A Mechanism for Iron Uptake by Transferrin

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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 × 104± 0.5 × 104 M−1 s−1; reverse second‐order‐rate constant, k−1, = 7.5 × 104± 0.5 × 104 M−1 s−1. The newly formed iron‐protein complex loses a single proton (proton‐dissociation constant, Ka= 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 × 10−2± 0.3 × 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 (K3a, = 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.