A MECHANISM FOR IRON UPTAKE BY TRANSFERRIN

Iron uptake by transferrin from iron nitrilotriacetate (FeNAc3) in the presence of bicarbonate has been investigated in the pH range 6.5-8. Apotransferrin, in interaction with bicarbonate, extracts iron from Fe NAc3 without the formation of an intermediate protein-iron-ligand mixe d complex (iron-exchange-equilibrium constant, K-1 = 1+/-0.05; direct second-order-rate constant, k(1) = 8.0x10(4)+/-0.5x10(4) M(-1) s(-1); reverse second-order-rate constant, k(-1) = 7.5x10(4)+/-0.5x10(4) M(-1 ) s(-1)). The newly formed iron-protein complex loses a single proton (proton-dissociation constant, K-a = 16+/-1.5 nM) and then undergoes a modification of its conformation followed by loss of two or three pro tons (first-order-rate constant, k(2) = 2.80+/-0.10 s(-1)). This induc es a new modification in the conformation (first-order-rate constant, k(3) = 6.2x10(-2)+/-0.3x10(-2) s(-1)). This second modification in con formation controls the rate of iron uptake by the N-site of the protei n and is followed by loss of one proton (K-3a = 6.80 nM). Finally, the holoprotein or the monoferric transferrin in its final equilibrated s tate is produced by a third modification in the conformation that occu rs after approximately 3000 s. Iron uptake by the N-site does not occu r when the apotransferrin interacts with bicarbonate. Nevertheless, it occurs with the monoferric transferrin, in which iron is bound to the C-site, in its final state of equilibrium by a mechanism similar to t hat of iron uptake by the C-site of apotransferrin. These modification s in the conformation of the protein occur after iron uptake by the C- site and may be important for the recognition of the protein by its re ceptor prior to iron delivery by endocytosis.