Anion-mediated Iron Release from Transferrins

Abstract

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 K 1 and K 2 (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 k 3 and the first order rate constant K 4 , 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.

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

“…The present study of C-oTf along with the previous N-lobe studies (39,43) demonstrate that the difference in the anion-binding structures can be well correlated with the differential kinetic pathways of anion-induced Fe 3ϩ release in the two lobes. The absence in C-oTf of SO 4 2Ϫ binding to the synergistic anion-binding groups Arg 460 -NE and -NH 2 and to Thr 461 -N can be closely related to the decelerated Fe 3ϩ release in the C-lobe.…”

“…When the second order rate constants for the rate-limiting, domain-opening reactions (the constants k 1 and k 3 for the C-and N-lobes, respectively) are compared for the two lobes, however, the value is 22-fold less in case of pyrophosphate for the C-lobe than for the N-lobe (Table I and Ref. 43). The apparent decelerated rate of Fe 3ϩ release from the C-lobe can be related, at least in part, to the decreased rate constant for the domain opening.…”

“…The fast kinetics was measured using fluorescence mode of SX.18MV stopped flow reaction analyzer (Applied Photophysics Ltd.) as described in detail previously (43). Measurement of complete iron release at pH 5.6 using different concentrations of anions were, however, unsuccessful within the maximum time limit of the instrument (1000 s) because of the slow rate iron release from C-oTf.…”

“…Although none of the anions yielded complete iron removal in the time span of 1000 s limited to the stopped flow equipment, the data points, including the ones at very early time after the stopped flow mixing, were best fitted to the single exponential of Equation 1. This was quite different from the Fe 3ϩ release kinetics of the isolated N-lobe of ovotransferrin, which displays clear biphasic progress curves under the same conditions (43). Monophasic progress curves have been also observed with the C-lobe monoferric human serum transferrin (47).…”

The Position of Arginine 124 Controls the Rate of Iron Release from the N-lobe of Human Serum Transferrin

“…This synergy is a unique characteristic of the transferrin family and is not observed in other metal-binding proteins. Although hTF, oTF, and LTF share identical iron-binding ligands and display high sequence homology, substantial differences in the binding affinity for iron both within and between the TFs are found and are not well understood (5)(6)(7)(8)(9)). An obvious experimental approach to explore these differences involves the mutation of specific residues followed by assessment of the changes in function.…”