Emerging pathogens and pathogens showing increasing levels of antibiotic resistance present a sizeable threat to global human health. One aspect of microbial physiology that is potentially targetable with therapeutics and is a potential mechanism of antibiotic resistance is the oxidative stress response. Microbial pathogens have multiple mechanisms in place for responding to oxidative stress, many of which are mediated by metalloproteins and other redox-active proteins. We are working to make use of multiple methods, including spectroscopy coupled to diffraction data collection, to probe the structure and function of oxidative stress response proteins from several bacterial pathogens. One of these proteins, rubrerythrin (Rbr), belongs to the ferritin-like superfamily and often functions physiologically in oxidative stress tolerance, especially in anaerobic bacteria. Most characterized Rbr proteins contain a di-iron site within a four-helix bundle with an N- or C-terminal rubredoxin domain. We are investigating Rbr and its partner proteins from multiple pathogens, both aerobic and anaerobic, including B. pseudomallei, C. difficiles and C. jejuni. We use X-ray crystallography experiments coupled to in crystallo UV-vis and energy dispersive X-ray fluorescence spectroscopy to directly probe metal speciation and peroxide binding. We have generated structural models of Rbr from B. pseudomallei in apo, Co-, Mn-, and Fe-bound forms, which lack the canonical rubredoxin domain and exhibit domain swapping in the functional dimer. We are also using neutron crystallography to investigate peroxide binding. Our structural and functional studies provide details about the structural role of multiple metals in these previously uncharacterized systems.