A mechanism for iron uptake by transferrin

Abstract

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 FeNAc3 without the formation of an intermediate protein-iron-ligand mixed complex (iron-exchange-equilibrium constant, K1 = 1 ± 0.05; direct second-order-rate constant, k1 = 8.0 x 104 ± 0.5 x 104 M-1 s-1; reverse second-order-rate constant, k-1 = 7.5 x 104 ± 0.5 x 104 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 protons (first-order-rate constant, k2 = 2.80 ± 0.10 s-1). This induces a new modification in the conformation (first-order-rate constant, k3 = 6.2 x 10-2 ± 0.3 x 10-2 s-1). This second modification in conformation controls the rate of iron uptake by the N-site of the protein and is followed by loss of one proton (K(3a) = 6.80 nM). Finally, the holoprotein or the monoferric transferrin in its final equilibrated state is produced by a third modification in the conformation that occurs after approximately 3000 s. Iron uptake by the N-site does not occur 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 that of iron uptake by the C-site of apotransferrin. These modifications 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 receptor prior to iron delivery by endocytosis.