The uptake and utilization of iron remains critical for the survival/virulence of the host/pathogens in spite of the limitations (low bioavailability/high toxicity) associated with this nutrient. Both the host and pathogens manage to overcome these problems by utilizing the iron repository protein nanocages, ferritins, which not only sequester and detoxify the free Fe(II) ions but also decrease the iron solubility gap by synthesizing/encapsulating the Fe(III)-oxyhydroxide biomineral in its central hollow nanocavity. Bacterial pathogens including Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, encode a distinct subclass of ferritins called bacterioferritin (BfrA), which binds heme, the versatile redox cofactor, via coaxial, conserved methionine (M52) residues at its subunit-dimer interfaces. However, the exact role of heme in Mtb BfrA remains yet to be established. Therefore, its coaxial ligands were altered via site-directed mutagenesis, which resulted in both heme-bound (M52C; ∼1 heme per cage) and heme-free (M52H and M52L) variants, indicating the importance of M52 residues as preferential heme binding axial ligands in Mtb BfrA. All these variants formed intact nanocages of similar size and iron-loading ability as that of wild-type (WT) Mtb BfrA. However, the as-isolated heme-bound variants (WT and M52C) exhibited enhanced protein stability and reductive iron mobilization as compared to their heme-free analogues (M52H and M52L). Further, increasing the heme content in BfrA variants by reconstitution not only enhanced the cage stability but also facilitated the iron mobilization, suggesting the role of heme. In contrary, heme altered the ferroxidase activity to a lesser extent despite facilitating the accumulation of the reactive intermediates formed during the course of the reaction. The current study suggests that heme in Mtb BfrA enhances the overall stability of the protein and possibly acts as an intrinsic electron relay station to influence the iron mineral dissolution and thus may be associated with Mtb's pathogenicity.

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