Although the presence of an exogenous anion is a requirement for tight Fe 3؉ binding by the bacterial (Neisseria) transferrin nFbp, the identity of the exogenous anion is not specific in vitro. nFbp was reconstituted as a stable iron containing protein by using a number of different exogenous anions [arsenate, citrate, nitrilotriacetate, pyrophosphate, and oxalate (symbolized by X)] in addition to phosphate, predominantly present in the recombinant form of the protein. Spectroscopic characterization of the Fe 3؉ ͞anion interaction in the reconstituted protein was accomplished by UV-visible and EPR spectroscopies. The affinity of the protein for Fe 3؉ is anion dependent, as evidenced by the effective Fe 3؉ binding constants (K eff) observed, which range from 1 ؋ 10 17 M ؊1 to 4 ؋ 10 18 M ؊1 at pH 6.5 and 20°C. The redox potentials for Fe 3؉ nFbpX͞ Fe 2؉ nFbpX reduction are also found to depend on the identity of the synergistic anion required for Fe 3؉ sequestration. Facile exchange of exogenous anions (Fe 3؉ nFbpX ؉ X 3 Fe 3؉ nFbpX ؉ X) is established and provides a pathway for environmental modulation of the iron chelation and redox characteristics of nFbp. The affinity of the iron loaded protein for exogenous anion binding at pH 6.5 was found to decrease in the order phosphate > arsenate ϳ pyrophosphate > nitrilotriacetate > citrate ϳ oxalate Ͼ Ͼ carbonate. Anion influence on the iron primary coordination sphere through iron binding and redox potential modulation may have in vivo application as a mechanism for periplasmic control of iron delivery to the cytosol.

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Virtually all organisms require iron, and iron-dependent cells of vertebrates (and some more ancient species) depend on the Fe(3+)-binding protein of the circulation, transferrin, to meet their needs. In its iron-donating cycle, transferrin is first captured by the transferrin receptor on the cell membrane, and then internalized to a proton-pumping endosome where iron is released. Iron exits the endosome to enter the cytoplasm via the ferrous iron transporter DMT1, a molecule that accepts only Fe(2+), but the reduction potential of ferric iron in free transferrin at endosomal pH (approximately 5.6) is below -500 mV, too low for reduction by physiological agents such as the reduced pyridine nucleotides with reduction potentials of -284 mV. We now show that in its complex with the transferrin receptor, which persists throughout the transferrin-to-cell cycle of iron uptake, the potential is raised by more than 200 mV. Reductive release of iron from transferrin, which binds Fe(2+) very weakly, is therefore physiologically feasible, a further indication that the transferrin receptor is more than a passive conveyor of transferrin and its iron.