Sulfate as a synergistic anion facilitating iron binding by the bacterial transferrin FbpA: the origins and effects of anion promiscuity

Abstract

The ferric binding protein, FbpA, has been demonstrated to facilitate the transport of naked Fe3+ across the periplasmic space of several Gram-negative bacteria. The sequestration of iron by FbpA is facilitated by the presence of a synergistic anion, such as phosphate or sulfate. Here we report the sequestration of Fe3+ by FbpA in the presence of sulfate, at an assumed periplasmic pH of 6.5 to form FeFbpA-SO4 with K'(eff) = 1.7 x 10(16) M(-1) (at 20 degrees C, 50 mM MES, 200 mM KCl). The iron affinity of the FeFbpA-SO4 protein assembly is 2 orders of magnitude lower than when bound with phosphate and is the lowest of any of the FeFbpA-X assemblies yet reported. Iron reduction at the cytosolic membrane receptor may be an essential aspect of the periplasmic iron-transport process, and with an E(1/2) of -158 mV (NHE), FeFbpA-SO4 is the most easily reduced of all FeFbpA-X assemblies yet studied. The variation of FeFbpA-X assembly stability (K'(eff)) and ease of reduction (E(1/2)) with differing synergistic anions X(n-) are correlated over a range of 14 kJ, suggesting that the variations in redox potentials are due to stabilization of Fe3+ in FeFbpA-X by X(n-). Anion promiscuity of FbpA in the diverse composition of the periplasmic space is illustrated by the ex vivo exchange kinetics of FeFbpA-SO4 with phosphate and arsenate, where first-order kinetics with respect to FeFbpA-SO4 (k = 30 s(-1)) are observed at pH 6.5, independent of entering anion concentration and identity. Anion lability and influence on the iron affinity and reduction potential for FeFbpA-X support the hypothesis that synergistic anion exchange may be an important regulator in iron delivery to the cytosol. This structural and thermodynamic analysis of anion binding in FeFbpA-X provides additional insight into anion promiscuity and importance.

Scheme 1

Range of reduction potentials for Fe³⁺ sequestered by FbpA, with various synergistic anions, FeFbpA-X. Data from this work and references and .

Figure 1

Ligand plots developed using kinemages displaying ferric binding protein, designated HitA (hFbpA) in H. influenzae and FbpA (nFbpA) in N. gonorrhoeae. The ligand interactions for FeFbpA-PO₄ and apo-HitA are shown between phosphate, iron and protein. A: Ligand interaction map using holo-FbpA backbone. The holo interactions for phosphate are Ser139, Ala141 amide N, and Gln58. The holo interactions for iron are water, Glu57, His9, Tyr195, Tyr196, and phosphate. B: Ligand interaction map using apo-HitA backbone. The apo interactions for phosphate are Ser139, Gly140 amide N, Ala141 amide N, Tyr196, and Asn193. Hydrogen bonds are shown by dashed blue lines with the bond length (Å) printed in blue. Coordinate Files: 1D9Y.pdb and 1D9V.pdb.

Figure 2

Representative plot for sulfate anion binding to FbpA obtained by difference UV spectroscopy at λ = 238 nm for apo-FbpA + SO₄²⁻ (difference spectra inset). Solid line represents a fit to a single site binding model with K_d = 4.1±0.5 mM. Conditions: [apoFbpA] = 15 μM, [SO₄²⁻] = 0.5 − 100 mM in 0.05 M MES, 0.1 M NaCl, pH 6.5, 25°C.

Figure 3

Representative Nernst plot obtained by spectroelectrochemistry for FeFbpA-SO₄ (spectral inset, λ_max = 495 nm). Solid line represents linear least squares fit to the data where slope and y-intercept correspond to Nernstian behavior with single electron transfer (n = 0.94) and E_½ = −158 mV. Conditions: [Fe³⁺FbpA-SO₄] = 0.7 mM (λ_max = 495 nm), [MV²⁺] = 4.9 mM (λ_max = 391 and 600 nm) in 0.05 M MES, 0.2 M KCl, pH 6.5, 20°C.

Figure 4

Absorbance change at 550 nm as a function of time for the reaction of FeFbpA-SO₄ with PO₄³⁻. Solid line represents a single exponential decay curve with k_obs = 30 s⁻¹. Conditions: [FeFbpA-SO₄] = 0.065 mM, [PO₄³⁻] = 0.65 mM, pH = 6.5, in 0.05 M MES, 0.2 M KCl at 20°C. Inset: Observed first-order rate constant for the reaction of phosphate (square) and arsenate (circle) with FeFbpA-SO₄. Data show no dependence on entering anion identity or concentration and are within one standard deviation of k_obs= 30 s⁻¹. Conditions: [FeFbpA-SO₄] = 0.065 mM, [Xⁿ⁻] = 0.65−65 mM, pH = 6.5, in 0.05 M MES, 0.2 M KCl at 20°C.

Figure 5

Plot of log of the stability constant (K′_eff) for the Fe³⁺FbpA-X assembly as a function of the corresponding E_½ values. The dotted line represents a best fit of a rearranged form of equation 17 with fixed slope of −0.017.

Figure 6

Correlations relating apo-FbpA binding to anions based on anion average charge (Z_ave) and hydration enthalpy (ΔH_hyd). (A) Plot of Δ(ΔG_FbpAX) as a function of the average charge on the anion (Z_ave), calculated using equation 6. Trend line represents linear least squares best fit to all data. (B) Plot of apo-FbpA anion binding energy corrected for electrostatic contributions (Δ(ΔG_FbpAX)/Z_ave) as a function of the anion hydration enthalpy (ΔH_hyd; Table 1). Trend line represents linear least squares fit to the data omitting data point for oxalate, for reasons of geometry. Δ(ΔG_FbpAX) is defined in ref .

Figure 7

Plot of Δ(ΔG_FeFbpAX) as a function of anion///apo-FbpA binding corrected for electrostatics (Δ(ΔG_FbpAX/Z_ave). Trend line represents linear least squares best fit to the data. Δ(ΔG_FeFbpAX) and Δ(ΔG_FbpAX) are defined in ref .

Figure 8

Plot of changes in free energy of the FeFbpA-X complex with different anions (Δ(ΔG_FeFbpAX)) as a function of anion enthalpy of hydration (ΔH_hyd; Table 1). Δ(ΔG_FeFbpAX) is defined in ref .