PROJECT SUMMARY: The nitrogenase enzyme complex is the only biological pathway for producing metabolically useful forms of nitrogen from atmospheric dinitrogen. This two component protein system made up of an obligate reductase, Fe-protein, and the catalytic protein, MoFe-protein, is only present in a small number of microorganisms, but produces nearly 50% of all bioavailable nitrogen. Thus, the biological nitrogen fixation mechanism has extremely important effects on global crop production and human health. Seminal works in the past half century have not only revealed an intricate kinetic pathway for nitrogenase, but also the most complex metallocluster found in nature within the MoFe-protein active site. This cluster, known as the FeMo-cofactor, has the composition [7Fe:9S:1C:1Mo]-R-homocitrate and is only coordinated by two residues within the active site. Electrons are shuttled through a [4Fe:4S] cluster in the Fe-protein to an [8Fe:7S] center in the MoFe-protein, known as the P- cluster. These reducing equivalents are then delivered to the active site FeMo-cofactor where substrate reduction occurs. Four consecutive cycles of electron transfer are required to bind dinitrogen, and four more are required to fully convert one dinitrogen molecule to two ammonia molecules. It is unknown how the FeMo-cofactor is primed for substrate binding, how the FeMo-cofactor binds or shuttles electrons to the substrate, or what role the surrounding active site residues play in substrate reduction. The primary hypothesis of this proposal is that physical rearrangements of the atoms in the FeMo-cofactor and in the surrounding protein during substrate reduction are necessary for substrate access and binding to the cofactor, and may further play a role in the reduction mechanism. The goal of this work is to determine what rearrangements occur in nitrogenase throughout turnover of substrate by harnessing the power of cryoEM and performing experiments outlined in two Aims. Aim 1: Structural characterization of resting states of nitrogenase: MoFe protein alone and the ADP-AlF4- stabilized complex with Fe-protein by single particle cryoEM. Aim 2: Structural characterization of nitrogenase turnover-related forms by single particle cryoEM. Through the pursual of Aims 1 and 2, I will obtain training in the field of single particle cryoEM, and each Aim will provide expertise in metalloenzyme chemistry. The research will be performed at the California Institute of Technology primarily in the well-known CryoEM Center with ample microscopes and expertise. These Aims will shed much needed light on transient intermediates within the nitrogenase turnover pathway thereby providing a better foundation for the rational design of efficient synthetic nitrogen fixation platforms. In addition, the insights and the methodology developed in this proposal can be applied to other poorly understood metalloenzymes related to human health.