EVIDENCE THAT THE SPECIFICITY OF IRON INCORPORATION INTO HOMOPOLYMERSOF HUMAN FERRITIN L-CHAIN AND H-CHAIN IS CONFERRED BY THE NUCLEATION AND FERROXIDASE CENTERS

Mammalian ferritins are iron-storage proteins made of 24 subunits of t wo types: the H- and L-chains. L-chains, in contrast with H-chains, la ck detectable ferroxidase activity. When ferritins were subjected to i ron loading in vitro with increments near the saturation limit of 4000 Fe atoms per molecule, the homopolymers of human H-chains formed inso luble aggregates, caused by non-specific iron hydrolysis, whereas the homopolymers of L-chains remained soluble and incorporated most of the available iron. To analyse the molecular reasons for the difference, Glu-57 and Glu-60, which are conserved and exposed on the cavity of L- chains, were substituted with His, as in H-chains. The double substitu tion made the L-homopolymers as sensitive as the H-homopolymers to the iron-induced aggregation, whereas the opposite substitution in the H- chain increased homopolymer resistance to the aggregation only margina lly. Millimolar concentrations of citrate and phosphate increased iron incorporation in H-homopolymers by reducing non-specific iron hydroly sis, but inhibited that in L-homopolymers by sequestering available ir on. The data indicate that the specific iron incorporation into L-homo polymers is mainly due to the iron-nucleation capacity of Glu-57, Glu- 60 and other carboxyl groups exposed on the cavity; in contrast, the s pecificity of iron incorporation into H-homopolymers is related to its ferroxidase activity, which determines rapid Fe(III) accumulation ins ide the cavity. The finding that ferroxidase centres are essential for the incorporation of iron in the presence of likely candidates of cel lular iron transport, such as phosphate and citrate, confirms their im portance in ferritin function in vivo.