The objective of the proposed research is to learn how iron is loaded and stored with ferritin. Our hypothesis is that loading is accomplished by ceruloplasmin in a process that protects ferritin from oxidation by iron. We propose that there is a specific association between ceruloplasmin and the heavy chain of ferritin which stimulates the ferroxidse activity of ceruloplasmin to place iron into ferritin. We propose that iron is transported into the core of ferritin through a channel in the four a-helix bundle of heavy rain. we further that iron is then safely stored in ferritin by forming ferric oxhydroxides around nucleation sites which are glutamic acids, mostly in the light chain of ferritin. Finally, we propose that phosphate is added to make complete, stable, terminal ends of polyferrioxyhydroxide. Many of the studies will include site-directed mutagenesis of recombinant homoplymers of the heavy and light proteins of human ferritin. These proteins are very similar both with respect to amino acid homology and three-dimensional structure so very few amino acid changes need to be made to study the different properties of the heavy and light homopolymers. Amino acids proposed to be involved in property of one hompolymer an be changed by site-directed mutagenesis to test for the establishment of that property in the other homopolymer. Conversely, we can change amino acids proposed to be involved in a property which resides in one homopolymer into the corresponding amino acid in the homopolymer that does not have a property of interest to see if the property is abolished by the change. Thus we can obtain both positive (the addition of a property) and negative (the deletion of a property) results. We propose to characterize the association of ferritin with ceruloplasmin and the stimulation of the ferroxidase activity of ceruloplsmin, the nature of the iron loading channel in ferritin, and the nature, importance and role of iron neculeation sites in ferritin. Finally, we will study the effect of phosphate on the stability of the iron core in ferritin. Site-directed mutagenesis will be guided by computer modeling using the published sequences.
