Anion-mediated Iron Release from Transferrins: The Kinetic And Mechanistic Model For N-Lobe Of Ovotransferrin

Iron release process of ovotransferrin N-lobe (N-oTf)
to anion/chelators has been resolved using kinetic and
mechanistic approach. The iron release kinetics of N-oTf
were measured at the endosomal pH of 5.6 with three
different anions such as nitrilotriacetate, pyrophosphate,
and sulfate using stopped flow spectrofluorimetric
method, all yielding clear biphasic progress curves.
The two observed rate constants and the corresponding
amplitudes obtained from the double exponential curve
fit to the biphasic curves varied depending on the type
and concentration of anions. Several possible models for
the iron release kinetic mechanism were examined on
the basis of a newly introduced quantitative equation.
Results from the curve fitting analyses were consistent
with a dual pathway mechanism that includes the competitive
iron release from two different protein states,
namely, X and Y, with the respective first order rate
constants of K1 and K2 (X, domain closed holo N-oTf; Y,
anion induced different conformer of holo N-oTf). The
reversible interconversions of X to Y and Y to X are
driven by the second order rate constant k3 and the first
order rate constant K4, respectively. The obtained rate
constants were greatly variable for the three anions
depending on the synergistic or nonsynergistic nature.
In the light of the anion-binding sites of N-oTf located
crystallographically, the compatible mechanistic model
that includes competitive anion binding to the iron coordination sites and to a specific anion site is suggested for the dual pathway iron release mechanism.