Mechanisms of anion exchange with Fe ³+-transferrin-anion complexes

Abstract

Transferrin, the serum iron transport protein, binds two Fe('3+) ions. Fe('3+) is not bound in the absence of carbonate or a substitute synergistic anion which serves to complete the metal binding site. In this study Fe('3+)-transferrin-glycine, Fe('3+)-transferrin-nitrilotriacetate, Fe('3+)-transferrin-salicylate, and Fe('3+)-transferrin-thioglycolate have been used as model systems to investigate the mechanisms by which a synergistic attacking anion A' is able to exchange for the resident anion A of an Fe('3+)-transferrin-A complex to form the product Fe('3+)-transferrin-A' complex. There appear to be several major mechanisms of anion exchange. These are: (1) direct exchange which does not appear to involve Fe('3+) release from the protein and (2) apotransferrin intermediate-I and -II mechanisms which involve formation of apotransferrin intermediates. In the apotransferrin intermediate-I mechanism, Fe('3+) is both removed from the Fe('3+)-transferrin-anion complex and subsequently donated to apotransferrin by the attacking anion; whereas the Fe('3+) transfer is mediated via the resident anion for the apotransferrin intermediate-II mechanism. The mechanism of exchange appears dependent upon the properties of both the resident and attacking anions. Bicarbonate, glycolate, lactate, phenyllactate, malate, pyruvate, salicylate, and thioglycolate react with Fe('3+)-transferrin-glycine via a direct exchange mechanism. Malonate and other dicarboxylates possessing the basic malonyl structure, such as ketomalonate and phenylmalonate, react with Fe('3+)-transferrin-glycine via the apotransferrin intermediate-I mechanism. Bicarbonate reacts with Fe('3+)-transferrin-nitrilotriacetate, Fe('3+)-transferrin-salicylate, and Fe('3+)-transferrin-thioglycolate via the apotransferrin intermediate-II mechanism. The chelating agent citrate appears to remove Fe('3+) from Fe('3+)-transferrin-glycine by a mechanism which is identical to the Fe('3+) removal phase of the apotransferrin intermediate-I mechanism. The initial step of the direct exchange, apotransferrin intermediate-I and -II, and citrate chelation mechanisms appears to be a rate-limiting conformational transition of the Fe('3+)-transferrin-anion complex resulting in exposure of the Fe('3+) and anion binding site to the attacking anion. The two sites of Fe('3+)-transferrin-glycine and Fe('3+)-transferrin-thioglycolate appear to react at different rates and differ in their susceptibility to attacking anions and chelating agents.