Intermediates in O2 Activation by Oxygenases at Non-heme Iron Centers

 描述（由申请人提供）：我们建议确定一组多样且通用的非血红素含Fe单加氧酶和双加氧酶的分子和调节机制，作为研究生物双氧活化机制的手段。一个主要的重点将是对反应周期中间体的检测和表征。在自然界中使用的最重要的O2活化策略在我们选择的酶组中有代表性，允许在广泛的战线上处理这个问题。含铁酶对O2的激活受到高度调节，因为活性氧（ROS）的逃逸会导致遗传物质和其他生物分子的损伤，导致细菌细胞死亡和人类疾病。因此，我们的研究将描绘的稳定和不稳定的力量在O2活化过程中的活性中间体。我们研究的单核含铁双加氧酶都使用芳香族底物，并代表了天然和人造芳香族化合物生物降解的主要手段。这提供了抵御这些化合物的毒性和致癌作用的第一道防线，因此这些酶对人类健康有另一个重大影响。提出用于研究的双加氧酶包括：含Fe（II）的二醇外双加氧酶、含Fe（III）的二醇内双加氧酶和氧化还原循环Rieske双加氧酶。正在研究的双核铁簇含单加氧酶是可溶性甲烷单加氧酶（MMO）和氯霉素，CmlA和CmlI的生物合成途径的两种酶。MMO是将强效温室气体甲烷释放到大气中的主要障碍。CmlA和CmlI在我们最有效的抗生素和化疗药物的天然产物生物合成途径中具有许多同源物，但仍然在机制上未被表征。我们已经发展了各种机制的假说，通过这些机制，我们研究的每种类型的加氧酶中的活性位点铁被用来促进催化。现在，我们将利用我们已经完善的方法来测试它们，以便在它们发生时逐步观察它们的反应。过去的研究已经导致每个酶类的单周转系统的发展。将通过协调使用停流瞬态动力学、快速混合冷冻淬灭（DIM）、定点诱变和使用慢速底物类似物来检测和捕获中间体。捕获的中间体将通过一系列光谱进行表征，包括光学，EPR，rRaman，NRVS，EXAFS，NIR CD，VTVH MCD，穆斯堡尔和新的时间分辨rRaman技术（TR3）。此外，将使用新的晶体技术，其中反应通过在酶晶体中进行而减慢（并且通常在中间体处停止）。晶体中捕获的中间体的3D结构将在晶体学上解决，并通过使用新开发的时间分辨技术，采用直线加速器相干光源。在正在进行的研究中，我们已经检测到并捕获了几个广泛假设的，但以前未观察到的，在每个酶类的中间体。这项工作将产生有关生物系统中的加氧酶，氧和金属化学的基本信息。出现的基本概念将是有用的，在这些领域的机制，类似的酶在哺乳动物和方法，以阻止生产有害的扩散活性氧在人类。

 DESCRIPTION (provided by applicant): We propose to determine the molecular and regulatory mechanisms of a varied and versatile set of non-heme Fe- containing mono- and dioxygenases as a means to study the mechanisms of biological dioxygen activation. A major focus will be on the detection and characterization of reaction cycle intermediates. The most important O2 activation strategies used in nature are represented within the group of enzymes we have chosen, allowing the problem to be approached on a broad front. The activation of O2 by Fe-containing enzymes is highly regulated because escape of reactive oxygen species (ROS) causes damage to genetic material and other biomolecules, leading to cell death in bacteria and diseases in humans. Consequently, our study will delineate the stabilizing and destabilizing forces for the reactive intermediates in the O2 activation process. The mononuclear Fe-containing dioxygenase enzymes we study all use aromatic substrates and represent the major means by which natural and man-made aromatics are biodegraded. This provides the first line of defense against the toxic and carcinogenic effects of these compounds, and thus these enzymes have another substantial impact on human health. The dioxygenase enzymes proposed for study include: Fe(II)-containing extradiol dioxygenases, Fe(III)-containing intradiol dioxygenases, and redox cycling Rieske dioxygenases. The dinuclear Fe cluster-containing monooxygenases being studied are soluble methane monooxygenase (MMO) and two enzymes from the biosynthetic pathway for chloramphenicol, CmlA and CmlI. MMO is the principal barrier to release of the potent greenhouse gas methane into our atmosphere. CmlA and CmlI have numerous homologs in the natural product biosynthetic pathways for some of our most effective antibiotic and chemotherapy drugs, but remain mechanistically uncharacterized. We have developed hypotheses for the mechanisms by which the active site iron(s) in each type oxygenase we study is used to promote catalysis. These will now be tested by exploiting methods we have perfected to observe their reactions step by step as they occur. Past studies have led to the development of single turnover systems for each of the enzyme classes. Intermediates will be detected and trapped by coordinated use of stopped-flow transient kinetics, rapid mixing freeze quench (RFQ), site directed mutagenesis, and use of slow substrate analogs. The trapped intermediates will be characterized by a range of spectroscopies including optical, EPR, rRaman, NRVS, EXAFS, NIR CD, VTVH MCD, Mössbauer and a novel time resolved rRaman technique (TR3). Also, novel in crystallo techniques will be used in which the reactions are slowed (and often stopped at intermediates) by carrying them out in enzyme crystals. The 3D structures of intermediates trapped in crystallo will be solved crystallographically and through the use of newly developed time resolved techniques that employ the Linac Coherent Light Source. In ongoing studies, we have detected and trapped several widely postulated, but previously unobserved, intermediates in each of the enzyme classes. This work will yield fundamental information about the chemistry of oxygenases, oxygen, and metals in biological systems. The basic concepts that emerge will be useful in such areas as the mechanisms of similar enzymes in mammals and methods to interdict the production of deleterious diffusible ROS in humans.