The Pseudomonas aeruginosa PhuS proximal ligand His-209 triggers a conformational switch in function from DNA binding to heme transfer.

Pseudomonas aeruginosa can acquire iron from heme via the heme assimilation system and Pseudomonas heme uptake (Phu) systems. Heme uptake is regulated at the metabolic level by the cytoplasmic protein PhuS that controls heme flux through a heme oxygenase HemO, releasing iron and biliverdin IXβ and IXδ. We have shown PhuS regulates extracellular heme flux, and in its apo-form transcriptionally regulates the iron and heme-dependent small RNAs (sRNAs) PrrF/PrrH. This mutual exclusivity of function is driven by conformational rearrangement of PhuS on heme binding. Herein, we show through a combination of EMSA and fluorescence anisotropy that mutation of the His-209 proximal ligand allows both apo- and holo-PhuS H209A to bind to the prrF1 promoter with significantly lower affinity when compared to PAO1 WT. Hydrogen deuterium exchange coupled to mass spectrometry revealed the apo- and holo-PhuS H209A structures are closer to each other than their WT counterparts and sample a conformational landscape between the apo- and holo-PhuS WT conformations, that is neither optimal for heme transfer nor DNA-binding. Furthermore, quantitative PCR and Western blot analysis of the phuSH209A allelic strain compared to PAO1 WT revealed an uncoupling of the PhuS-HemO dependent regulation of heme flux into the cell that abrogates the heme dependent regulation of the PrrF/PrrH sRNAs. The data supports a model where heme coordination through His-209 drives the conformational switch that determines mutual exclusivity in function of apo- and holo-PhuS. This dual function of PhuS is central to integrating extracellular heme utilization into the PrrF/PrrH sRNA regulatory network critical for P. aeruginosa adaptation within the host.