Rapid Ferroxidase Activity and Iron Mobilization in Ferritin: Impact of Intrinsic Electron Relay Stations

Abstract

Despite being an indispensable co-factor for myriad essential life functions, excess iron is toxic. To maintain the balance between its essentiality and toxicity, nature has devised a spherical nanocaged protein - ‘ferritin’ - that can store up to ~4500 iron atoms, reversibly, in the form of hydrated ferric oxyhydroxide mineral and facilitate controlled iron release to support physiological processes. The ferroxidase/mineralization activity of ferritin and its mineral dissolution involves a complex interplay of redox reactions, possibly through long range electron transfer (ET), in multiple steps, via various electron relay stations (i.e., heme and intrinsic redox active amino acids of protein cage). Till date the mechanism and ET pathways in ferritin are not well-defined. Therefore, to better understand these ET pathways, we attempt to reveal the critical amino acids associated with its rapid ferroxidase and iron-mineralization activity by site directed mutagenesis (SDM) and stopped flow kinetics. Ferritin mutants with rational substitutions of its conserved/semi-conserved redox active amino acids have been successfully designed to study their impact on the iron oxidation/mineralization ability. All the synthesized ferritin variants were seen to retain their self-assembled form and iron-loading ability, but a drastic difference was seen in the rapid iron oxidation kinetic profiles of certain variants which indicates the possible role of these specific redox active amino acid residues in ET pathways. These findings not only helped to reveal critical amino acids residues and to understand the underlying mechanisms of ferritin iron mineralization but may also help in understanding the ET pathways associated with several other biological processes