Elucidating the mechanism of particulate methane monooxygenase

描述（由申请人提供）：拟研究的目的是阐明整体膜金属酶颗粒甲烷单加氧酶（pMMO）的机制。pMMO在环境条件下有效地催化甲烷选择性氧化制甲醇。我们的中心假设是pMMO通过一种新的机制途径催化甲烷的选择性氧化。催化途径的核心是氧桥接的二铜种，与先前表征的二铜酶和模型化合物有些相似。然而，pMMO迪铜中心的配位环境与所有其他已知的迪铜酶明显不同，可能定义了一类全新的酶。新的活性位点和新的O2活化化学可能会出现，影响生物无机化学和催化。pMMO机制将由三种方法定义。初步表征将使用各种光谱技术研究pMMO在diccopper位点的O2结合和可溶性pmoB结构域（spmoB）的重组结构。spmoB将在研究中作为pMMO的功能模型，位点特异性变异体将进一步探索活性位点的特性。一旦O2结合被表征，酶动力学使用气相色谱和停止流动光谱将被确定。这些数据将确定膜在pMMO中的作用，并捕获反应途径上的快速时间尺度中间体。在生化研究的同时，还将采用氧化和还原pMMO和spmoB的高分辨率晶体结构。所有拟议的研究都将在有或没有合适的底物的情况下进行，以调查甲烷进入和氧化的位置。该提案通过制定新的战略来减少引起癌症的环境污染和气候变化引起的疾病，与美国国立卫生研究院的使命相关。pMMO在环境条件下可以分解惰性烃甲烷，因此在开发绿色催化剂以实现生物修复和减少温室气体排放方面是一个有吸引力的目标。对人类健康构成威胁的卤代烃污染物，如三氯乙烯（TCE）和氯乙烯（VC），可通过pMMO有效降解。根据疾病控制中心的数据，氯化碳氢化合物与内分泌紊乱和多种癌症有关。此外，pMMO是最大限度减少温室气体排放的目标，这些温室气体通过增加地球气候对人类健康构成威胁。温室气体排放造成的气候变化增加了水媒疾病和通过昆虫传播的疾病，如腹泻、营养不良、疟疾和登革热。

DESCRIPTION (provided by applicant): The objective of the proposed research is to elucidate the mechanism of the integral membrane metalloenzyme particulate methane monooxygenase (pMMO). pMMO efficiently catalyzes the selective oxidation of methane to methanol under ambient conditions. Our central hypothesis is that pMMO catalyzes the selective oxidation of methane using a novel mechanistic pathway. Central to the catalytic pathway is an oxo-bridged dicopper species somewhat similar to that in previously characterized dicopper enzymes and model compounds. However, the coordination environment of the pMMO dicopper center is significantly different from that in all other known dicopper enzymes, and likely defines a completely new class of enzymes. A novel active site and new O2 activation chemistry will likely emerge, impacting both bioinorganic chemistry and catalysis. The pMMO mechanism will be defined by three approaches. Initial characterization will investigate the O2 binding at the dicopper site of pMMO and a recombinant construct of the soluble pmoB domain (spmoB) using various spectroscopic techniques. spmoB will be used in the studies as a functional model for pMMO and site-specific variants will be made to further probe the properties of the active site. Once the O2 binding has been characterized, enzyme kinetics using gas chromatography and stopped-flow spectroscopy will be determined. These data will define the role of the membrane in pMMO and trap fast timescale intermediates on the reaction pathway. In parallel to the biochemical studies, the high-resolution crystal structures of oxidized and reduced pMMO and spmoB will be employed. All the proposed studies will be run in the presence and absence of a suitable substrate to investigate the site of methane entry and oxidation. This proposal is relevant to the mission of the NIH by developing new strategies to diminish both cancer causing environmental contaminates and diseases induced by climate change. pMMO breaks down the most inert hydrocarbon, methane, under ambient conditions and therefore represents an attractive target in the development of green catalysts to target bioremediation and minimize greenhouse gas emissions. Halogenated hydrocarbon pollutants, such as trichloroethylene (TCE) and vinylchloride (VC) that pose a threat to human health are effectively degraded by pMMO. According to the Centers of Disease Control, chlorinated hydrocarbons are implicated in endocrine disorders and many forms of cancer. Additionally, pMMO represents a target for minimizing greenhouse gas emissions that pose a threat to human health by increasing the earth<s climate. Climate changes due to greenhouse gas emissions increase water borne diseases and diseases transmitted through insects such as diarrhea, malnutrition, malaria, and dengue. PUBLIC HEALTH RELEVANCE: The studies proposed in this work are significant because they address the growing concern in our society on the effect environmental contamination and the global climate change has on human health. The development of greener, safer catalysis is essential in diminishing these environmental health concerns. The strategies proposed here are the first steps in developing these catalysts and minimizing the effects environmental stressors pose on human health and wellbeing.