Anion-mediated Fe3+ Release Mechanism in Ovotransferrin C-lobe

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 SO4 2_ 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 SO4 2_ binding structure supported the single-pathway kinetic model.