| Ferritin is a ubiquitous iron-storage protein that is comprised of 24 H and L subunits in various ratios. The physiological mechanisms for iron incorporation into ferritin as well as iron release from ferritin remain uncertain.;Recombinant human ferritin heteromers and homomers, as well as various variants, were produced in E. coli. Iron was loaded into these ferritins using their own ferroxidase activity and the oxidative consequences of loading were determined. The ferroxidase center in the alpha-helix bundle of the H subunit was inactivated, most likely by oxidation of histidine 65, in proportion to the amount of iron loaded.;Purified recombinant ferritin heteromers were separated according to subunit content, resulting in ferritins with a range of H to L subunit ratios, in order to determine the channel of iron release. The number of alpha-helix bundle channels is H-subunit dependent while the number of hydrophilic channels is subunit independent. Ferritins loaded with iron were assayed for their ability to release iron using different reductants. The kinetic data demonstrated that iron release occurred through both the alpha-helix bundle channels and the hydrophilic channels.;A ferritin variant, with the alpha-helix bundle channel opened in the L-subunit, was also prepared. The data obtained from studies of the ferritin variant supported the conclusion that iron can be released through the alpha-helix bundle channel. In addition the proposed redox center (tryptophan residue at position 93) was not found to affect iron release from ferritin.;A bacterial two-hybrid system was used to search cDNA libraries for proteins that might interact with ferritin, most importantly for either a ferroxidase to load iron, or a reductase to release iron from ferritin. Several proteins were identified by the two-hybrid system, as candidates to interact with ferritin in vivo including NADH dehydrogenase subunits, cytochrome oxidase, complement proteins, and lipoprotein. Additionally, when the H and L ferritin subunits were used as bait to screen both a liver and brain cDNA library, the H and L ferritin subunits in the library were found at nearly the same ratio as contained in ferritin purified from liver and brain tissues, respectively. |
