Transferrins: Iron release from lactoferrin

Iron loss in vitro by the iron scavenger bovine lactoferrin was investigated in acidic media in the presence of three different monoanions (NO₃/^-, Cl^- and Br^-) and one dianion (SO₄/^2-). Holo and monoferric C-site lactoferrins lose iron in acidic media (pH ≤ 3.5) by a four-step mechanism. The first two steps describe modifications in the conformation affecting the whole protein, which occur also with apolactoferrin. These two processes are independent of iron load and are followed by a third step consisting of the gain of two protons. This third step is kinetically controlled by the interaction with two Cl^-, Br^- and NO₃/^- or one SO₄/^2-. In the fourth step, iron loss is under the kinetic control of a slow gain of two protons; third-order rate-constants k₂, 4.3(±0.2) x 10³, 3.4(±0.5) x 10³, 3.3(±0.5) x 10³ and 1.5(±0.5) x 10³ M^-2 s^-1 when the protein is in interaction with SO₄/^2-, NO₃/^-, Cl^- or Br^-, respectively. This step is accompanied by the loss of the interaction with the anions; equilibrium constant K₂, 20 ± 5 mM, 1.0(±0.2) x 10^-1, 1.5(±0.5) x 10^-1 and 1.0(±0.3) x 10^-1 M², for SO₄/^-, NO₃/^-, Cl^- and Br^-, respectively. This mechanism is very different from that determined in mildly acidic media at low ionic strength (μ < 0.5) for the iron transport proteins, serum transferrin and ovotransferrin, with which no prior change in conformation or interaction with anions is required. These differences may result from the fact that in the transport proteins, the interdomain hydrogen bonds that consolidate the closed conformation of the iron-binding cleft occur between amino acid side-chain residues that can protonate in mildly acidic media. With bovine lactoferrin, most of the interdomain hydrogen bonds involved in the C-site and one of those involved in the N-site occur between amino acid side-chain residues that cannot protonate. The breaking of the interdomain H-bond upon protonation can trigger the opening of the iron cleft, facilitating iron loss in serum transferrin and ovotransferrin. This situation is, however, different in lactoferrin, where iron loss requires a prior change in conformation. This can explain why lactoferrin does not lose its iron load in acidic media and why it is not involved in iron transport in acidic endosomes. (C) 2000 Academic Press.