In situ mechanisms of biological nitrogen fixation - PROJECT SUMMARY. Nitrogen fixation by the metalloenzyme nitrogenase supports nearly 50% of the global population and is the only biological pathway for nitrogen reduction. Nitrogenase consists of two proteins, the obligate reductase Fe-protein and the catalytic MoFe-protein, both of which are rapidly inactivated by oxygen. Biochemical, crystallographic, and spectroscopic studies have yielded seminal insights into the structures and functions of purified nitrogenase proteins, but a unified understanding of the enzymatic mechanism is still unrealized. These proteins are expressed in a small subset of prokaryotes termed diazotrophs, including aerobes and anaerobes, which inhabit a diverse array of environments. Despite this evolutionary demonstration of compatibility between nitrogen fixation and a variety of metabolisms, robust heterologous expression of the nitrogenase proteins has not been achieved. Instead, endogenous expression within the free-living soil bacterium Azotobacter vinelandii remains the most widely used system for the purification and study of nitrogenase. Several peculiar features of this obligate aerobe have been annotated under nitrogen-limited conditions, to wit, the formation of an intracytoplasmic membrane network, but a comprehensive investigation of these features and their relationship to the nitrogenase proteins is lacking. The primary hypothesis of this proposal is that interactions with cellular ultrastructures and as yet unidentified binding partners in vivo significantly regulate and promote nitrogenase activity. My goal is to identify and characterize these states of nitrogenase in diazotrophic organisms by training in and applying emerging cryoelectron tomography (cryoET) methodologies and performing experiments outlined in two Aims. Aim 1: Revealing the architecture of the nitrogenase interactome in A. vinelandii with mass spectrometry and single particle (SP) cryoEM. Aim 2: Determining in situ structures of nitrogenase complexes in conjunction with cellular features using cryogenic correlative light and electron microscopy (cryoCLEM/ET) and sub-tomogram averaging (STA). As a postdoctoral fellow I have gained expertise in anaerobic SP cryoEM to obtain high resolution structures of nitrogenase. These skills will provide a foundation for the proposed research. However, I seek training in the growing field of cryoET for the study of in situ structures. Throughout the outlined aims, I will train in genetic manipulation of non-model organisms, mass spectrometry, and cryoET from my advisory committee, the Caltech CryoEM and proteomic facilities, and national facilities while continuing to expand my mastery of SP cryoEM and metalloenzyme chemistry. In my independent career, I will apply this training to the continued study of nitrogenase and other anaerobic systems relevant to environmental and human health. The proposed aims will yield the first comprehensive investigation of nitrogenase in situ with the increasingly high resolution technique of cryoET, and will provide insight not only into cellular regulation of nitrogen fixation, but also structures of true turnover states within the cell thus fundamentally moving our understanding of this important biogeochemical process forward.
