Impact of Phosphate on Iron Mineralization and Mobilization in Nonheme Bacterioferritin B from <i>Mycobacterium tuberculosis</i>

Ferritins are supramolecular nanocage proteins, which synthesize hydrated ferric oxyhydroxide mineral via protein mediated rapid Fe 2+ sequestration and ferroxidase reactions. Ferritin minerals are also associated with a significant amount of phosphate, which contribute toward their structure and reactivity. Like iron, phosphate also regulates the pathogenesis of Mycobacterium tuberculosis ( Mtb ), which expresses two types of ferritin: heme binding bacterioferritin A (BfrA) and nonheme binding bacterioferritin B (BfrB). Unlike Mtb BfrA, the rapid kinetics and mechanism of ferroxidase activity, and the mineral core formation/dissolution in Mtb BfrB are not well explored. Moreover, the effect of physiological levels of phosphate (0-10 mM) on the synthesis, structure, and reactivity of ferritin mineral core also require investigation in detail. Therefore, the stopped-flow rapid kinetics of ferroxidase activity (Δ A 650 /Δ t ) of Mtb BfrB was carried out, which detected a transient intermediate similar to diferric peroxo species as observed in frog and human ferritins. Increasing phosphate concentration increased the initial rate of iron mineralization (Δ A 350 /Δ t ) and dissolved O 2 consumption (both ∼1.5-2-fold). Phosphate not only decreased the amount of iron loading and size of the BfrB mineral core (both up to 2-fold) but also decreased its crystallinity, resembling the variations observed in the core morphology of different native ferritins. In addition, phosphate inhibited the kinetics of reductive iron mobilization (∼6-8-fold) indicating its influence on the stability of the iron mineral core. Hence, the current work provides the kinetic/mechanistic insight toward the ferroxidase activity in Mtb BfrB, apart from demonstrating the role of phosphate toward the structure/reactivity of its iron mineral.