Elucidating the mechanism of particulate methane monooxygenase

描述(申请人提供)：建议研究的目的是阐明完整的膜金属酶颗粒甲烷单加氧酶(PMMO)的机制。在常温下，pMMO能有效地催化甲烷选择氧化制甲醇。我们的中心假设是，pMMO通过一种新的机理途径催化甲烷的选择性氧化。催化途径的中心是一种氧桥联的双铜物种，与以前表征的双铜酶和模型化合物有些相似。然而，pMMO双铜中心的配位环境与所有其他已知的双铜酶有显著不同，可能定义了一类全新的酶。一个新的活性中心和新的氧活化化学可能会出现，对生物无机化学和催化都有影响。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的双铜位置上的O2结合以及可溶性pmob结构域的重组结构(SpmoB)。SpmoB将被用作pMMO的功能模型，并将进行特定部位的变体，以进一步探索活性部位的性质。一旦确定了O2结合的特征，将使用气相色谱和停流光谱分析来确定酶的动力学。这些数据将确定膜在pMMO中的作用，并在反应途径上捕获快速时间尺度的中间产物。在生化研究的同时，将使用氧化和还原的pMMO和spmoB的高分辨率晶体结构。所有拟议的研究都将在有或没有合适底物的情况下进行，以调查甲烷进入和氧化的位置。这项建议与国家卫生研究院的使命相关，它制定了新的战略，以减少导致环境污染的癌症和由气候变化引起的疾病。PMMO在环境条件下分解最惰性的碳氢化合物甲烷，因此是开发针对生物修复和最大限度减少温室气体排放的绿色催化剂的一个有吸引力的目标。三氯乙烯(TCE)、氯乙烯(VC)等对人类健康构成威胁的卤代烃污染物可被pMMO有效降解。根据疾病控制中心的说法，氯代烃与内分泌紊乱和多种形式的癌症有关。此外，PMMO还代表了一个目标，即通过增加地球和S的气候来最大限度地减少温室气体排放，这些温室气体对人类健康构成威胁。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。

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.