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

 描述(由申请人提供)：我们建议确定一组不同和多功能的非血红素含铁单加氧酶和双加氧酶的分子和调控机制，作为研究生物氧气激活机制的一种手段。重点将放在反应循环中间体的检测和表征上。自然界中使用的最重要的氧气激活策略代表了我们选择的一组酶，使这个问题得以在广泛的战线上解决。含铁酶对O2的激活受到高度调控，因为活性氧物种(ROS)的逃逸会对遗传物质和其他生物分子造成损害，导致细菌中的细胞死亡和人类的疾病。因此，我们的研究将描绘出O2活化过程中活性中间体的稳定和不稳定力量。我们研究的单核含铁双加氧酶都使用芳香族底物，代表了天然和人造芳烃生物降解的主要途径。这为这些化合物的毒性和致癌作用提供了第一道防线，因此这些酶对人类健康有另一个实质性的影响。拟研究的双加氧酶包括：含Fe(II)的外源性双加氧酶、含Fe(III)的内源性双加氧酶和氧化还原循环Rieske双加氧酶。所研究的含双核铁簇的单加氧酶是可溶性甲烷单加氧酶(MMO)和氯霉素生物合成途径的两种酶CmlA和CmlI。MMO是将强有力的温室气体甲烷释放到我们大气中的主要障碍。CmlA和CmlI在我们一些最有效的抗生素和化疗药物的天然产物生物合成途径中有许多同系物，但在机制上仍未确定。我们对我们研究的每一种加氧酶中的活性部位铁(S)用于促进催化的机制提出了假说。现在将利用我们已经完善的方法来测试这些方法，以便在它们发生时一步一步地观察它们的反应。过去的研究已经导致了每种酶类的单一周转系统的发展。通过协调使用停流瞬变动力学、快速混合冷冻淬火(RFQ)、定点突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、rmann、NRVS、EXAFS、NIR CD、VTVH MCD、Mössbauer和一种新的时间分辨rmann技术(TR3)。此外，还将使用新的结晶技术，通过在酶晶体中进行反应来减缓反应速度(并经常停止在中间体)。捕获在晶体中的中间体的3D结构将通过使用直线加速器相干光源的新开发的时间分辨技术，通过结晶学和使用时间分辨技术来解决。在正在进行的研究中，我们在每一种酶类中检测并捕获了几个广泛假设的、但以前没有观察到的中间体。这项工作将提供有关生物系统中加氧酶、氧和金属的化学的基本信息。出现的基本概念将在哺乳动物中类似酶的机制以及在人类中阻断有害的、可扩散的ROS产生的方法等领域有用。

 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.