The influence of the synergistic anion on iron chelation by ferric binding protein, a bacterial transferrin

Abstract

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 x 10(17) M(-1) to 4 x 10(18) M(-1) at pH 6.5 and 20 degrees C. The redox potentials for Fe(3+)nFbpXFe(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' --> 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 approximately pyrophosphate > nitrilotriacetate > citrate approximately oxalate 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.

Figure 1

X-band EPR spectra of Fe³⁺nFbpX complexes. X is arsenate, NTA, oxalate, pyrophosphate, and citrate in A–E, respectively.

Figure 2

Nernst plot for recombinant nFbp in the presence of various exogenous anions, Fe³⁺nFbpX. X is phosphate, arsenate, pyrophosphate, citrate, oxalate, and NTA in A–F, respectively. Data were obtained by spectroelectrochemistry, using solutions consisting of ≈0.63–1.2 mM Fe³⁺nFbpX, 4.4–8.4 mM methyl viologen mediator in 0.05 M Mes, and 0.2 M KCl at pH 6.5 and 20°C.

Figure 3

Plot of log of the stability of the Fe³⁺nFbpX complex expressed as K′_eff (Upper) and [Xⁿ⁻]₅₀ (Lower) as a function of the corresponding E_1/2 values. Data are from Table 1.

Figure 4

Schematic representation of a model for outer membrane to cytosol transport of iron in pathogenic Neisseria based on data presented here. (1) Docking of ferric transferrin (Fe³⁺hTf) at the outer membrane receptor and reduction of bound Fe³⁺ to Fe²⁺. (2) Release of Fe²⁺ from transferrin and subsequent movement to interior side of the membrane receptor. (3) Sequestration of Fe²⁺ by nFbp in the presence of phosphate. (4) Oxidation of Fe²⁺nFbp(PO₄). (5) Movement of Fe³⁺nFbp(PO₄) or Fe³⁺nFbp(cit) across the periplasmic space. (6) Docking of Fe³⁺nFbp(PO₄) at the inner membrane receptor and reduction of bound Fe³⁺ to Fe²⁺. (7) Release of Fe²⁺ and passage through the cytoplasmic membrane receptor into the cytosol. (8) Exchange of phosphate exogenous anion by citrate to form Fe³⁺nFbp(cit). (9) Docking of Fe³⁺nFbp(cit) at the inner membrane receptor and reduction of bound Fe³⁺ to Fe²⁺. (10) Release of Fe²⁺ and passage through the cytoplasmic membrane receptor into the cytosol.