Mechanism of ferritin iron uptake: activity of the H-chain and deletion mapping of the ferro-oxidase site. A study of iron uptake and ferro-oxidase activity of human liver, recombinant H-chain ferritins, and of two H-chain deletion mutants.
To study the functional differences between human ferritin H- and L-chains and the role of the protein shell in the formation and growth of the ferritin iron core, we have compared the kinetics of iron oxidation and uptake of ferritin purified from human liver (90% L) and of the H-chain homopolymer overproduced in Escherichia coli (100% H). As a control for iron autocatalytic activity, we analyzed the effect of Fe(III) on the iron uptake reaction. The results show that the H-chain homopolymer has faster rates of iron uptake and iron oxidation than liver ferritin in all the conditions analyzed and that the difference is reduced in the conditions in which iron autocatalysis in high: i.e. at pH 7 and in presence of iron core. We have also analyzed the properties of two engineered H-chains, one lacking the last 22 amino acids at the carboxyl terminus and the other missing the first 13 residues at the amino terminus. These mutant proteins assemble in ferritin-like proteins and maintain the ability to catalyze iron oxidation. The deletion at the carboxyl terminus, however, prevents the formation of a stable iron core. It is concluded that the ferritin H-chain has an iron oxidation site which is separated from the sites of iron transfer and hydrolysis and that either the integrity of the molecule or the presence of the amino acid sequences forming the hydrophobic channel is necessary for iron core formation.
Levi
S
S
Department of Biomedical Sciences and Technology, University of Milano, Italy.
Luzzago
A
A
Cesareni
G
G
Cozzi
A
A
Franceschinelli
F
F
Albertini
A
A
Arosio
P
P
D002872:Chromosome Deletion; D005293:Ferritins / Q000235:genetics / Q000378:metabolism*; D005796:Genes; D006801:Humans; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D009154:Mutation; D010088:Oxidoreductases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D005293:Ferritins; D007501:Iron; D010088:Oxidoreductases