Toward a molecular understanding of iron homeostasis in the opportunistic pathogen Pseudomonas aeruginosa. The role of Bacterioferritin B.

Bacterioferritin B is an important protein for P. aeruginosa as it is capable of storing about 4000 Fe3+ ions in the form of ferric hydroxy phosphate in its core and release when it is required by the cell. Our research was aimed at understanding the mechanisms and pathways of iron uptake and mobilization from Pa BfrB which are considered as two vital processes of this protein molecule. The recombinant Pa-BfrB was overexpressed and purified to homogeneity. The structural studies of Pa BfrB were carried out by solving four crystal structures: (1) crystals grown from Pa BfrB without a mineral core (as-isolated), (2) mineralized with ~600 iron atoms (mineralized), (3) crystals soaked in a Fe2+ solution and (Fe soak) and (4) Fe soaked crystals soaked in a crystallization solution (double soak). Crystal structures revealed that it is composed of 24 subunits arranged as a sphere and can bind 12 heme molecules per protein. Further, crystallographic studies elegantly showed that iron ions to be stored inside the cavity are taken into the inner cavity of the protein via the ferroxidase pores on the surface. Both crystallographic and solution experiments provide evidence to show that these Fe2+ ions are oxidized at the ferroxidase center and subsequently internalized via a gated mechanism operated by the amino acid residue His130 at the ferroxidase center. An interaction between BfrB and apo Bfd (Bcaterioferritin associated ferredoxin) was found to be necessary for efficient mobilization of iron from the mineral core of BfrB. In addition, the thus far mysterious role of heme in bacterioferritins was revealed for the first time and was experimentally demonstrated that ferric ion mineral core of BfrB is reduced by the heme mediated efficient transfer of electrons from the reductase to the core. Additional experiments show that Bfd binds to BfrB with a ratio of 12:1 to support the efficient mobilization of iron from BfrB and it was found that Bfd is not functioning as a typical ferredoxin in the iron mobilization process of BfrB as it is the apo Bfd not the holo Bfd which is involved in the iron mobilization from the inner cavity of BfrB. Crystallographic data suggest that four-fold pores may be the dominant route of iron release from the inner cavity of BfrB. Results of the studies carried out with site directed mutagenesis and Elastic Network Model calculations provided experimental evidence suggesting that the main path way of iron release is through the four fold pores of BfrB