Rational pore engineering reveals the relative contribution of enzymatic sites and self-assembly towards rapid ferroxidase activity and mineralization: Impact of electrostatic guiding and cage-confinement in bacterioferritin

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 (Fe2+) and develops a concentration gradient across the protein shell. This phenomenon fuels the rapid ferroxidase activity by an upsurge in the substrate concentration at the catalytic sites. However, it is difficult to segregate the relative contribution 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 was rationally altered by site-directed mutagenesis to generate self-assembled (E121A, E121Q) and assembly-defective (E121K, E121F) variants. In comparison to autoxidation of Fe2+ in buffer, the assembly-defective variants exhibited a significantly faster ferroxidase/mineralization activity and O2 consumption kinetics due to their functional catalytic sites, but failed to level-up with the self-assembled variants even at 100-fold higher Fe2+ concentration. Only the self-assembled variants exhibited cooperativity in iron oxidation, maintained biomineral solubility, and protected DNA against Fenton reaction. This report highlights the concerted effect of self-assembly and ferroxidase sites that propels the rapid Fe2+ 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 property. 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 influences ferritin’s natural functions. Manipulation of hot-spot residues can be further extended towards encapsulation of cargo, for various bio-medical applications, by strategically inducing its disassembly and subsequent reassembly by adjustments in the ionic strength. This would bypass the need for extreme/harsh reaction conditions and minimize the loss of cargo/protein.