Role of the P Clusters and FeMo-Cofactors in Nitrogenase Catalysis

拟议的工作涉及实施一种多学科的方法，使用基础生物化学、光谱学和X射线结晶学技术来探索关于固氮关键酶--固氮酶的一些关键悬而未决的问题。我们将探讨这一假设，即依赖于氧化还原的结构变化是调节中点电位和确定P团簇在门控分子间电子转移中的作用的关键。结合使用关键的定点突变体和Eu络合物氧化还原介体，将有助于捕获与催化机制相关的酶的定义状态，而这些状态在自然状态下是不可能观察到的，并将回答有关底物与FEMO辅因子结合的问题。生物固氮的基础研究有助于减少对能源的需求，建立更无害环境的机制，以满足与全球人口增长相关的对固定氮日益增长的需求，因为可用于植物生长的固定氮源是全球营养的一个限制因素，而氮肥的生产非常耗能，并可能对环境产生负面影响。更广泛的影响包括学生培训和通过开发农业和能源在线课程以及针对包括部落学校在内的偏远农村地区的教师的其他努力，对培训K-12教师作出重大贡献。

The proposed work involves implementing a multidisciplinary approach using basic biochemistry, spectroscopy, and x-ray crystallography techniques to probe some of the key outstanding issues regarding the mechanism of the key enzyme in nitrogen fixation, nitrogenase. We will probe the hypothesis that the redox-dependent structural changes are key to modulating the midpoint potential and defining the role of the P cluster in gated intermolecular electron transfer. The combined use of key site-directed variants and a europium complex redox mediator will facilitate the capture of defined states of the enzyme relevant to the catalytic mechanism that are not possible to observe in the native state, and will answer questions regarding substrate binding to the FeMo-cofactor. Basic research on biological nitrogen fixation could contribute to less energy demanding and more environmentally sound mechanisms to meet the growing demands for fixed nitrogen associated with global population growth, as the availability of fixed nitrogen sources for plant growth is a limiting factor in global nutrition and the production of nitrogenous fertilizers is very energy demanding and can have negative environmental implications. Broader Impacts include student training and significant contributions to training K-12 teachers through the development of an online course on Agriculture and Energy along with other efforts aimed at teachers in remote rural areas that include tribal schools.