The positions and components of the iron nucleation site in H-ferritin

Ferritin (Ft) is the iron storage protein of the cell. Ft is heterooligomeric consisting of nonstoichiometric ratios of two homologous chains: L (liver) and H (heart). Despite their structure and sequences similarity (55% identical) the L and H-Ft subunits fulfill distinct functions. H-Ft has ferroxidase activity via a di-nuclear iron-binding site (E62, H65), oxidating Fe+2 into Fe+3. L-Ft has no ferroxidase activity and is thought to provide the nucleation site for iron sedimentation. Consequently, it is assumed that the initial formation of iron sediments once iron is recruited to the oligomer involves several steps: oxidation by H-Ft followed by migration to the nucleation site in L-Ft and sedimentation at this site. However, in-vitro, not only L-Ft but also H-Ft can nucleate iron sedimentation. Only the nucleation site of L-Ft (E60, E61, E64) has been determined by structural and mutational studies. Consequently, the location of the nucleation site on H-Ft, and the effects of having the ferroxidase center located on the same chain as the nucleation site is unclear. The goal of this project is to determine the position and morphology of nascent iron sediments in H-Ft. The proteins will be recombinantly expressed and purified in Escherichia coli BL21. Purification using heat treatment, followed by FPLC steps. Each of the purified proteins will be incubated in several concentrations of iron to allow the formation of sediments. Compare H-Ft and L-Ft iron sediments to see if ferroxidase center on the same chain affects nascent iron sediment formation.