Effect of Ligand Structure on the Pathways for Iron Release from Human Serum Transferrin

Abstract

Rate constants for the removal of iron from N-terminal monoferric transferrin have been measured for a series of phosphate and phosphonocarboxylic acids in pH 7.4 0.1 M hepes buffer at 25 degrees C. The bidentate ligands pyrophosphate and phosphonoacetic acid (PAA) show a combination of saturation and first-order kinetics with respect to the ligand concentration. Similar results are observed following a single substitution at the 2-position of PAA to give 2-benzyl-PAA and phosphonosuccinic acid. In contrast, disubstitution at the 2-position to form 2,2-dibenzyl-PAA leads to a marked reduction in iron removal via the first-order pathway. Rate constants were also measured for tripolyphosphate and phosphonodiacetic acid, which are elongated versions of PP(i) and PAA. In both cases, this elongation completely eliminates the first-order component for iron release while having relatively little impact on the saturation pathway. The sensitivity of the first-order component to the structure of the ligand strongly indicates that this pathway involves the binding of the ligand to a specific site on the protein and cannot be attributed to changes in the overall ionic strength of the solution as the ligand concentration increases. It is proposed that this structural sensitivity reflects steric restrictions on the ability of the incoming ligand to substitute for the synergistic carbonate anion to form a relatively unstable Fe-ligand-Tf ternary intermediate, which then dissociates to FeL and apoTf.

Pyrophosphate-Mediated Iron Acquisition from Transferrin in Neisseria meningitidis Does Not Require TonB Activity

“…According to these results, pyrophosphate can bind iron loaded on transferrin and deliver it to ferritin [26] . Other authors demonstrated transfer from iron-loaded transferrin to pyrophosphate [37] , [38] . The results described in these reports prompted us to check for the effect of pyrophosphate on the use of iron-loaded transferrin as an iron source.…”

Allosteric effects of sulfonate anions on the rates of iron release from serum transferrin

“…These kinetic effects do not correlate well with simple changes in the ionic strength of the solutions [35]. Nor do they correlate well with the Hoffmeister series [53,54], which describes general anion affects on protein structure.…”

“…We have proposed that the first-order term represents a separate reaction pathway for iron removal that involves the replacement of the synergistic carbonate by the incoming ligand and is not subject to a rate-limiting protein conformational change [32,[35][36][37][38]. The disulfonates show very distinct effects on the saturation and linear components of iron release.…”

“…Several ligands, particularly pyrophosphate [31][32][33][34][35] and phosphonic acids [23,[35][36][37][38], appear to follow saturation kinetics at low concentrations, but at higher ligand concentrations, the rate of iron release does not level off. Instead, it continues to increase linearly with the ligand concentration.…”

“…(6) [32,33,35]. This laboratory has proposed that k′ is the second order rate constant for a reaction pathway that somehow avoids the conformational gating and removes iron from the closed conformation of the holoprotein [35].…”