Pseudomonas aeruginosa, a notorious multi-drug resistant pathogen, sequesters heme from the host as its primary iron source during chronic infections. This occurs through the combined action of the non-redundant Pseudomonas heme uptake (Phu) system and heme assimilation system (Has). Once in the cytoplasm heme is bound by the cytoplasmic heme binding protein, PhuS, that selectively transfers it to the heme oxygenase, HemO, for cleavage and release of iron, carbon monoxide (CO), and biliverdin-β/δ (BVIX-β/δ). We have previously shown that PhuS is critical to maintaining iron and heme homeostasis through its coupled interaction with HemO and also modulates the expression of the PrrF sRNA system which is critical to iron homeostasis and virulence. This dual function allows extracellular heme acquisition to be integrated into the regulation of the PrrF sRNAs enabling P. aeruginosa to fine-tune its response to varying nutrient availability. However, the biophysical and biochemical properties of PhuS that allow it to mediate its dual functions, and the ramifications on global cellular processes in P. aeruginosa if they are disrupted are not yet fully understood. In this study, through a combination of biophysical, biochemical, proteomic, and metabolic approaches we characterized the properties of a PhuS variant, PhuS H209A, that no longer retained its mutually exclusive functions and the implications on global processes once this functionality was lost. The substantially different conformational states displayed by the C-terminal helices in the presence and absence of heme in the WT are lost in PhuS H209A, with the C-terminal helices in the variant exhibiting a conformational intermediate incapable of adopting either the optimal heme or DNA binding states. This revealed that heme coordination to the proximal ligand, His-209, is the driving factor in catalyzing the conformational rearrangement that enables both functions to remain separate. Proteomic and metabolic studies on a phuSH209A allelic variant in static conditions showed significant changes to phenazine (PHZ), pyochelin (PCH), and Pseudomonas quinolone signaling (PQS) biosynthetic pathways as well as changes to major metabolic intermediates including the shikimate pathway. Additionally, qPCR and bioinformatic approaches revealed that relative PrrF expression was increased in the phuSH209A variant and that identified genes from the proteomic analysis may reflect direct PrrF targets or indirect downstream effects on several metabolic pathways. Finally, proteomic analysis of the ΔphuS strain in static conditions revealed many of the same proteomic changes observed for phuSH209A further substantiating the effects seen are a result of disrupting iron and heme homeostasis and PrrF expression. This report adds to our existing knowledge of how PhuS is able to maintain its mutually exclusive heme transfer and DNA binding functions and the ramifications on metabolic and virulence networks in P. aeruginosa if these processes are disrupted. By further understanding how P. aeruginosa is able to adapt and respond to extracellular heme availability, particularly in static conditions, we hope to illuminate new networks and relationships in P. aeruginosa that better inform the development of novel therapeutic strategies.