The structural characterization of bacterioferritin, BfrA, from Mycobacterium tuberculosis

Tuberculosis is a deadly disease caused by Mycobacterium tuberculosis. Like most bacterial pathogens, iron acquisition, regulation, and storage are critical for its survival. Due to the poor solubility of iron under physiological conditions, both eukaryotes and prokaryotes possess ferritins, large protein complexes that store iron and keep it bioavailable. Mtb encodes for two ferritin homologs: a heme-containing bacterioferritin (Mtb-BfrA) and a non-heme eukaryotic-like ferritin (Mtb-BfrB). A conserved feature of bacterioferritins is the presence of a heme group at the interface between two subunits of each dimer that is related by a non-crystallographic two-fold axis. The structure of a selenomethionine derivative of Mtb-BfrA was previously reported (PDB ID: 2WTL); however, a proposed heme degradation product was modeled into the heme-binding site, as electron density for intact heme was not observed. Here, the purification and structure determination of recombinant Mtb-BfrA is reported. As-isolated Mtb-BfrA from Escherichia coli is not fully heme loaded. However, the absorption spectrum features suggest binding of intact heme. In an attempt to fully complement Mtb-BfrA with heme, two different methodologies are described. Electronic spectroscopy and structure determination were used to confirm varying amounts of intact bis-methionine coordinated heme to Mtb-BfrA. We also report that increased heme incorporation only slightly increases Mtb-BfrA ferroxidase activity. Finally, the cognate partner of Mtb-BfrA is proposed to be a putative encoded gene which is located approximately 300 bps upstream of Mycobacterium tuberculosis bfrA, homologous to the cognate partner of Pseudomonas aeruginosa bacterioferritin, a 7 kDa ferrodoxin Bfd.