Proteins containing iron-sulfur (FeS) clusters within facultative anaerobes, organisms that can transition between aerobic and anaerobic metabolism, can provide a window into the evolutionary transition of life before oxygenation of the planet to after. However, the experimental design necessary to study oxygen-sensitive FeS cluster containing proteins, is often nontrivial. Performing experiments to study detailed molecular mechanisms in the absence of oxygen requires significant and diligent effort. This thesis will focus on two FeS proteins from facultative anaerobes, Escherichia coli and Saccharomyces cerevisiae, and the development and application of advanced structural techniques, such as serial crystallography, small-angle X-ray scattering (SAXS), and cryo-electron microscopy (cryoEM). Towards room-temperature crystallography studies of metalloproteins, I describe the development of a fixed-target sample preparation and mounting system for serial synchrotron crystallography in collaboration with MiTeGen and the Cornell High Energy Synchrotron Source (CHESS). I then describe the development of anoxic SAXS at CHESS station ID7A and apply the system to perform the first structural characterization of the E. coli transcription factor, Fumarate and Nitrate Regulation (FNR) protein, and its use of oxygen-sensing [4Fe-4S] clusters. Finally, I describe a model of the S. cerevisiae diphthamide biosynthesis complex Dph123-EF2 obtained by cryoEM and AlphaFold Multimer that hints at the mechanism by which a catalytically essential [4Fe-4S] cluster is rescued in the presence of oxygen. Altogether, the advances described within this thesis have contributed to the field of structural biology by providing two new avenues of experimental techniques that can be utilized by researchers throughout the world to characterize samples that were previously very difficult if not impossible to characterize. The results presented in this thesis also provide insight into how facultative anaerobes may have overcome or taken advantage of the oxygen sensitivity of FeS clusters.