Pseudomonas aeruginosa an opportunistic pathogen requires iron for survival and virulence. Within the host heme is the preferred source of iron, particularly in chronic infection where P. aeruginosa switches to utilize heme at the expense of iron-siderophores. Heme enters the cell through two non-redundant heme uptake systems, the heme assimilation system (Has) and the Pseudomonas heme uptake (Phu) system. Once in the cytoplasm, heme is bound by PhuS, which selectively transfers heme to heme oxygenase (HemO) for metabolism and release of iron and the biliverdin (BVIX)β and BVIXδ metabolites. We have recently shown that in its apo-form PhuS binds to the promoter upstream of the prrF1,F2 operon encoding the iron-regulated sRNAs PrrF1 and PrrF2. Previous hydrogen deuterium exchange mass spectrometry (HDX-MS) studies showed significant differences in dynamics and flexibility between apo- and holo-PhuS, as well as long-range conformational rearrangement and allostery, providing significant insight into the mutual exclusivity of the apo- and holo- forms and their respective functions. Herein utilizing a combination of bacterial genetics, biochemical and biophysical analyses we have characterized mutants disrupted in either heme transfer or prrF regulation. We will present data to show the mutual exclusivity in function of apo- and holo-PhuS is critical for integrating extracellular heme uptake into the iron-regulatory networks governing virulence in P. aeruginosa.