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 × 10¹⁶ M⁻¹ (at 20°C, 50 mM MES, 200 mM KCl). The iron affinity of the FeFbpA-SO₄ protein assembly is two 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 Xⁿ⁻ 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 Xⁿ⁻. 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, with first order kinetics with respect to FeFbpA-SO₄ (k = 30 s⁻¹) 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.

Introduction

Iron is an essential element for nearly every living organism. The survival of each cell is dependent on its ability to acquire and sustain a necessary quantity of this transition metal.¹ However, the iron acquisition processes of even the most rudimentary organism are quite complex, with strict requirements due to the paradoxical properties of iron in biology: necessity, insolubility and toxicity.² While iron is essential to sustain life, under certain conditions the metal can act as a redox center facilitating the production of harmful reactive oxygen species through Fenton chemistry and the Haber-Weiss cycle.¹ Also, while iron is the second most abundant metal on the earth's surface, it predominantly exists as biologically unavailable iron hydroxide polymers, requiring that microorganisms develop specialized means of acquiring sufficient concentrations of this element.²

For pathogenic bacteria, the ability to successfully acquire iron from the environment is directly related to virulence.^3-5 There are two common means of microbial iron uptake. The first is accomplished through the synthesis of a low molecular weight organic chelator molecule, known as a siderophore, which is released to the environment to selectively sequester iron and return to the microbial colony.^6-11 The second method of iron acquisition is a protein mediated system.³ In either case, it is critical for bacteria to gain control of the first-coordination shell of iron to inhibit its toxic effects, including the synthesis of reactive oxygen species and resulting oxidative damage. Therefore, iron-acquisition is not just simple recognition and transport at the bacterial cell surface; it is also an issue of “iron-management”.

Certain gram-negative bacteria, including the genera Neisseria and Haemophilus, express surface receptors specific for host iron transport proteins (e.g., lactoferrin and transferrin), which bind the host iron carrier and pilfer the precious metallic cargo.^3,5 A series of proteins mediate the transfer of the iron from these host-iron binding proteins into the bacterial cytoplasm.^12,13 In these cases, a single periplasmic iron-binding protein, ferric binding protein (FbpA), designated hFbpA (HitA) in Haemophilus influenzae and nFbpA (FbpA) in Neisseria gonorrhoeae ¹⁴ effectively mediates iron transport from the inside of the outer membrane to the outside of the inner membrane.^3,5,16-19 As the only means of shuttling iron across the periplasmic space, FbpA plays a pivotal role in the iron uptake process of these pathogens, perhaps as the rate-limiting step in bacterial iron acquisition.³

FbpA is a member of the transferrin superfamily of proteins that traces its ancestry to an anion binding protein.²⁰ FbpA is referred to as bacterial transferrin; a title attributed to both functional and structural similarities to mammalian transferrin.^20-23 FbpA acts as a transport protein, shuttling iron between membrane receptors, analogous to its human transferrin (hTf) counterpart. Structurally, FbpA from Haemophilus influenzae and Neisseria gonorrhoeae contains a single functional site and exhibits protein-folding similar to a single lobe of the bilobal hTf. In all three proteins, iron is hexa-coordinate, bound by two tyrosines, a histidine and a carboxylic acid, provided by either aspartate (hTf) or glutamate (FbpA), with the final two coordination sites occupied by an exogenous anion, bidentate carbonate for hTf ^23,24 and from the reported crystal structure, a monodentate phosphate anion and water for FbpA.²⁰ The anion and iron binding sites are illustrated in Figure 1 for apo-HitA and holo-FbpA, relating both FbpA and Tf to the anion binding superfamily of proteins.²⁰

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.pdb³⁸ and 1D9V.pdb.²¹

An exogenous (or synergistic) anion is essential for both FbpA and Tf to facilitate tight iron binding. In FbpA, numerous anions have been found to serve the essential function of the synergistic anion in vitro.^25-28 These anions are readily exchangeable upon in vitro manipulation and, at pH 6.5, modulate the iron affinity of the FeFbpA-X assembly over the log K′_eff range 17 −18 and the Fe³⁺/Fe²⁺ redox potential over the range −300 to −180 mV.^25,26,29 Given the diverse composition of the periplasm,³⁰ this synergistic anion promiscuity may be biologically significant. Since the anion dependent redox potentials are in the range where reduction may be driven by NADH/NADPH cofactors, a possible role for the anion has been proposed in modulating the delivery of iron to the cytosol.^25,26 While the significance of the synergistic anion to the in vivo function of FbpA has been questioned based on crystallographic interpretations of FbpA mutant data,³¹ the anion binding influence on biologically relevant physico-chemical properties of FbpA in solution is clearly demonstrable. The role of the synergistic anion in vivo remains an open biological question and may be important in understanding the mechanism of iron assimilation by pathogenic bacteria utilizing FbpA orthologs.

In this work, we study both the thermodynamic and kinetic ability of sulfate to act as the synergistic anion in iron sequestration by FbpA at the periplasmic pH of 6.5.^30,32,33 By producing the least thermodynamically stable of the synergistic anion-protein assemblies, sulfate expands the range with which FbpA can modulate the ease of iron reduction simply through anion exchange. These findings have broader implications for a reductase driven iron delivery to the cytosol ²⁶ and expand our previous findings by demonstrating that various synergistic anions produce thermodynamically stable, kinetically labile protein assemblies. A structural analysis is described to define the role of the anion in complex stabilization and the relationship to the larger anion-binding protein superfamily. In this analysis, we examine the anion influence in modulating the stability of these assemblies through iron-anion (Fe³⁺///Xⁿ⁻), protein-anion (FbpA///Xⁿ⁻) and iron-protein (Fe³⁺///FbpA) interactions.

Materials and Methods

Reagents

Solutions were prepared in distilled water using the highest grade of commercially available reagents. Buffer solutions of 0.05 M MES (Fisher Biotech, enzyme grade)/background electrolyte were made, with the pH adjusted to 6.5 using an Orion pH-meter model 230 with the appropriate electrolyte.

Preparation of anion-substituted holo-ferric binding protein, FeFbpA-X

Recombinant FbpA from N. gonorrhoeae was overexpressed in Escherichia coli and isolated as previously described.³⁴ Anion-substituted FeFbpA-X assemblies were prepared for sulfate, phosphate and arsenate anions as follows. FeFbpA-SO₄ was prepared by the addition of 1.2 equivalents of ferrous sulfate (Allied Chemical) and 10 equivalents of sulfate (sodium sulfate, Fisher Scientific) to 1 equivalent of apo-FbpA ([apo-FbpA] = 0.1−1.1 mM). FeFbpA-PO₄ and FeFbpA-AsO₄ were prepared by mixing 10 equivalents of phosphate (potassium phosphate, monobasic, United States Biochemical) or arsenate (arsenic acid, sodium salt, ICN Biomaterials) with 1 equivalent of apo-FbpA, then adding 1.2 equivalents of FeCl₂ (ICN Biomedicals) while stirring gently for 30 minutes and then storing at 4°C overnight. An UV-vis spectrum was taken before and after storage, with additional appropriate ferrous salt added to ensure complete Fe³⁺ loading of the protein, indicated by no changes in the spectrum. The identity of the FeFbpA-X species was determined by the λ_max in the visible region: FeFbpA-PO₄ λ_max = 481 nm (ε₄₈₁ = 2430 M⁻¹ cm⁻¹);²⁶ FeFbpA-AsO₄ λ_max = 476 nm (ε₄₇₆ = 2280 M⁻¹ cm⁻¹);²⁶ FeFbpA-SO₄ λ_max = 495 nm (ε₄₉₅ = 2460 M⁻¹ cm⁻¹).³⁵ Excess iron present as insoluble Fe(OH)₃ was removed by using a syringe-driven 0.45 μm filter unit (Millex, Millipore). The resulting solution was dialyzed three times against the appropriate buffer at pH 6.5 and stored at 4°C. The final protein concentration was determined by monitoring the absorbance at λ = 280 nm (ε₂₈₀ = 5.11 × 10⁴ M⁻¹ cm⁻¹ for holo-FbpA).²⁶ All spectra were obtained using a CARY 100 Bio UV-Vis-NIR spectrophotometer (Varian) at 20.0 °C ± 0.1°C.

Sulfate anion binding to apo-FbpA

Apo-FbpA was dialyzed five times against a buffer of 50 mM MES, 100 mM NaCl at pH 6.5 and diluted with additional fresh buffer to a final concentration of 15 μM. A stock solution of 1.01 M sulfate was prepared from sodium sulfate (Fisher Scientific) in buffer, and adjusted to pH 6.5 with minimal NaOH. Divided quartz cuvettes (Wilmad Labglass) were placed in the sample and reference beam of a CARY 100 Bio UV-Vis-NIR spectrophotometer, with the compartments arranged in series, with each beam passing through a protein and buffer compartment, each of path length = 0.4375 cm. Sulfate was added via a 25 μL microsyringe to the protein compartment of the sample cell and the buffer compartment of the reference cell, incrementally increasing the sulfate concentration from 0.5 to 100 mM. An equivalent volume of buffer was added to the reference protein compartment to correct for dilution. For each sulfate addition, spectra were recorded from 300 to 230 nm at 12 nm/min with 1 nm spectral band width. Difference absorbance spectra were generated by subtracting the spectrum of [SO₄²⁻] = 0 mM from all scans. Intensities at λ = 238 nm were plotted against sulfate concentration and analyzed by fitting to a non-linear regression for single site saturation ligand binding (equation 1) using SigmaPlot 9.0 software,

$$\Delta {\mathrm{A}}_{\lambda =238}=\frac{A_{\max }\left[S{O}_4\right]}{K_d+\left[S{O}_4\right]}$$ (1)

where ΔA_λ=238 is the change in absorbance at 238 nm on sulfate binding, A_max is the maximum absorbance of the curve (when [FbpA]_tot = [FeFbpA-SO₄]), [SO₄] is the molar free sulfate concentration and K_d is the equilibrium constant for the dissociation of apo-FbpA-SO₄. Identical methodology was used to determine all of the anion binding K_d values listed in Table 1.

Table 1.

Anion binding constants for apo-FbpA (K_d), anion average charge at pH 6.5 (Z_ave), and calculated anion hydration enthalpies (ΔH_hyd)

Anion	Kd (mM)a	Zaveb	ΔHhyd (kJ mol−1)c
Sulfate	4.1 (5)d	−2.0	−1130
Arsenate	2.1 (4)e	−1.4	−521
Phosphate	1.6 (4)e	−1.4	−498
NTA	3.0 (4)e	−2.0	---
Oxalate	0.21 (5)e	−2.0	−1080
Citrate	0.06 (1)e	−2.5	---
Pyrophosphate	0.018 (7)e	−2.8	−845

^aConditions: [FbpA] = 15 mM with [Xⁿ⁻] varying from 0.5 to 100 mM, in 50 mM MES/100 mM NaCl, pH 6.5 via difference spectroscopy. Values are determined as an average of three independent experiments. Number in parentheses indicates the standard error in three independent determinations of K_d.

^bAverage anion charge at pH 6.5. See Experimental Section.

^cHydration enthalpy calculated values using equation 7. Calculated values agree with experimental values where available for sulfate and oxalate.⁴³,⁴⁵

^dThis work

^eRef ⁴⁷

Spectroelectrochemistry

Spectroelectrochemistry of FeFbpA-SO₄ was carried out using an in-house anaerobic optically transparent thin layer electrode (OTTLE) cell with an optical path length of 0.025 ± 0.005 cm, connected to an EG & G Princeton Applied Research Potentiostat Model 263, and CARY 100 Bio UV-Vis-NIR spectrophotometer at 20.0°C ± 0.1°C as previously described.^25,26 Methyl viologen (MV²⁺) mediator solution was prepared by dissolving methyl viologen dihydrate (Aldrich) in 0.05 M MES/0.2 KCl, adjusting the pH to 6.5 and diluting to generate a final MV²⁺ concentration of 35 mM. A portion of the MV²⁺ solution was added to the FeFbpA-SO₄ in buffer to obtain a 1:7 protein:mediator mol ratio, with a final concentration of ∼0.2−0.4 mM FbpA. All potential values are reported relative to the normal hydrogen electrode (NHE). Spectral scans were performed from 350−750 nm, monitoring at λ = 495 nm for FeFbpA-SO₄ and at λ = 391 and 598 nm for the reduced methyl viologen signal. After each experiment, the OTTLE cell was opened to air to allow reoxidation of the holo-protein, with 85−95% recovery after 12 h.

Determination of Fe³⁺ binding constant

A FeFbpA-SO₄ stability constant, K′_eff, was determined from a competition equilibrium between FeFbpA-SO₄ and EDTA at pH 6.5 in 0.05 M MES, 0.2 M KCl and 20°C using methods described previously.²⁶ The equilibrium position of reaction 2 was monitored spectrophotometrically at the λ_max for FeFbpA-SO₄ (495 nm) at different protein:EDTA mol ratios from 1:0.5 to 1:5. Aliquots of a 5 mM EDTA (Acros Organics) solution in buffer were added to a FeFbpA-SO₄ solution (0.159 mM as determined from the initial absorbance at 280 nm).

$$\mathrm{FeFbpA}\text{-}{\mathrm{SO}}_4+\mathrm{EDTA}\leftrightarrows \mathrm{FeEDTA}+\mathrm{apo}\text{-}\mathrm{FbpA}\text{-}{\mathrm{SO}}_4$$ (2)

The FeFbpA-SO₄ stability constant (K′_eff) was calculated using equation 3 where K is the experimentally determined equilibrium constant for reaction 2 and ${\beta }_{110}^{FeEDTA}$ was calculated at these conditions from known equilibrium constants for the Fe³⁺/EDTA system³⁶ by using mass balance equations involving the usual Ringbom's coefficients.³⁷

$$K=\frac{\left[FeEDTA\right][FbpA-S{O}_4]}{[FeFbpA-S{O}_4]\left[EDTA\right]}=\frac{{\beta }_{110}^{FeEDTA}\left(pH6.5\right)}{{K^{\prime }}_{eff}}$$ (3)

The value of K′_eff reported is an average of three independent determinations.

Kinetic measurements by stopped-flow spectrophotometry

The anion exchange reactions of FeFbpA-SO₄ with phosphate and arsenate were studied using an Applied Photophysics stopped-flow (SX.18MV) spectrophotometer equipped with both a photomultiplier and photodiode array detector. All reactions were performed at 25°C in 0.05 M MES/0.2 M KCl at pH 6.5 under pseudo-first order conditions with the entering anion in at least 10 fold excess of the protein ([FeFbpA-SO₄] = 130 μM). Reactions were monitored in rapid-scan absorbance mode, using the photodiode array detector recording from λ = 350 to 700 nm to confirm direct conversion from FeFbpA-SO₄ (λ_max = 495 nm) to FeFbpA-PO₄ (λ_max = 481 nm) or FeFbpA-AsO₄ (λ_max = 476 nm) and to determine the wavelengths of the greatest absorbance change in the exchange of FeFbpA-SO₄ with phosphate and arsenate (λ = 550 and 450 nm). Both wavelengths were then used to monitor the change in absorbance as a function of time for each anion at concentrations of 10 to 1000 fold excess over FeFbpA-SO₄. Data were analyzed using Applied Photophysics kinetics software.

Structural analysis of ligand binding sites in FbpA and HitA

Structures for ferric binding proteins FbpA and HitA were obtained from the RCSB Protein Data Bank, http://www.rcsb.org/pdb/Welcome.do. The two pdb files used were 1D9Y, holo-FbpA from N. gonorrhoeae, and 1D9V, apo-HitA from Haemophilus influenzae.^21,38 The apo form of FbpA has not been deposited into the databank so the apo-form of a homologous protein, HitA was used to map the iron-free protein interactions. Holo-FbpA and holo-HitA share 71% sequence identity and exhibit identical assembled iron coordination sites.¹⁵

In order to investigate H-bonding and electrostatics, the pdb files were structurally validated using the MolProbity web service, with 3D interactive graphics (kinemages) rendered using Prekin, Mage, and Probe, all available at http://kinemage.biochem.duke.edu/.^39,40 The MolProbity web service was used on the pdb files in order to add explicit hydrogens and flip certain amino acids⁴¹ (histidines, asparagines, and glutamines) where it was indicated that such adjustment resulted in an improved structure.⁴² “Prekin” was used to generate three-dimensional kinemages from the revised pdb files. “Probe” was then used to generate a pattern of dots representing the van der Waals surface of amino acid residues that are within hydrogen bonding distances and orientation. The kinemages were rendered in “Mage” and each residue was examined for bonding interactions with phosphate and iron. Figure 1 depicts a cartoon illustration of the kinemages displaying the conserved residues found in FbpA and HitA, and bonding interactions with phosphate and iron.

Calculation of anion average charge (Z_ave) and hydration enthalpy (ΔH_hyd)

The average charge of an anion (Z_ave) is defined as the sum of the species relative abundance (x_i) multiplied by its charge (Z_i) for all possible species (equation 4).

$${\mathrm{Z}}_{\mathrm{ave}}=\Sigma {\mathrm{x}}_{\mathrm{i}}{\mathrm{Z}}_{\mathrm{i}}$$ (4)

$${\mathrm{Z}}_{\mathrm{i}}=\mathrm{h}\text{-}\mathrm{m}$$ (5)

Z_i is defined in terms of m and h (equation 5), with m equal to the maximum possible charge of an anion (e.g. m = 3 for PO₄³⁻), and h equal to the number of bound protons (e.g. h = 2 for H₂PO₄¹⁻). The value of Z_ave is dependent on two factors, the anion pK_a values and the solution pH. Rearranging equation 4 to determine x_i in terms of K_a values and pH (as [H⁺]), Z_ave can be calculated by the following equation 6.

$${\mathrm{Z}}_{\mathrm{ave}}=\sum _{h=0}^m(h-m)\frac{{\left[H^{+}\right]}^{\left(h\right)}{K}_{a_{m-h}}{K}_{a_{m-(h+1)}}...K_{a_0}}{\sum _{h=0}^m{\left[H^{+}\right]}^{\left(h\right)}{K}_{a_{m-h}}{K}_{a_{m-(h+1)}}...K_{a_0}}$$ (6)

Average charge calculations are used as a way to describe the distribution of anion species as a result of the degree of protonation at pH 6.5.

Z_ave values are subsequently used to calculate anion hydration enthalpies (ΔH_hyd) according to equation 7, where r_i is defined as the thermochemical radius of the anion.^43,44 Calculated ΔH_hyd values for sulfate and oxalate anions where Z_ave = 2.0

$$\Delta H_{hyd}=\frac{-700{Z_{ave}}^2}{r_i+0.3}$$ (7)

were found to agree with experimental values in the literature.^45,46

Results

Sulfate anion binding to apo-FbpA

Anions are known to bind the apo form of FbpA at the anion binding site while simultaneously preorganizing the iron binding site tyrosines for Fe³⁺ chelation.²¹ UV-difference spectroscopy was used to determine the sulfate anion binding constant for apo-FbpA, expressed below as a dissociation reaction.

$$\mathrm{apo}\text{-}\mathrm{FbpA}\text{-}{\mathrm{SO}}_4\leftrightarrows \mathrm{apo}\text{-}\mathrm{FbpA}+{{\mathrm{SO}}_4}^{2\text{-}}$$ (8)

Representative data are shown in Figure 2, where K_d for reaction 8 is 4.1 mM at pH 6.5, determined by fitting data to equation 1 for single site binding. Higher order binding stoichiometries were considered and subsequently rejected due to an inability to fit an equation with differing binding constants to the data, although our method is unable to definitively eliminate the possibility of multiple binding sites of equivalent affinity. However, based on crystallographic evidence for a single phosphate binding to the active site of the apo-protein²¹ with a K_d value similar to sulfate (Table 1), the stoichiometry of reaction 8 is considered appropriate. Table 1 is a compilation of sulfate and other anion binding constants for apo-FbpA at pH 6.5.⁴⁷ These K_d values vary with the identity of the anion and are in the range observed for other periplasmic anion binding proteins.^20,48-50 The binding of sulfate to FbpA is the weakest observed compared with all of the other anions tested.

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.

Thermodynamic Stability of FeFbpA-SO₄

As previously observed,^{27, 35} sulfate anion will satisfy the role of synergistic anion and produce a stable iron-protein assembly, FeFbpA-SO₄, with λ_max = 495 nm at pH 6.5. An effective stability constant (K′_eff) was determined for the FeFbpA-SO₄ assembly by spectrophotometrically monitoring the equilibrium between the protein complex and the competing chelator EDTA (reaction 2). From these data, K′_eff = 1.7 × 10¹⁶ M⁻¹ was calculated for equilibrium reaction 9 at pH 6.5, 50 mM MES, in 200 mM KCl at 20°C. This represents a significant affinity for Fe³⁺, but is the lowest iron affinity of any FeFbpA-X assembly studied to date.^23-25

$${{\mathrm{Fe}}^{3+}}_{\mathrm{aq}}+\mathrm{FbpA}\text{-}{\mathrm{SO}}_4\leftrightarrows \mathrm{FeFbpA}\text{-}{\mathrm{SO}}_4$$ (9)

The redox potential of FeFbpA-SO₄ was determined by spectroelectrochemistry using an anaerobic OTTLE cell and methyl viologen as a mediator. Well behaved Nernst plots were obtained (Figure 3) with a slope corresponding to a single electron transfer and intercept E_1/2 = −158 mV, corresponding to reaction 10 uncorrected for any Fe²⁺

$${\mathrm{Fe}}^{3+}\mathrm{FbpA}\text{-}{\mathrm{SO}}_4+e^{\text{-}}\leftrightarrows {\mathrm{Fe}}^{2+}\mathrm{FbpA}\text{-}{\mathrm{SO}}_4$$ (10)

dissociation. This makes FeFbpA-SO₄ the most easily reduced of all of the FeFbpA-X assemblies investigated (Scheme 1).²⁶

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.

Scheme 1.

Range of reduction potentials for Fe³⁺ sequestered by FbpA, with various synergistic anions, FeFbpA-X. Data from this work and references 25 and 26.

Kinetics of sulfate anion exchange

The kinetics of anion exchange for FeFbpA-SO₄ were studied at periplasmic pH (6.5), under pseudo-first order conditions with the exchanging anions (PO₄³⁻ or AsO₄³⁻) in excess. The spectral changes observed were consistent with the following exchange reaction, where X = PO₄³⁻ or AsO₄³⁻. Kinetic data for both anion exchange reactions

$$\mathrm{FeFbpA}\text{-}{\mathrm{SO}}_4+{\mathrm{X}}^{\mathrm{n}\text{-}}\to \mathrm{FeFbpA}\text{-}\mathrm{X}+{{\mathrm{SO}}_4}^{2\text{-}}$$ (11)

followed a single exponential decay with a first-order rate constant of k_obs = 30 s⁻¹ (Figure 4). The observed rate constant for reaction 11 is independent of anion concentration over the range of 10−1000 fold excess over FeFbpA-SO₄ (Figure 4 inset).

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.

These kinetic data are consistent with the following mechanism, where Xⁿ⁻ = PO₄³⁻, AsO₄³⁻.

$$\mathrm{FeFbpA}\text{-}{\mathrm{SO}}_4\leftrightarrows \mathrm{FeFbpA}+{{\mathrm{SO}}_4}^{2\text{-}}{\mathrm{k}}_1∕{\mathrm{k}}_{\text{-}1}$$ (12)

$$\mathrm{FeFbpA}+{\mathrm{X}}^{\mathrm{n}\text{-}}\to \mathrm{FeFbpA}\text{-}\mathrm{X}{\mathrm{k}}_2$$ (13)

Under pseudo-first-order conditions with excess Xⁿ⁻, the rate law may be expressed as:

$$\text{Rate}=\frac{d[FeFbpA-X]}{dt}={\mathrm{k}}_{\mathrm{obs}}\left[\mathrm{FeFbpA}\text{-}{\mathrm{SO}}_4\right]$$ (14)

Applying the steady state assumption to the intermediate FeFbpA, k_obs may be expressed as follows:

$${\mathrm{k}}_{\mathrm{obs}}=\frac{k_1{k}_2\left[X\right]}{k_{-1}\left[S{O}_4^{2-}\right]+k_2\left[X\right]}$$ (15)

At our experimental conditions where [SO₄²⁻] << [X] and making the reasonable assumption that k₋₁ ∼ k₂, equation 15 reduces to:

$${\mathrm{k}}_{\mathrm{obs}}={\mathrm{k}}_1$$ (16)

This is consistent with the observed lack of dependence of k_obs on either the identity or concentration of Xⁿ⁻ (Figure 4). Consequently, we conclude that the rate constant k₁ for the dissociation of SO₄²⁻ from FeFbpA-SO₄ is 30 s⁻¹ and the bound sulfate anion is readily exchanged.

Discussion

Periplasmic anion composition and FbpA

The importance of synergistic anion promiscuity in FbpA can be seen from the modulation of physico-chemical properties of this periplasmic iron transport protein by exogenous anions, which are both available and otherwise essential to the bacterium. The anion composition of the periplasm is not specifically defined, but is rich in diversity.³⁰ This diverse anion population results from the porin-laced outer membranes of gram-negative bacteria, through which ions of less than 1000 daltons diffuse. The periplasm represents the intervening space between the porous outer membrane and relatively impervious cytoplasmic membrane. This space is not insignificant, comprising about 30% of the bacterial total volume. It is this space through which low molecular weight anionic nutrients must transiently pass from the environment to the cytosol. Proteins such as FbpA and other high molecular weight molecules such as membrane derived oligosaccharides, trapped within this space, facilitate the accumulation and uptake of these nutrients.

The anionic composition of the periplasm is complex, dynamic, and heavily influenced by the external environment. Commensal and pathogenic species of the genus Neisseria are obligate human parasites, and as such are restricted to growth in biological fluids. The phosphate, citrate, and sulfate concentrations within serum are between 0.1 and 1.0 mM^51-53 and therefore are expected to be normal constituents of the low molecular weight composition of the periplasmic space.

Sulfate as a synergistic anion for Fe³⁺ sequestration by FbpA from Neisseria is the focus of this manuscript. There are definitive species of pathogenic bacteria that use inorganic sulfate as a sulfur source, including Neisseria meningitides, which can grow using sulfate as a sole source of this nutrient.⁵⁴ Most other Neisseria strains have been shown to grow with only sulfate or thiosulfate as the lone sulfur source.^54,55 Thus, while our studies are performed in vitro, these experiments are supported by physiological evidence for the accumulation of sulfate within the periplasm, facilitating interaction between this low molecular weight anion and FbpA.

Synergistic anion influence on FeFbpA-X biophysical properties

Our physico-chemical characterization of FeFbpA-SO₄ shows that of all of the iron/FbpA/anion assemblies studied to date,^25,26 the sulfate anion containing assembly exhibits the lowest binding affinity for Fe³⁺ (log K′_eff = 16.2) and the highest E_1/2 for Fe^3+/2+ reduction (E_1/2 = −158 mV). The relative stability of FeFbpA-SO₄ reported here is consistent with qualitative results obtained previously by SUPREX methods.²⁷

Under our conditions, FeFbpA-SO₄ exchange with the biologically important phosphate anion has an ∼20 ms half-life, which is consistent with rapid adjustment of the synergistic anion binding with changes in periplasmic conditions. Apparently, the kinetics are controlled by rate limiting sulfate release, followed by rapid coordination of the entering anion at the iron and anion binding site. Consequently, FbpA is likely to be responsive to periplasmic anion composition on a kinetic and thermodynamic basis through synergistic anion exchange.

These physico-chemical characteristics have significant implications with respect to the possible in vivo mechanisms for periplasm-to-cytosol delivery of the essential nutrient iron. Tight Fe³⁺ binding (K_d = 10^−16.2 M) in the presence of sulfate ensures delivery of iron to the cytosolic membrane receptor FbpB/FbpC where release to the cytosol could be facilitated by a relatively facile reduction (E_1/2 = −158 mV). A modulation of this facile reduction can be brought about by anion exchange to form, for example, FeFbpA-PO₄, where the E_1/2 (−300 mV)²⁶ is less favorable (Scheme 1).

The different synergistic anions have an equivalent influence on the observed range for K′_eff and E_1/2 (∼14 kJ range for both, as a function of the synergistic anion). This suggests that the variation in E_1/2 comes largely from differences in ground state stabilization of the Fe³⁺ oxidation state in FeFbpA-X. Figure 5 illustrates the trend in log K′_eff plotted as a function of E_1/2 for the various anion containing forms of FeFbpA-X. The dashed line represents the best fit of a rearranged form of the Nernst equation (equation 17) to the data with the slope of the plot fixed at –n/58 = −0.017 mV⁻¹. Small

$${\mathrm{E}}_{1∕2}={{\mathrm{E}}^{\mathrm{o}}}_{\mathrm{aq}}+(\text{-}0.058∕\mathrm{n})\log ({{\mathrm{K}}^{\prime }}_{\mathrm{eff}}∕{\mathrm{K}}^{\mathrm{II}})$$ (17)

deviations of the data points from this line most likely represent small fluctuations in the Fe²⁺ binding to FbpA (i.e. K^II) and experimental uncertainty.

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.

Structural analysis of FbpA and synergistic anion interactions

It is important to undertake a careful analysis of the nature of both iron and anion binding by FbpA due to: 1) the requirement of an exogenous anion for tight Fe³⁺ sequestration; 2) the fact that several anions can satisfy this requirement; and 3) the observation that varying the anion identity can modulate several physico-chemical characteristics of the FeFbpA-X assembly. For this analysis, it is useful to consider the crystal structures of holo-FbpA-phosphate and apo-HitA-phosphate as models in evaluating the effects of different synergistic anions on the kinetic and thermodynamic stability of FeFbpA-X. In doing this, we note the similar size and shape of phosphate, sulfate and arsenate. Illustrated in Figure 1A is a simplified version of phosphate anion interactions with the protein anion binding site and backbone, as well as coordination to iron. Phosphate directly interacts with the N-terminus (N-cap) of an α-helix that is adjacent to the iron binding site through the backbone amide of Ala141 and the hydroxyl of Ser139 (both from the C-terminal domain), the iron, and Gln58 from the N-terminal domain of the protein. Glu57 and His9 from the N-terminal domain both bind to the iron. Tyr195 and Tyr196 from the C-terminal domain complete the protein binding complement to iron. In contrast, the bonding network of the apo protein shown in Figure 1B is simplified and consists of only C-terminal domain interactions with phosphate oriented at a three-point docking site on the N-cap of the aforementioned α-helix where directional hydrogen bonds from the N-cap confer affinity for tetrahedral anions. The location of the binding site of phosphate in both the apo and holo-FbpA structures is similar to that of the sulfate anion in the periplasmic binding protein, “sulfate binding protein (SBP)” ⁵⁶ and phosphate (and arsenate) in “phosphate binding protein (PBP)”.⁵⁷ While SBP and PBP exhibit selectivity for specific tetrahedral anions, FbpA does not discriminate and will bind sulfate, phosphate and arsenate.

Several attributes of synergistic anions must be compared in order to determine which contributions are most likely to significantly influence FeFbpA-X ternary assembly stability. One factor in synergistic anion selectivity is shape. Phosphate, arsenate and sulfate anions are all tetrahedral, thus FbpA should show no spatial preference between them as discussed above.

The protonation state of the anion should also be considered as electrostatic interactions appear to play some role in anion selection (c.f. K_d values in Table 1). Examining this charge selectivity within anion binding proteins, SBP binds only fully ionized tetrahedral oxydianions (sulfate, selenate and chromate),⁵⁷ while PBP exhibits a preference towards the partially protonated phosphate and arsenate (dibasic and monobasic) species. Hydrogen bond arrays reported for SBP and PBP may provide the key for understanding (1) why FbpA will bind sulfate and phosphate and (2) why sulfate binding to FbpA is weaker in comparison to phosphate and arsenate. Phosphate is held tightly (K_d = 1 μM) in PBP by twelve strong hydrogen bonds and sulfate (K_d = 0.1 μM) is held in SBP by seven.^48,57 Only one residue in PBP (Asp56) is responsible for discriminating between sulfate and phosphate. The fully ionized sulfate dianion is repulsed by the negatively charged PBP-Asp56 whereas either a monobasic (H₂PO₄⁻) or dibasic (HPO₄²⁻) phosphate will bind with this hydrogen bond acceptor.⁴⁸ All of the hydrogen bonding interactions with sulfate in SBP are with polar residues and there is no group in the binding site that works as an hydrogen bond acceptor.

A similar hydrogen bonding activity defines the ability of anions to bind to FbpA at the N-cap of the anion binding helix, specifically the Ser139 hydroxyl residue. This residue plays a vital role in anion binding affinity due to rotational degrees of freedom and the ability to donate or accept hydrogen bonds.⁴⁸ Elimination of this interaction in FbpA and SBP greatly reduces anion binding affinity.^58,59 The anion interactions in FbpA are reduced in comparison (Figure 1) and sulfate and phosphate binding is weaker than that observed for the SBP and PBP anions (i.e K_d of apo-FbpA-X on the order of mM) (Table 1). The presence of Glu57 (Figure 1) in FbpA should play a role similar to that of Asp56 in PBP, resulting in discrimination against sulfate. This is not the case (as shown in Figure 1) as a result of (1) a distance of greater than 7Å between Glu57 and the anion binding site on the other domain of apo-FbpA, (2) the presence of a water molecule between Glu57 and the anion in holo-FbpA, and (3) the presence of Fe³⁺ in holo-FbpA which favorably accepts negative charge from Glu57 through oxygen lone pair donation to a coordinate covalent bond. Although the effect is muted somewhat by the presence of Fe³⁺ and water, the proximity of Glu57 and its negative charge may be the predominate factor in the differences between sulfate and phosphate in FbpA. Mismatched hydrogen-bonding pairs such as sulfate and Glu57 have been reported to represent a binding energy barrier of 6−7 kcal/mol.⁶⁰ While the interactions shown in Figure 1 for FbpA and discussed for SBP and PBP are structural in nature,^21,38 they clearly form a platform from which the relative effects of these interactions and their possible origins can be discussed further in the following paragraphs.

In addition to shape and protonation state, a third factor in anion selection is electrostatic field strength. A higher charge density correlates to a more negative hydration enthalpy, such as with sulfate, which results in a stabilization of the primary hydration shell (Table 1). More strongly hydrated ions tend to remain in the bulk solvent. These aspects of synergistic anion interactions with FeFbpA will be discussed further below with the help of extra-thermodynamic relationships. In doing so, it is helpful to break down the analysis of the variations in FeFbpA-X stability with differing Xⁿ⁻ into the component parts of binding interactions: iron-anion (Fe³⁺////Xⁿ⁻); iron-protein (Fe³⁺////FbpA); and protein-anion (FbpA///Xⁿ⁻).

The influence of iron-anion, iron-FbpA and FbpA-anion interactions on the biophysical properties of the FeFbpA-X assembly

The Fe³⁺///Xⁿ⁻ interaction alone is apparently not responsible for the variations in FeFbpA-X assembly stability with the identity of Xⁿ⁻. No correlation was observed between log K′_eff and pFe, where pFe was taken as a comparative measure of the relative affinity of Xⁿ⁻ anions of different denticities for Fe³⁺ (Figure S1, Supporting Information).⁶¹ Fe³⁺///Xⁿ⁻ interaction is evidently not as important as Fe³⁺///FbpA or FbpA///Xⁿ⁻ interactions in determining the observed trends in FeFbpA-X stability.

The protein-anion, FbpA///Xⁿ⁻, interactions are reflected in the K_d values measured for various anions bound to apo-FbpA, which span several orders of magnitude (Table 1). In analyzing trends for anion binding to apo-FbpA, Figure 6A illustrates that while electrostatics play a strong role in determining the variations of FbpA affinity for different anions, the situation is more complicated.⁶² This complication is evident in the significant deviation of SO₄²⁻ from the correlation, which may be due to its very negative hydration enthalpy (Table 1). The influence of hydration enthalpy on anion binding to apo-FbpA is illustrated by the trend in Figure 6B, where Δ(ΔG) for anion binding (Δ(ΔG_FbpAX), associated with K_d⁻¹)⁶² corrected for anion charge (Z_ave) is plotted as a function of the anion hydration enthalpy calculated using Z_ave at pH 6.5.

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 62.

In addressing the electrostatic influence on variations in FeFbpA-X assembly stability, we find that combining the influence of both anion charge distribution (Z_ave) and protein–anion affinity (K_d) on K′_eff provides a useful correlation. This is illustrated in Figure 7, where the relative anion binding affinity for apo-FbpA is corrected for variations in anion charge (at pH 6.5) by expressing the x-axis as Δ(ΔG_FbpAX)/Z_ave.⁶² This correlation suggests that variations in FeFbpA-X assembly stability are influenced by protein–anion interactions corrected for electrostatics; that is, variations in FeFbpA-X stability correlates with FbpA///Xⁿ⁻ interactions due to van der Waals and H-bond interactions. The lack of a correlation between log K′_eff and Z_ave or log K_d⁻¹ (Supporting Information Figure S2A and S2B) indicate that simple electrostatic binding variations and/or apo-FbpA-anion binding alone are not responsible for the anion identity dependent fluctuations in K′_eff. One factor that may contribute to the observed difference in Δ(ΔG_FbpAX)/Z_ave between phosphate and sulfate (Figure 7) may result from the mismatched hydrogen-bond pairing involving Glu57, mentioned above. Figure 8 illustrates that variations in FeFbpA-X assembly stability correlate with calculated anion hydration enthalpy, where data are available.

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 62.

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 62.

As a result of this analysis of iron///anion///protein interactions, we can speculate on FbpA binding properties for other anions. Of particular interest is carbonate, the physiological synergistic anion of hTf. While carbonate is able to facilitate stable iron sequestration at the analogous iron binding site in hTf, this anion is unable to bind iron in FbpA at pH 6.5,²⁶ although there is evidence that it will bind at higher pH.^28,63 We attribute these observations to the fact that at pH 6.5, carbonate Z_ave is −0.6, less negative than at pH 8 and considerably less negative than the other anions that function as synergistic anions for FbpA. From the data in Figure 6A, for example, we would predict that apo-FbpA affinity for carbonate at pH 6.5 to be at least an order of magnitude less than for phosphate.

Summary and conclusions

In conclusion, we have verified the ability of sulfate to act as a synergistic anion to facilitate thermodynamically stable sequestration of Fe³⁺ by FbpA through determination of the FeFbpA-SO₄ binding constant and reduction potential. These sulfate anion findings extend the free energy range by which the synergistic anion can modulate FeFbpA-X complex stability to ∼14 kJ (Δ(ΔG_FeFbpAX)),⁶² while also extending the range of anion influence on (Fe³⁺/Fe²⁺)FbpA-X reduction free energies to ∼14 kJ. An examination of the apo-HitA and holo-FbpA crystal structures reveal details of the anion binding interactions, and with further thermodynamic analysis of the protein assemblies, suggest that the difference in stability of the FeFbpA-X assemblies with varying anions is predominately attributed to the inherent properties of the anion (e.g. ΔH_hyd, Z_ave), along with the anion-protein interactions. Although variations in K_d for apo-FbpA-X with variation in anion also extend over a ∼14 kJ range (Δ(ΔG_FbpAX)), similar to that noted above for K′_eff and E_1/2, the complexity of these anion interactions, the extent to which they modulate the stability of the FeFbpA-X ternary complex, and their role in the overall delivery of iron in pathogenic bacteria, are yet to be fully understood. In conjunction with the diverse composition of the periplasmic space, our observed kinetics of anion exchange and the anion influence on K′_eff and E_1/2 support the hypothesis of redox facilitated iron delivery to the cytosol, along with the implication of a crucial role for anion identity in FbpA mediated bacterial iron acquisition.