MUTATIONAL ANALYSIS OF THE 4 ALPHA-HELIX BUNDLE IRON-LOADING CHANNEL OF RAT-LIVER FERRITIN

We previously reported that the heavy chain of ferritin was required f or loading it with iron using ceruloplasmin as a ferroxidase [J.-H. Gu o, M. Abedi, and S. D. Aust (1996) Arch. Biochem. Biophys. 335, 197-20 4], Site-directed mutagenesis, K58E and G61H, on recombinant rat liver L chain ferritin (rL-Ft) was performed to construct a proposed iron-l oading channel in the alpha-helix bundle similar to rat liver H chain ferritin (rH-Ft), Conversely, the channel in rH-Ft was closed by mutat ions E62K and H65G to form a K62 to E107 salt bridge, which is believe d to exist in the L chain. Both variants were expressed in insect cell s and were soluble and able to form multi-subunit homopolymers. The rH -Ft mutant homopolymer could not be loaded, whereas the rL-Ft mutant h omopolymer could be loaded with iron by ceruloplasmin. However, we fou nd that the initial rate of iron loading into the rL-Ft mutant homopol ymer by ceruloplasmin was less than that into the rH-Ft homopolymer. W hen 500 atoms of iron per ferritin were used for loading, 98% was load ed into the rH-Ft homopolymer by ceruloplasmin in 15 min, but only 30% was loaded into the rL-Ft mutant homopolymer in the same time. Moreov er, the ferroxidase activity of ceruloplasmin was enhanced in the pres ence of the rH-Ft and the rH-Ft mutant homopolymers, but not in the pr esence of the rL-Ft or the rL-Ft mutant homopolymers. These observatio ns suggested that the four alpha-helix bundle channel of ferritin is r equired for iron loading, but an additional factor, i.e., a site which stimulate the ferroxidase activity of ceruloplasmin, is also essentia l. (C) 1998 Academic Press.