CONSTRUCTION OF A FERROXIDASE CENTER IN HUMAN FERRITIN L-CHAIN

Ferritins are 24-mer proteins which store and detoxify intracellular i ron. Mammalian ferritins are made of two subunit types, the H- and L-c hains, with different functional specificity. The H-chain has a metal- binding site (the ferroxidase center) which confers ferroxidase activi ty to the protein and accelerates iron incorporation. In the L-chain t he center is substituted by a salt bridge. We performed several site d irected mutageneses in the L-chain with the aim to construct the cente r and confer ferroxidase activity to the protein. Most variants were i nsoluble and did not refold into homopolymers, probably due to electro static repulsion introduced by the substitutions. However, they formed hybrids when they were renatured together with the L- or H-chains. Th e 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 ferroxidas e activity of the H-chain homopolymer. This corresponds to the activit y of an H/L heteropolymer with 7% H-chain. It is concluded that: (i) i t 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 subunit s with large differences of refolding rates.