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

本研究将采用多学科方法，运用基础生物化学、光谱学和x射线晶体学技术，探讨固氮过程中关键酶——固氮酶的作用机制。我们将探讨氧化还原依赖的结构变化是调节中点电位和确定P簇在门控分子间电子转移中的作用的关键的假设。关键位点导向变体和铕络合物氧化还原介质的联合使用将有助于捕获与催化机制相关的酶的定义状态，这些状态在天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能源需求和更环保的机制，以满足与全球人口增长相关的对固定氮不断增长的需求，因为植物生长的固定氮源的可用性是全球营养的限制因素，而氮肥的生产非常需要能源，并可能产生负面的环境影响。更广泛的影响包括培训学生，并通过开发农业和能源在线课程为培训K-12教师做出重大贡献，同时还为包括部落学校在内的偏远农村地区的教师开展了其他工作。

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.