Iron Mineralization in Mycobacterial Ferritins: Impact of Protein Cage; Pores; and Phosphate

Abstract

Iron is essential for the growth of almost all organisms including pathogens. Despite its importance, free Fe2+ can mediate cellular toxicity by generating reactive oxygen species. To maintain the balance between its essentiality and toxicity, appropriate cellular levels of iron are tightly regulated by a self-assembled spherical protein nanocage called ferritin, which synthesizes ferric oxyhydroxide mineral in its central cavity. These minerals in native ferritins are also associated with significant amount of phosphate (Fe/P ~ 1-2 in bacteria vs. >10 in animals). Like iron, phosphate also regulates the pathogenesis of Mycobacterium tuberculosis (Mtb), which expresses two types of ferritin: non-heme binding ferritin (BfrB) and heme binding bacterioferritin (BfrA). The mechanism of mineral core formation and the impact of phosphate towards its structure and reactivity in BfrB are not explored and thus investigated herein. The study confirms that phosphate alters the kinetics of iron oxidation and decreases the size/crystallinity of the mineral core. Iron mineralization commences with the rapid influx of Fe2+ via different pores, formed during self-assembly process. Investigation of these pores as Fe2+ uptake routes in ferritins remain a subject of intense research, in iron metabolism, toxicity and bacterial pathogenesis. As BfrA expression is upregulated during iron deprivation, its Fe2+ uptake/storage mechanism must be efficient and crucial for its survival and pathogenesis. Therefore, the electrostatics at/along its pores are altered to unravel the Fe2+ uptake pathways. While the 4-fold and B-pores are involved in rapid Fe2+ uptake/oxidation, alteration of 3-fold pores abolished the self-assembly process, thereby exhibiting impaired ferroxidase activity. The current dissertation helps to understand iron-phosphate solution chemistry occurring inside the ferritin nanocage and unravels the Fe2+ entry pathways along with the significance of self-assembly phenomena in Mtb ferritins; these findings may provide a future platform to engineer ferritin cage as nanosink/nanoreactor and regulate Mtb pathogenesis.