Mutational analysis of the four alpha-helix bundle iron-loading channel of rat liver ferritin.
We previously reported that the heavy chain of ferritin was required for loading it with iron using ceruloplasmin as a ferroxidase [J.-H. Guo, M. Abedi, and S. D. Aust (1996) Arch. Biochem. Biophys. 335, 197-204]. Site-directed mutagenesis, K58E and G61H, on recombinant rat liver L chain ferritin (rL-Ft) was performed to construct a proposed iron-loading channel in the alpha-helix bundle similar to rat liver H chain ferritin (rH-Ft). Conversely, the channel in rH-Ft was closed by mutations E62K and H65G to form a K62 to E107 salt bridge, which is believed to exist in the L chain. Both variants were expressed in insect cells and were soluble and able to form multi-subunit homopolymers. The rH-Ft mutant homopolymer could not be loaded, whereas the rL-Ft mutant homopolymer could be loaded with iron by ceruloplasmin. However, we found that the initial rate of iron loading into the rL-Ft mutant homopolymer by ceruloplasmin was less than that into the rH-Ft homopolymer. When 500 atoms of iron per ferritin were used for loading, 98% was loaded 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. Moreover, the ferroxidase activity of ceruloplasmin was enhanced in the presence of the rH-Ft and the rH-Ft mutant homopolymers, but not in the presence of the rL-Ft or the rL-Ft mutant homopolymers. These observations suggested that the four alpha-helix bundle channel of ferritin is required for iron loading, but an additional factor, i.e. , a site which stimulate the ferroxidase activity of ceruloplasmin, is also essential.
Guo
J H
JH
Biotechnology Center, Utah State University, Logan, Utah 84322-4705, USA.
Juan
S H
SH
Aust
S D
SD
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D002570:Ceruloplasmin / Q000378:metabolism; D004247:DNA / Q000235:genetics; D005293:Ferritins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007501:Iron / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera
D011994:Recombinant Proteins; D004247:DNA; D005293:Ferritins; D007501:Iron; D002570:Ceruloplasmin