Abstract
The self-assembled ferritin protein nanocage plays a pivotal role during oxidative stress, iron metabolism, and host-pathogen interaction by executing rapid iron uptake, oxidation and its safe-storage. Self-assembly creates a nanocompartment and various pores/channels for the uptake of charged substrates (Fe(2+)) and develops a concentration gradient across the protein shell. This phenomenon fuels rapid ferroxidase activity by an upsurge in the substrate concentration at the catalytic sites. However, it is difficult to segregate the relative contributions of the catalytic sites and self-assembly towards rapid ferroxidase/mineralization activity owing to the inherent self-assembly propensity of ferritins. In the current work, 3-fold pore electrostatics of bacterioferritin from Mycobacterium tuberculosis were rationally altered by site-directed mutagenesis to generate self-assembled (E121A and E121Q) and assembly-defective (E121K and E121F) variants. In comparison to the autoxidation of Fe(2+) in buffer, the assembly-defective variants exhibited significantly faster ferroxidase/mineralization activity and O(2) consumption kinetics due to their functional catalytic sites, but failed to level-up with the self-assembled variants even at 100-fold higher Fe(2+) concentration. Only the self-assembled variants exhibited cooperativity in iron oxidation, maintained biomineral solubility, and protected DNA against the Fenton reaction. This report highlights the concerted effect of self-assembly and ferroxidase sites that propels the rapid Fe(2+) uptake, its oxidation and biomineralization in bacterioferritin. The findings also establish the importance of electrostatic guiding and nanoconfinement offered by ferritin self-assembly towards its enzymatic activity and antioxidative properties. Moreover, this work identifies the key electrostatic interactions ("hot-spots") at the subunit contact points that control the cage/pore formation, impart cage stability and influence ferritin's natural functions. Manipulation of hot-spot residues can be further extended towards the encapsulation of cargo, for various bio-medical applications, by strategically inducing its disassembly and subsequent reassembly through adjustments in ionic strength. This would bypass the need for extreme/harsh reaction conditions and minimize the loss of cargo/protein.