The kinetics of iron removal from human serum transferrin by diphosphonates and phosphonocarboxylate ligands

Serum transferrin is the iron transport protein in higher organisms. It folds into two distinct lobes (designated N-terminal and C-terminal) and binds a single Fe3+ ion in each lobe. The mechanism for iron release from transferrin has not been conclusively determined, and more than one mode for iron release is proposed. This study has focused on iron release from transferrin by a series of phosphonocarboxylic acids at pH 7.4. A series of monosubstituted phosphonoacetic acid derivatives all show similar rates of iron release, but disubstitution at the alpha-carbon of phosphonoacetic acid significantly reduced the iron release rate. Iron release shows two kinetic pathways. Some ligands show simple saturation kinetics, whereas others show combination kinetics with both a saturable and a 1st order component. It is proposed that the 1st order pathway reflects the ability of ligands to replace the carbonate synergistic anion within the closed form of transferrin. The proposed Fe-L-Tf intermediate has been prepared under carbonate-free conditions with benzylphosphonoacetic acid, ethylphosphonoacetic acid and 3-phosphonopropionic acid.; Two transferrin mutants, R124E and T120D, were produced in an attempt to attach an endogenous carbonate to transferrin. Although R124E was unable to bind iron even in the presence of carbonate, stable Fe-L-Tf ternary complexes were formed with a series of alternative synergistic anions. Some ligands (e.g. glycine and oxalate) form ternary complexes with both wild type transferrin and the R124E and T120D mutants. These anions bind to the iron as 1,2-bidentate ligands to form a stable 5-membered chelate ring. Several other anions that were nonsynergistic anions with wild type transferrin acted as synergistic anions with the mutants. It is proposed that these ligands can function as synergistic anions by coordinating to iron via a carboxylate group and using other functional groups to form hydrogen bonds and/or salt bridges to the protein.