Iron release from recombinant N-lobe and single point Asp63 mutants of human transferrin by EDTA.
Transferrins bind ferric ion and deliver the iron to cells. The mechanism of the iron release has been studied kinetically, in vitro, with the aid of single point mutants in which the iron-binding ligand, Asp63 (aspartic acid-63, D63), has been changed to Ser, Asn, Glu and Ala. Iron release from the unmutated N-lobe of human serum transferrin (hTF/2N) by EDTA is influenced by a variety of factors. The rate-determining conformational-change mechanism may be a major pathway for iron release from hTF/2N's having a 'closed' conformation, which leads to a saturation kinetic mode with respect to ligand concentration. The effect of chloride depends on the protein conformation, showing a negative action in the case of tight binding and a positive action when the protein has an 'open' or 'loose' conformation. The negative effect of chloride could originate from the binding competition between chloride and the chelate to the active site for iron release, and the positive effect could derive from the synergistic participation of chloride in iron removal. The 'open' conformation may be induced by decreasing pH: the 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 D63, which result in the loss of a key linking component in interdomain interactions of the protein. In the latter case, structural factors dominate over other potential negative effects because the weak interdomain contacts derived from the mutation of D63 cause the binding site to open easily, even at pH 7.4. Therefore chloride exhibits an accelerating action on iron release by EDTA from all the D63 mutants.
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Department of Biochemistry, College of Medicine, University of Vermont, Burlington, VT 05405, USA.
Mason
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Woodworth
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D001224:Aspartic Acid / Q000235:genetics*; D001667:Binding, Competitive; D004492:Edetic Acid / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D011189:Potassium Chloride / Q000494:pharmacology; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014168:Transferrin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D010446:Peptide Fragments; D011994:Recombinant Proteins; D014168:Transferrin; D001224:Aspartic Acid; D011189:Potassium Chloride; D004492:Edetic Acid; D007501:Iron