The differential properties of anion-mediated Fe₃⁺ release between the N- and C-lobes of transferrins have been a focus in transferrin biochemistry. The structural and kinetic characteristics for isolated lobe have, however, been documented with the N-lobe only. Here we demonstrate for the first time the quantitative Fe₃⁺ release kinetics and the anion-binding structure for the isolated C-lobe of ovotransferrin. In the presence of pyrophosphate, sulfate, and nitrilotriacetate anions, the C-lobe released Fe₃⁺ with a decelerated rate in a single exponential progress curve, and the observed first order rate constants displayed a hyperbolic profile as a function of the anion concentration. The profile was consistent with a newly derived single-pathway Fe₃⁺ release model in which the holo form is converted depending on the anion concentration into a "mixed ligand" intermediate that releases Fe₃⁺. The apo C-lobe was crystallized in ammonium sulfate solution, and the structure determined at 2.3 Å resolution demonstrated the existence of a single bound SO₄²⁻ in the interdomain cleft, which interacts directly with Thr⁴⁶¹-OG1, Tyr⁴³¹-OH, and His⁵⁹²-NE2 and indirectly with Tyr⁵²⁴-OH. The latter three groups are Fe₃⁺-coordinating ligands, strongly suggesting the facilitated Fe₃⁺ release upon the anion occupation at this site. The SO₄²⁻ binding structure supported the single-pathway kinetic model.
Mizutani et al. (Fri,) studied this question.