Anion-mediated Fe3+ release mechanism in ovotransferrin C-lobe - A structurally identified SO42- binding site and its implications for the kinetic pathway

The differential properties of anion-mediated Fe2+ release between the N- a nd C-lobes of transferrins have been a focus in transferrin biochemistry. T he structural and kinetic characteristics for isolated lobe have, however, been documented with the N-lobe only. Here we demonstrate for the first tim e the quantitative Fe3+ release kinetics and the anion-binding structure fo r the isolated C-lobe of ovotransferrin. In the presence of pyrophosphate, sulfate, and nitrilotriacetate anions, the C-lobe released Fe3+ with a dece lerated rate in a single exponential progress curve, and the observed first order rate constants displayed a hyperbolic profile as a function of the a nion 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 F e3+. The apo C-lobe was crystallized in ammonium sulfate solution, and the structure determined at 2.3 A resolution demonstrated the existence of a si ngle bound SO42- in the interdomain cleft, which interacts directly with Th r(461) -OG1, Tyr(431)-OH, and HiS(592)-NE2 and indirectly with Tyr(524)-OH. The latter three groups are Fe3+-coordinating ligands, strongly suggesting the facilitated Fe3+ release upon the anion occupation at this site. The S O42- binding structure supported the single-pathway kinetic model.