The chloride effect is related to anion binding in determining the rate ofiron release from the human transferrin N-lobe

The major function of human transferrin is to deliver iron from the bloodst ream to actively dividing cells. Upon iron release, the protein changes its conformation from 'closed' to 'open'. Extensive studies in vitro indicate that iron release from transferrin is very complex and involves many factor s, including pH, the chelator used, an anion effect, temperature, receptor binding and intra-lobe interactions. Our earlier work [He, Mason and Woodwo rth (1997) Biochem. J. 328, 439-445] using the isolated transferrin N-lobe (recombinant N-lobe of human transferrin comprising residues 1-337; hTF/2N) has shown that anions and pH modulate iron release from hTF/2N in an inter dependent manner: chloride retards iron release at neutral pH, but accelera tes the reaction at acidic pH. The present study supports this idea and fur ther details the nature of the dual effect of chloride: the anion effect on iron release is closely related to the strength of anion binding to the ap oprotein. The negative effect seems to originate from competition between c hloride and the chelator for an anion-binding site(s) near the metal centre . With decreasing pH, the strength of anion binding to hTF/2N increases lin early, decreasing the contribution of competition with the chelator. In the meantime, the 'open' or 'loose' conformation of hTF/2N, induced by the pro tonation of critical residues such as the Lys-206/Lys-296 pair at low pH, e nables chloride to enter the cleft and bind to exposed side chains, thereby promoting cleft opening and synergistically allowing removal of iron by th e chelator, leading to a positive anion effect. Disabling one or more of th e primary anion-binding residues, namely Arg-124, Lys-206 and Lys-296, subs tantially decreases the anion-binding ability of the resulting mutant prote ins. In these cases, the competition for the remaining binding residue(s) i s increased, leading to a negative chloride effect or, at most, a very smal l positive effect, even at low pH.