Protein-Protein Interactions Regulate Iron Homeostasis in Pseudomonas aeruginosa

Abstract

Our lab had previously shown that the interaction between bacterioferritin (BfrB) and its associated ferredoxin (Bfd) enables mobilization of iron stored in BfrB. My research was aimed at understanding the mechanism of the BfrB-Bfd interaction and how this interaction facilitates electron transfer into the ferric core in BfrB for subsequent mobilization of Fe2+ from the core. Results from biochemical and structural studies allowed us to conclude that the BfrB-Bfd interaction enables heme-mediated electron transfer from the [2Fe-2S] cluster in Bfd to the Fe3+ mineral stored in BfrB. The Bfd fold was revealed for the first time in the X-ray crystal structure of BfrB-Bfd complex. The crystal structure revealed that Bfd has a helix-turn-helix fold, which is distinct from the canonical ferredoxin fold. Analysis of the structure, followed by studies in solution showed that the [2Fe-2S] cluster in Bfd is important to stabilizing the Bfd fold. In addition, evidence from X-ray crystallography, in vitro assays, and in silico experiments, showed that Bfd is also stabilized by the binding of a phosphate anion. The crystal structure of the BfrB-Bfd complex showed that 12 Bfd molecules bind a 24-mer BfrB, with each heme binding almost immediately above each of the 12 hemes in BfrB. The crystal structure of the BfrB/Bfd complex also allowed us to identify the residues from each of the two proteins that participate at the complex interface, and to analyze specific interactions among these residues. The interaction between BfrB and Bfd was also characterized in solution by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). The results show Bfd binds at each of the sites on BfrB with Kd of ~ 3 M, in an association that is driven entropically. In addition, contributions of the residues that participate at the BfrB-Bfd complex interface were evaluated in solution, which led to the identification of the hotspot for the BfrB-Bfd interaction.