Segregation of The Relative Contribution of Enzymatic Sites and Self-Assembly Towards Rapid Ferroxidase Activity and Bio-Mineralization

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 nano compartment and pores/channels for Fe 2 uptake and to develop a concentration gradient across the protein shell, which is proposed to fuel rapid ferroxidase activity However, it is difficult to segregate the relative contribution of the catalytic sites and nanocompartment towards rapid ferroxidase/mineralization activity owing to ferritin’s inherent self assembly propensity In this work, 3 fold pore electrostatics of bacterioferritin was rationally altered by site directed mutagenesis to generate self assembled and assembly defective variants In comparison to autoxidation of Fe 2 in buffer, the assembly defective variants exhibited faster ferroxidase/mineralization activity 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 Fenton reaction The findings highlight the importance of electrostatic guiding and nanoconfinement (increased effective substrate concentration) offered by ferritin self assembly towards its enzymatic 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 influences ferritin’s natural functions.