Construction of a ferroxidase center in human ferritin L-chain.
Ferritins are 24-mer proteins which store and detoxify intracellular iron. Mammalian ferritins are made of two subunit types, the H- and L-chains, with different functional specificity. The H-chain has a metal-binding site (the ferroxidase center) which confers ferroxidase activity to the protein and accelerates iron incorporation. In the L-chain the center is substituted by a salt bridge. We performed several site-directed mutageneses in the L-chain with the aim to construct the center and confer ferroxidase activity to the protein. Most variants were insoluble and did not refold into homopolymers, probably due to electrostatic repulsion introduced by the substitutions. However, they formed hybrids when they were renatured together with the L- or H-chains. The heteropolymers made of 90% L-chain and 10% of an L-variant with all the ligand residues of the H-chain center had 25-30% of the ferroxidase activity of the H-chain homopolymer. This corresponds to the activity of an H/L heteropolymer with 7% H-chain. It is concluded that: (i) it is possible to construct a ferroxidase center in the L-chain with an activity equivalent to that of the H-chain, (ii) the residues of the center interfere with the folding/assembly of the L-, but not of the H-chain, (iii) heteropolymers can be made even between ferritin subunits with large differences of refolding rates.
Levi
S
S
Department of Biological and Technological Research, San Raffaele Scientific Institute, Milano, Italy.
Corsi
B
B
Rovida
E
E
Cozzi
A
A
Santambrogio
P
P
Albertini
A
A
Arosio
P
P
D000595:Amino Acid Sequence; D002570:Ceruloplasmin / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D005293:Ferritins / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D019791:Guanidine; D006146:Guanidines; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry
D006146:Guanidines; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D005293:Ferritins; D002570:Ceruloplasmin; D019791:Guanidine