Ligand Induced Allostery in Pseudomonas Aeruginosa Cytoplasmic Heme Binding Protein (Phus) Drives the Protein-Protein Interaction with Heme Oxygenase

Abstract

Integral to its essential role in the metabolism of extracellular heme, Pseudomonas Aeruginosa's Phus binds incoming heme and tranfers it to the iron-regulated heme oxygenase (HemO). Previous studies have shown that heme binding induces conformational changes required to interact with HemO. Here, we used mass spectrometry and spectroscopic techniques to probe heme binding, characterize the heme-induced conformational changes in Phus and map the holo-Phus-HemO interaction. Hydrogen-Deuterium exchange (HDX-MS) of apo- and holo-PhuS reveals that heme binding results in significant rearrangements of the heme-binding pocket. Notably, C-terminal proximal helices α6/α7/α8 (residues 198-233) are highly labile in the apo-Phus and become largely protected from exchange upon heme binding only to subsequently undergo EX1-type cooperative unfolding within 1hr. Also, N-terminal α1/α2 helices (residues 1-36) undergo cooperative unfolding in apo-Phus which is significantly slowed in holo-Phus. Remarkably, mapping the interface of the holo-PhuS-HemO complex using chemical crosslinking and MALDI-TOF-MS revealed that these same regions, the α7/α8 helices (residues 217-236) and α1 helix (residues 1-29) contact HemO. Together, HDX-MS and crosslinking data is consistent with a heme-binding induced conformational rearrangement of the C-terminal domain driving the initial PhuS-HemO interaction. A binding-competent yet transfer-incompetent mutant, H212R-Phus was used to further interrogate the conformational changes of Phus in response to heme binding. Spectroscopic studies show H212R-Phus binds heme with approximately 2-fold faster kinetics than wt-Phus. In accordance, HDX-MS shows, in the apo form, the C-terminal binding pocket helices α6/α7/α8 of H212R-Phus are slightly but significantly more protected from exchange than wt-Phus. In holo-H212R-Phus, whereas the large conformational rearrangements in the heme binding pocket is still observable, the cooperative unfolding of the C-terminal and N-terminal helices is largely abolished, suggesting a possible role for such cooperative unfolding in heme transfer.