Elucidating the mechanism of particulate methane monooxygenase

描述（由申请人提供）：拟议研究的目的是阐明完整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为甲醇。我们的中心假设是，pMMO催化甲烷的选择性氧化使用一种新的机制途径。中央的催化途径是一个氧桥的双铜物种有点类似于先前表征的双铜酶和模型化合物。然而，pMMO双铜中心的配位环境与所有其他已知的双铜酶中的配位环境显著不同，并且可能定义了一类全新的酶。一个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机制将通过三种方法来定义。初步表征将调查在双铜网站的pMMO和重组构建的可溶性pmoB域（spmoB）使用各种光谱技术的O2结合。spmoB将在研究中用作pMMO的功能模型，并将制备位点特异性变体以进一步探测活性位点的性质。一旦O2结合的特点，酶动力学使用气相色谱法和停流光谱将被确定。这些数据将定义膜在pMMO中的作用，并捕获反应途径上的快速时标中间体。在生化研究的同时，将采用氧化和还原的pMMO和spmoB的高分辨率晶体结构。所有拟议的研究将在存在和不存在合适的底物的情况下进行，以研究甲烷进入和氧化的位点。 这项建议与NIH的使命有关，即制定新的战略，减少导致癌症的环境污染和气候变化引起的疾病。pMMO在环境条件下分解最惰性的烃，甲烷，因此代表了开发绿色催化剂以实现生物修复和最小化温室气体排放的有吸引力的目标。pMMO可有效降解威胁人类健康的卤代烃污染物，如三氯乙烯（TCE）和氯乙烯（VC）。根据疾病控制中心的说法，氯化碳氢化合物与内分泌失调和多种癌症有关。此外，pMMO代表了通过增加地球气候来最大限度地减少对人类健康构成威胁的温室气体排放的目标。温室气体排放导致的气候变化增加了水传播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。 公共卫生相关性：这项工作中提出的研究是重要的，因为它们解决了我们社会对环境污染和全球气候变化对人类健康影响的日益关注。开发更环保、更安全的催化剂对于减少这些环境健康问题至关重要。这里提出的战略是开发这些催化剂和最大限度地减少环境压力对人类健康和福祉的影响的第一步。

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.