EFFECTS OF MUTATIONS OF ASPARTIC-ACID-63 ON THE METAL-BINDING PROPERTIES OF THE RECOMBINANT N-LOBE OF HUMAN SERUM TRANSFERRIN

Mutations of the aspartic acid residue at position 63 of the N-lobe of human serum transferrin substantially alter the metal ion- and anion- binding properties of the protein. Substitution of serine, asparagine, glutamic acid, or alanine results in the loss of a key component of t he interface in the interdomain cleft and the metal-binding ligand, as partic acid, leading in all cases to an increased preference for NTA r ather than carbonate as the ''synergistic'' anion relative to the wild -type protein. Excess bicarbonate is required to eliminate the NTA and obtain the ''correct'' visible spectrum. Carbonate replaces NTA via a n intermediate. Blue shifts for the characteristic absorption band of each mutant show a range of effects on the Fe-O (Tyr) interaction. Tit ration with Co(III) yielded the molecular absorption coefficient for e ach mutant except D63A, where Co(III) appeared to oxidize the tyrosine residues and damage the ability of the mutant to bind metal. The chel ator, Tiron, removes iron from hTF/2N with a simple saturation kinetic mode with respect to the ligand concentration. Chloride inhibits the release in an interesting manner: the effect is initially sharp and th en levels off with a minimum k(obs) at [KCl] = 0.5 M. However, the rea ction of the D63 mutants with Tiron results in the formation of the te rnary complexes Fe-hTF/ 2N-Tiron. Significant red shifts for the chara cteristic absorption bands of these complexes suggest a different liga tion of Tiron in the mutants from that in wild-type hTF/2N.