The mechanism of iron release from transferrin. Slow-proton-transfer-induced loss of nitrilotriacetatoiron(III) complex in acidic media.

Abstract

The role of protonation of amino acid ligands involved in iron release from human serum transferrin, previously saturated with nitrilotriacetatoiron(III) complex, has been elucidated in acidic media. Iron loss occurs first from the N-terminal site at pH < 6 and is followed at pH < 4 by iron release from the C-terminal iron-binding site. Nitrilotriacetatoiron(III) release from the N-terminal site is controlled by the slow protonation of the mixed protein/nitrilotriacetatoiron(III) complex; the second-order rate constant was k3a = 9.95 +/- 0.35 x 10(4) M-1.s-1. Protonation of an amino acid ligand in the C-terminal site leads to a new protein-site-C-loaded mixed complex with dissociation constant K4 = 0.300 +/- 0.025 mM. Nitrilotriacetatoiron(III) release is the result of mixed complex dissociation and the slow rate-limiting protonation of the iron-free protein with a proton dissociation constant K5a = 0.100 +/- 0.010 mM and a second-order rate constant k5a = 4.20 +/- 0.40 x 10(3) M-1.s-1. The mechanism of iron uptake and release seems to imply that slow proton transfers can induce complex formation between iron and the amino acid ligands of each of the protein iron-binding sites. These slow proton transfers may be controlled by the change of conformation of the binding sites upon iron loss.