Pseudomonas aeruginosa (Pa) is a ubiquitous Gram-negative bacterium that is a major cause of chronic nosocomial infections, especially when Pa forms biofilms. These recalcitrant extracellular networks provide an advantageous protective quality for Pa by significantly increasing its antibiotic resistance, making Pa a major health threat. Previous work has uncovered a two-component signal transduction system, BqsRS, that regulates biofilm formation and decay in Pa through the sensing of Fe2+. Homology suggests that PaBqsS is a transmembrane sensor kinase while PaBqsR is a DNA-binding cytosolic response regulator that alters the transcription of genes involved in biofilm formation. Neither of these proteins have been structurally characterized, and the details of how they interact with Fe2+ remain unknown. Using X-ray crystallography, we have solved the structure of the N-terminal phosphorylation domain of PaBqsR to 1.3 Å resolution, which reveals a canonical (βα)5 response regulator assembly. Using NMR, we have initially characterized intact PaBqsR and its DNA-binding domain. Interestingly, phosphorylation mimics of PaBqsR (D51E and BeF3--activated) show a concentration-dependent oligomerization based on gel filtration studies, and the quaternary state appears to alter DNA-binding capabilities. Importantly, we have found that the PaBqsR DNA-binding consensus sequence is located upstream of the feo operon, the primary Fe2+ transporter in Pa. Electrophoretic mobility shift assays (EMSAs) confirm that PaBqsR binds upstream of the feo operon, but only in the pseuodo-phosphorylated and/or dimeric state. Distinct from other characterized response regulators, we show that PaBqsR binds a single Fe2+ per protein monomer, and we have characterized this binding site using X-ray absorption spectroscopy (XAS), demonstrating an important functional divergence in this response regulator from others. Regarding the intact His kinase, we have cloned, expressed, and purified detergent-solubilized PaBqsS. We have shown that PaBqsS binds 1 Fe2+ ion per protein dimer, and using site-directed mutagenesis and XAS, we have characterized the His kinase Fe2+ binding site for the first time. These results provide the first biophysical characterization of the Bqs system and demonstrate its connection the Feo system, an important Pa virulence factor that could be targeted for future therapeutic developments.