THE EFFECT OF PUTATIVE NUCLEATION SITES ON THE LOADING AND STABILITY OF IRON IN FERRITIN

The L chain of the iron storage protein ferritin contains more putativ e nucleation sites in the core (Glu53, 56, 57, 60, and 63) than does t he H chain (Glu61, 64, and 67). Recombinant DNA techniques were used t o investigate the role of these putative nucleation sites on iron load ing by ceruloplasmin and on the stability of the iron core. Recombinan t rat liver ferritin H chain homopolymer and the two mutants (E61A and EB1A-E64A), containing three, two and one nucleation sites, respectiv ely, loaded up to 2010 +/- 50, 2010 +/- 40, and 1950 +/- 40 atoms of i ron per ferritin, respectively. However, the mutations resulted in a 5 0% decrease in the rate of iron loading by ceruloplasmin. The ferritin variants incorporated the same amount of phosphate after iron loading (410 +/- 20, 400 +/- 30, and 420 +/- 20 atoms per ferritin, respectiv ely). The stability of the iron cores prior to phosphate incorporation , assessed by the rate of iron release by 10 mM EDTA and the paraquat cation radical, corresponded to numbers of proposed nucleation sites. The subsequent incorporation of phosphate seemed to stabilize the iron core and minimized the effect of numbers of putative nucleation sites in ferritin on the rate of iron release by EDTA and the paraquat cati on radical. After incorporation of phosphate the ferritins behaved sim ilarly to the native rat liver ferritin with respect to the rate of ir on release by the paraquat cation radical. (C) 1998 Academic Press.