis.

机译：细胞色素P450催化中活性血红素氧中间体的分离和表征。

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摘要

The cytochrome P450 enzymes constitute a ubiquitous superfamily of heme monooxygenases responsible for an extensive range of chemical reactivities in host organisms. The potent, and almost indiscriminant, oxidative prowess of these enzymes is attributable to an active site which can accommodate a wide variety of chemical moieties, and the production of a series of highly reactive heme-oxygen adducts. These active intermediates are not exclusive to the P450 catalytic scheme, and thus divulging the intrinsic formation and reactivity of dioxygen derived intermediates in the P450 cytochromes provides important mechanistic details for a plethora of heme and non-heme containing metalloenzymes.; The following work describes the application of cryogenic irradiation as an essential tool in the formation, isolation, and characterization of intermediates in the P450 catalytic cycle. Furthermore, the use of active site mutants, in which the reactivity of these intermediates is dramatically altered, has enabled the deconvolution of their participation in the proton and electron assisted pathway of P450 catalyzed dioxygen cleavage. The result is the first rigorous spectroscopic assignment of the ferric-peroxoanion and ferric-hydroperoxo species in P450 monoxygenation, and the direct assessment of the role of the distal pocket in their formation and resultant reactivity.; The P450 monooxygenases, and specifically the intermediate species formed within the course of their reaction cycle, are formidable oxidants, capable of a diverse range of chemical reactions. As the cryoradiolytic methodologies prove a convenient tool for the isolation and stabilization of heme-oxygen intermediates, a range of substrate probes has been coupled to these studies in order to assess their role in known P450 reactions. This provides a template not only for the assorted reactivity of the P450 superfamily, but the utilization of related intermediates in other metalloenzyme systems. In order to assess the commonality of these intermediates, the ferric-peroxo species have also been generated in a number of other heme enzyme systems in order to assess the role of proximal ligand and distal pocket in the proton mediated functionalization of these reactive intermediates, and thus, to compare these to the reaction mechanism associated with the P450 monoxygenases.
机译：细胞色素P450酶构成了血红素单加氧酶的一个普遍存在的超家族，负责在宿主生物中进行广泛的化学反应。这些酶的强力和几乎不加区别的氧化能力归因于可容纳多种化学部分的活性位点，以及一系列高反应性血红素-氧加合物的产生。这些活性中间体不是P450催化方案所独有的，因此，揭示P450细胞色素中双氧衍生中间体的内在形成和反应性为大量血红素和不含血红素的金属酶提供了重要的机理细节。以下工作描述了低温辐射作为P450催化循环中中间体的形成，分离和表征的基本工具的应用。此外，使用其中这些中间体的反应性显着改变的活性位点突变体，已使它们参与P450催化的双氧裂解的质子和电子辅助途径的解卷积成为可能。结果是在P450单加氧作用中首次对铁-过氧阴离子和铁-氢过氧物种进行了严格的光谱指配，并直接评估了远端囊袋在其形成和所得反应性中的作用。 P450单加氧酶，特别是在其反应循环过程中形成的中间物种，是强大的氧化剂，能够进行多种化学反应。由于超低温分解方法学被证明是分离和稳定血红素氧中间体的便捷工具，因此一系列底物探针已与这些研究相结合，以评估其在已知P450反应中的作用。这不仅为P450超家族的各种反应性提供了模板，而且为其他金属酶体系中相关中间体的利用提供了模板。为了评估这些中间体的通用性，还已经在许多其他血红素酶系统中生成了铁-过氧物种，以评估近端配体和远端口袋在质子介导的这些反应性中间体的功能化中的作用，并且因此，将其与与P450单加氧酶相关的反应机理进行比较。

著录项

  • 作者

    Makris, Thomas Michael.;

  • 作者单位

    University of Illinois at Urbana-Champaign.;

  • 授予单位 University of Illinois at Urbana-Champaign.;
  • 学科 Biophysics General.; Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2004
  • 页码 167 p.
  • 总页数 167
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物物理学;生物化学;
  • 关键词

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