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

Abstract

The differential properties of anion-mediated Fe 3؉ 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 Fe 3؉ 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 Fe 3؉ 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 Fe 3؉ release model in which the holo form is converted depending on the anion concentration into a "mixed ligand" intermediate that releases Fe 3؉ . 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 SO

Ovotransferrin: Structure, bioactivities, and preparation

“…It was recently reported that the protonation of iron-binding residues of ovotransferrin at acidic pH in the absence of anionic chelators is slow (Abdallah & Chahine, 1999). Studies suggested the release of iron ion from ferric transferrin requires the presence of a simple anion such as pyrophosphate, sulfate, or chloride; however, the rate of anion-mediated iron release is much slower for the C-lobe than for the N-lobe (Mizutani, Muralidhara, et al, 2001;Muralidhara & Hirose, 2000), as there are two anion binding sites in the N-lobe but only one anion-binding site in the C-lobe. Anion binding to the lobe is recognized as the key to iron ion release through multiple mechanisms.…”

Release of iron from transferrin by phosphonocarboxylate and diphosphonate chelating agents

“…The ligand-dependence of k obs was evaluated in terms of a mechanism similar to that in Scheme 4 except that there was no K 1 pathway [59]. We also attempted to fit our data on MDP to this mechanism by including an explicit ligand term in the various reaction steps, but no satisfactory fit was obtained for the combination of saturation and first-order kinetics observed with this ligand.…”

“…Iron release from the C-terminal site of ovotransferrin at pH 5.6 showed simple saturation kinetics with respect to the concentrations of the ligands NTA, PP i , and sulfate [59]. The ligand-dependence of k obs was evaluated in terms of a mechanism similar to that in Scheme 4 except that there was no K 1 pathway [59].…”

Structure of diferric hen serum transferrin at 2.8 Å resolution

“…rabbit and porcine, is only 51%. All cellbinding studies (Mason et al, 1987(Mason et al, , 1996Mason & Woodworth, 1984) have utilized ovotransferrin, with the assumption that it is identical to its serum counterpart, but an in vivo irontransport function characteristic of serum transferrin has not been demonstrated for hOT (Mizutani, Muralidhara et al, 2001). The fact that ovotransferrin does not have an irontransport role may, however, be a consequence of its location in egg white.…”