IRON RELEASE FROM RECOMBINANT N-LOBE AND SINGLE-POINT ASP(63) MUTANTSOF HUMAN TRANSFERRIN BY EDTA

Transferrins bind ferric ion and deliver the iron to cells. The mechan ism of the iron release has been studied kinetically, in vitro, with t he aid of single point mutants in which the iron-binding ligand, Asp(6 3) (aspartic acid-63, D63), has been changed to Ser, Asn, Glu and Ala. Iron release from the unmutated N-lobe of human serum transferrin (hT F/2N) by EDTA is influenced by a variety of factors. The rate-determin ing conformational-change mechanism may be a major pathway for iron re lease from hTF/2N's having a 'closed' conformation, which leads to a s aturation kinetic mode with respect to ligand concentration. The effec t of chloride depends on the protein conformation, showing a negative action in the case of tight binding and a positive action when the pro tein has an 'open' or 'loose' conformation. The negative effect of chl oride could originate from the binding competition between chloride an d the chelate to the active site for iron release, and the positive ef fect could derive from the synergistic participation of chloride in ir on removal. The 'open' conformation may be induced by decreasing pH: t he transitional point appears to be at about pH 6.3 for the wild-type hTF/2N; the 'loose' conformation may be facilitated by mutations at D6 3, which result in the loss of a key linking component in interdomain interactions of the protein. In the latter case, structural factors do minate over other potential negative effects because the weak interdom ain contacts derived from the mutation of D63 cause the binding site t o open easily, even at pH 7.4. Therefore chloride exhibits an accelera ting action on iron release by EDTA from all the D63 mutants.