A kinetically active site in the C-lobe of human transferrin.

Release of iron from transferrin, the iron-transporting protein of the circulation, is a concerted process involving remote amino acid residues as well as those at the two specific iron-binding sites of the protein. Previous studies of fluoresceinated transferrin have suggested Lys 569 as a kinetically active site in the C-terminal lobe of the protein. We have therefore turned to site-directed mutagenesis to investigate the role of Lys 569 in the release process at pH 5.6, the pH of the endosome where iron is transferred from transferrin to the iron-dependent cell. Mutation of positively charged Lys 569 to an uncharged Gln results in a protein in which release of iron from the mutated lobe to pyrophosphate is slowed by a factor of 15-20 and in which release kinetics switch from a complex saturation-linear to a simple saturation function. Acceleration of release by chloride is also substantially less than in native transferrin. When Lys 569 is replaced by a positively charged Arg, in contrast, observed release rates and chloride dependence are close to those of the native protein. The mechanism of release from the C-lobe site therefore appears to be sensitive to positive charge at position 569. Binding of chloride or other simple anion accelerates and is essential for release from the C-lobe; a muted response of K569Q to chloride concentration suggests that Lys 569 may function as a kinetically active anion-binding residue in the C-lobe. Despite the kinetic effects of the K569 mutation on iron release, rates of iron uptake by K562 cells from the C-lobes of native, K569Q, and K569R proteins are almost identical. In contrast to the C-lobe, iron release from the N-lobe is insensitive to charge at residue 233, the site in that lobe homologous to residue 569, with chloride retarding rather than accelerating release. K233, therefore, is not a kinetically active anion-binding site in the N-lobe. Release mechanisms differ substantially in the two lobes of transferrin despite the identity of ligands and their nearly identical arrangements in the lobes.