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.
