Transferrins, the mechanism of iron release by ovotransferrin

Iron release from ovotransferrin in acidic media (3 < pH < 6) occurs in at least six kinetic steps. The first is a very fast (≤ 5 ms) decarbonation of the iron-loaded protein. Iron release from both sites of the protein is controlled by what appear to be slow proton transfers. The N-site loses its iron first in two steps, the first occurring in the tenth of a second range with second order rate constant k₁ = (2.30 ± 0.10) x 10⁴ M^-1·S^-1, first order rate constant k_-1 = (1.40 ± 0.10) s^-1 and equilibrium constant K(1a) = (60 ± 6) μM. The second step occurs in the second range with a second order rate constant k₂ = (5.2 ± 0.15) x 10³ M^-1·s^-1, first order rate constant k_-2 = (0.2 ± 0.02) s^-1 and equilibrium constant K(2a) = (39 ± 5) μM. Iron is afterward lost from the C-site of the protein by two different pathways, one in the presence of a strong Fe(III) ligand such as citrate and the other in the presence of weak ligands such as formate or acetate. The first step, common to both paths, is a slow proton uptake which occurs in the tens of second range with a second order rate constant k₃ = (1.22 ± 0.03) x 10³ M^-1·S^-1 and equilibrium constant K(3a) = (1.0 ± 0.1) mM. In the presence of citrate, this step is followed by formation of an intermediate complex with monoferric ovotransferrin; stability constant K(LC) = (0.435 ± 0.015) mM. This last step is rate- controlled by slow proton gain which occurs in the hundred second range with a second order rate constant k₄ = (1.05 ± 0.05) x 10⁴ M^-1·S^-1, first order rate constant k_-4 = (1.0 ± 0.1) x 10^-2 s^-1 and equilibrium constant K(4a) = (0.95 ± 0.15) μM. In the presence of a weak iron(III) ligand such as acetate or formate, formation of an intermediate complex is not detected and iron release is controlled by two final slow proton uptakes. The first occurs in the hundred to thousand second range, second order rate constant k₅ = (6.90 ± 0.30) x 10⁶ M^-1·S^-1. The last step occurs in the thousand second range. Iron release by ovotransferrin is similar but not identical to that of serum-transferrin. It is slower and occurs at lower pH values. However, as seen for serum-transferrin, it seems to involve the protonation of the amino acid side-chains involved in iron co-ordination and perhaps those implicated in interdomain H-bonds. The observed proton transfers are, then, probably controlled by the change in conformation of the binding lobes from closed when iron-loaded to open in the apo-form.