Fe3+ Coordination and Redox Properties of a Bacterial Transferrin*

Céline H. TaboyDagger , Kevin G. Vaughan§, Timothy A. Mietzner§, Philip Aisen, and Alvin L. CrumblissDagger ||

From the Dagger  Department of Chemistry, Duke University, Durham, North Carolina 27708, the § Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, and the  Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461

Received for publication, June 1, 2000, and in revised form, October 11, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The Fe3+ binding site of recombinant nFbp, a ferric-binding protein found in the periplasmic space of pathogenic Neisseria, has been characterized by physicochemical techniques. An effective Fe3+ binding constant in the presence of 350 µM phosphate at pH 6.5 and 25 °C was determined as 2.4 × 1018 M-1. EPR spectra for the recombinant Fe3+nFbp gave g' = 4.3 and 9 signals characteristic of high spin Fe3+ in a strong ligand field of low (orthorhombic) symmetry. 31P NMR experiments demonstrated the presence of bound phosphate in the holo form of nFbp and showed that phosphate can be dialyzed away in the absence of Fe3+ in apo-nFbp. Finally, an uncorrected Fe3+/2+ redox potential for Fe-nFbp was determined to be -290 mV (NHE) at pH 6.5, 20 °C. Whereas our findings show that nFbp and mammalian transferrin have similar Fe3+ binding constants and EPR spectra, they differ greatly in their redox potentials. This has implications for the mechanism of Fe transport across the periplasmic space of Gram-negative bacteria.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Iron is an essential nutrient for almost all living cells (1). Different organisms have developed specialized mechanisms to acquire, transport, and assimilate iron. The sequestration of iron is critical because aqueous unchelated iron in an aerobic environment will precipitate as an insoluble Fe3+ hydroxide (2) and can catalyze chemical reactions leading to the production of reactive oxygen species via the Haber-Weiss cycle (1). It is therefore essential for biological systems to control the chemical environment of iron.

Disease-causing bacteria are in constant battle with their hosts for essential iron. Humans and other mammals sequester extracellular iron in serum using transferrin and in secretions using lactoferrin. Pathogenic bacteria have developed efficient iron-uptake systems to compete with these host iron-binding proteins. For many pathogenic bacteria, iron-uptake efficiency is directly related to virulence (3, 4). Two fundamentally different strategies are used by pathogenic bacteria to scavenge iron (3). The first strategy is to synthesize low molecular weight organic Fe3+ chelators (siderophores) that are secreted into the surrounding environment for iron uptake. Using this mechanism iron is transported into the bacterial cell in the form of an iron-siderophore chelate. This strategy has the advantage of being nonspecific and therefore easily used for diverse sources of iron. Some pathogenic organisms have developed multiple siderophore-based systems to allow for a more efficient iron uptake from a wide range of situations. Siderophores are generally polydentate chelators with high affinity Fe3+-specific binding groups such as hydroxamic acids or catechols (5). However, siderophore-mediated iron acquisition processes are energetically demanding because of the constant need for synthesis and metabolism of these low molecular weight ligands.

The second strategy employed by some pathogenic organisms, more specific with regard to iron and energetically more favorable, is an uptake mechanism based on ferric-binding proteins that sequester iron. Gram-negative pathogenic bacteria such as Neisseria and Haemophilus, which cause ailments ranging from gonorrhea to respiratory tract infections to meningitis, acquire iron from the host proteins lactoferrin and transferrin using surface receptors. Iron is then released from these proteins and transported across the bacterial outer membrane and into the periplasmic space where it is bound by the ferric-binding protein and subsequently transported across the cytoplasmic membrane. Ferric-binding proteins (Fbps)1 are found in Haemophilus influenzae (hFbp), Neisseria gonorrhoeae, and Neisseria meningitidis (nFbp) (3). Although these bacteria express several different transferrin and lactoferrin receptors, Fbps appear to be the nodal point for transport of iron across the periplasmic space to the cytoplasmic membrane. hFbp and nFbp have been shown to possess 80% amino acid sequence identity (3).

The transferrins are a class of iron-binding proteins that are characterized by a high thermodynamic affinity for Fe3+ (3, 6, 7). Human transferrin (hTf) is a bilobal protein with a single Fe3+ binding site per lobe. Each hTf binding site contains two tyrosines, a histidine, and an aspartate. The fifth and sixth Fe3+ coordination sites are occupied by exogenous CO32- (8, 9). Fbps and transferrin lobes are functionally and structurally homologous. The recently published x-ray crystal structure of hFbp (also referred to as HitA) from H. influenzae (10) and of nFbp (also referred to as FbpA) from N. meningitidis (11) provides a convincing argument that these proteins function as bacterial transferrins. From previous work reported on nFbp (3), it is clear that structural and functional similarities exist between the transferrins and the Fbps. The inner coordination shell around iron in both nFbp (11) and hFbp (10) show a striking resemblance with the inner coordination sphere of iron in mammalian transferrins. These Fbps contain only a single Fe3+ binding site composed of four donor groups from protein side chains: two tyrosyl oxygens, one histidyl nitrogen, and one glutamyl oxygen. The fifth and sixth positions are also occupied by exogenous ligands and have been found to be PO43- (monodentate) and H2O in both Fbps. The Fe3+ binding constant for hFbp and nFbp was estimated to be comparable to that for each hTf binding site (6, 12, 13). A comparison of Fbp sequences with each domain of the transferrins reveals no remarkable primary structure similarities, yet both proteins reversibly bind Fe3+ with substantial affinity and play related roles in the transport of Fe3+ within the spaces between two membranes.

The mechanism of Fe3+ acquisition at the outer membrane/periplasm interface and the mechanism of Fe3+ deposition at the periplasm/cytoplasmic interface in Gram-negative bacteria is of general scientific interest and of medical significance. Although it is postulated that the release of Fe3+ from Fbps in vivo is facilitated by binding to a permease on the bacterial cytoplasmic membrane and that release is accelerated by a pH effect (3), essentially no work has been done in this area at the molecular mechanism level.

We have embarked on a series of experiments that directly relate to a further chemical characterization of the Fe3+ binding site in nFbp and the ability, in a thermodynamic sense, of this protein to tightly bind and release Fe3+. These results are presented against a background of similar data from the literature for mammalian transferrins. Here, we report how subtle differences in the Fbp binding site profoundly influence the Fe3+/2+ redox potential (E1/2) for Fe-nFbp when compared with hTf. The E1/2 is a relevant parameter for iron transport because high-spin Fe2+ exchanges ligands more rapidly than Fe3+ and forms less stable complexes with hard donor ligands (14). By determining the redox properties of this nFbp system, we are able to establish if reduction within the periplasm at the cytoplasmic membrane is thermodynamically feasible, and consequently whether redox may play a role in the transport mechanism of Fe3+ across the periplasmic space to the cytosol. These results, along with relevant EPR data and the Fe3+ binding constant for nFbp, allow us to chemically characterize the Fe3+ binding site of nFbp. Comparison of these data with corresponding data for mammalian transferrin studies enable us to develop a molecular paradigm for iron transport by Fbps in terms of a possible Fe3+/2+ couple that could trigger iron release in vivo.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation and Purification of Ferric-binding Protein-- Recombinant nFbp was prepared by a variation of the method described previously (15). Briefly, Escherichia coli strain DH5alpha MCR (Life Technologies, Inc.) containing the plasmid pSBGL (16), which encodes nFbp as a fusion protein with lacZalpha were grown to mid-log phase in liquid medium at 37 °C and then plated on 20 × 40-cm plates of Luria Broth agar containing 100 µg/ml ampicillin and grown for 3 days at room temperature. The bacteria were harvested by scraping the plate and washing in phosphate-buffered saline. The bacteria were pelleted, suspended in 500 mM Tris/2% hexadecyltrimethylammonium bromide (Sigma) at pH 8.0, and shaken at 37 °C for 2 h. Lysates were centrifuged, stored overnight at 4 °C to precipitate detergent, and again centrifuged. The cleared lysate was diluted with 6 volumes of distilled H2O, loaded onto a 2.5 × 6-cm CM-Sepharose CL-6B column (Sigma) equilibrated with 10 mM Tris, pH 8.0, and eluted using a NaCl gradient. To further concentrate the protein, peak fractions were combined, diluted, and loaded onto a 1 × 3 cm CM-Sepharose column, then eluted with a sharp NaCl gradient (5-column volumes from 0 to 1 M NaCl). Iron-free protein was prepared by washing the second column containing bound protein with 10 mM citrate at pH 6. The column was then eluted with a sharp NaCl gradient. In each case, purified protein was dialyzed against 50 mM MES, 200 mM KCl, pH 6.5, and stored frozen at -80 °C.

Determination of Fe3+ Binding Constant-- Conditional or effective stability constants for Fe3+ binding were determined by an ultrafiltration method, a variation on dialysis procedures (6, 17). As in these previous studies, citrate was used as a competing chelator with known stability constants for Fe3+ binding to permit evaluation of free Fe3+ concentrations in equilibrium with Fe3+nFbp. Briefly, 30-35 µM solutions of iron-free nFbp were incubated at 25 °C in 80 mM KCl, 20 mM MES, 350 µM phosphate, 30 mM citrate, pH ~6.5 with varying concentrations of 59Fe- or 55Fe-labeled FeCl3. These buffer conditions were chosen based on the reported slightly acidic nature of the periplasmic space (18). Aliquots of preparations were taken at 5 days, a time amply sufficient for equilibria to be attained in human serum transferrin. Aliquots were ultrafiltered through a cellulose membrane with a 10-kDa cutoff (Centricon 10, Millipore) for determinations of total and bound iron, the latter derived from the differences between filtrates and retentates. pH measurements of the final equilibrated solutions were taken and calculations of effective binding constants (for the experimental conditions selected to mimic the periplasmic space) were based on equilibria previously presented (6) and are given in Table I.

EPR Methods-- A Bruker 200D EPR spectrometer with ESP 300 upgrade was used to record EPR spectra at 77 K using a liquid nitrogen insert Dewar and standard rectangular cavity. Instrumental settings were as follows: microwave power, 10 milliwatt; microwave frequency, 9.297 GHz; receiver gain, 6.3 × 105; conversion time and time constant, 655 ms; sweep time, 671 s. The X-band EPR spectrum of recombinant nFbp and reconstituted nFbp in the presence of bicarbonate are presented in Fig. 1, A and B, respectively.

31P NMR Methods---A Varian Inova-400 NMR spectrometer was used to record 31P spectra using 20 mM (CH3)4PCl as a standard. 31P spectra of apo- and holo-nFbp (~ 0.8-1.1 mM) were obtained in 0.05 M MES, 0.2 M KCl at pH 6.5 with 3 drops of D2O added as an instrument lock. Equilibrium dialysis experiments were performed using a Slide-A-Lyzer® cassette (Pierce).

Spectroelectrochemistry-- The electrochemical mediator, methyl viologen (MV2+) dichloride hydrate (Aldrich, 98%), was dissolved in a 0.05 M MES (Aldrich, 98%) and 0.2 M potassium chloride (KCl, Fisher Scientific) buffer solution adjusted to pH 6.5 to give a concentration of 4.5-5.5 mM. MES was selected as the buffer for its noncomplexing properties and stability, as well as the absence of spectral and electrochemical interferences. A concentration of 0.2 M KCl was used as the background electrolyte during the spectroelectrochemical experiments. Nanopure water was used at all times, and all solutions were stored under a N2 atmosphere at 4 °C.

For each experiment, a solution containing 0.2 M KCl, 2.5 mM MV2+, and 0.05 M MES at pH 6.5 in a 5-ml pear-shaped flask was connected to a vacuum line for repeated pump-purging with N2, followed by addition of ~0.4 mM nFbp with 1:7 protein/mediator mol ratio with additional pump-purging and gentle swirling to minimize bubbling. All chemicals were free of any electrochemically detectable impurities and used as received.

Spectroelectrochemical experiments were carried out in an anaerobic optically transparent thin layer electrode (OTTLE) cell made of two 1 × 2-cm pieces of 52 mesh gold gauze placed between the inside wall of a 1-cm path length cuvette and a piece of silica glass held in place by a small Tygon spacer positioned so as not to interfere with the spectral measurement (19). This setup results in an optical path length of 0.025 ± 0.005 cm, as calculated from the absorbance of the fully oxidized spectrum of nFbp at 481 nm (epsilon  = 2430 mM-1 cm-1) (12). The cell was kept anaerobic by capping the cuvette with a septum that allowed no air to enter but permitted a continuous flow of N2. A gold wire connected to the gold gauze working electrode was inserted through the septum. A salt bridge constructed from a Pasteur pipette plugged at the bottom with an agar gel (Aldrich) was prepared so as to connect the Ag+/AgCl reference (Bioanalytical Systems Inc.) electrode to the working electrode. The salt-bridge solution was composed of 0.2 M KCl in 0.05 M MES at pH 6.5 and was degassed and then flushed with N2 for 1 h. The gold auxiliary electrode was in a separate compartment as well, connected to the working solution through an agar gel. The OTTLE cell was purged with N2 for 15 min prior to injecting the protein solution. A continuous flow of N2 was maintained over the cell during the spectroelectrochemical experiment to prevent O2 contamination.

Spectroelectrochemical experiments were conducted using solutions consisting of ~0.4 mM nFbp, 2.5 mM mediator in 0.05 M MES, 0.2 M KCl at pH 6.5. In a typical experiment, ~0.5 ml of the working solution was injected at the bottom of the OTTLE cell via a gas-tight syringe. The cell was then placed in the temperature-controlled cell holder at 20.0 ± 0.1 °C of a CARY 100 Bio UV-Vis-NIR spectrophotometer (Varian) linked to a PAR model 75 potentiostat. Spectral scans were made from 360 to 700 nm at different applied potentials, with specific absorbance changes monitored at the following wavelengths; lambda  = 392 and 598 nm (mediator signal) and lambda  = 481 nm (Fe3+nFbp). No detectable change in the absorbance of fully oxidized nFbp (Ao) was observed between +100 and -200 mV. The decrease in concentration of Fe3+nFbp by reduction at various fixed electrode potentials was monitored from -200 to -400 mV in increments of 10 mV. At each potential, the equilibrium was reached within 30 min. The signal of the mediator was present at low potentials but did not interfere with the actual absorbance reading at 481 nm (Fig. 3). After each experiment, the OTTLE cell was opened to the air to allow for reoxidation of the protein, with 85-95% of the protein recovered after 12 h.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Fe3+nFbp Stability Constant-- Effective stability constants for Fe3+ binding to nFbp in the presence of PO43- were determined by an equilibrium dialysis method using citrate as a competing chelator. Data obtained at various Fe3+/nFbp ratios are summarized in Table I. The average effective binding constant (K'eff) at pH 6.5, 350 µM phosphate, and 25 °C is 2.4 (±0.9) × 1018 M-1 for the equilibrium reaction is shown in Reaction 1. 


                              
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Table I
Effective (Keff) thermodynamic constants for binding of Fe3+ to nFbp at 25 °C


<UP>Fe</UP><SUP><UP>3+</UP></SUP><SUB><UP>aq</UP></SUB>+n<UP>Fbp</UP>+<UP>HPO</UP><SUP><UP>3−</UP></SUP><SUB><UP>4</UP></SUB>⇌<UP>Fe</UP><SUP><UP>3+</UP>⋅</SUP>n<UP>Fbp<SUP>⋅</SUP>PO<SUB>4</SUB><SUP>⋅</SUP>H<SUB>2</SUB>O</UP>

<UP><SC>Reaction</SC> 1</UP>

EPR Characterization-- Recombinant nFbp gave a g' = 4.3 signal and a weaker signal centered near g' = 9 (Fig. 1A), both characteristic of high-spin Fe3+ in a strong ligand field of low (orthorhombic) symmetry. A poorly resolved splitting was evident in the g' = 4.3 line. Fe3+nFbp reconstituted from the apo protein and freshly prepared Fe(NH4)2(SO4)2 showed a similar spectrum, but addition of bicarbonate to a concentration of 17 mM enhanced the splitting in the spectrum of the reconstituted protein to 5.4 mT, producing a line similar to that given by native hTf (Ref. 20, Fig. 1B) and indicating increased axial perturbation in the zero-field tensor. A similar concentration of bicarbonate added to buffer increased the pH from 7.4 to 7.55, a change that does not alter the EPR spectrum of recombinant nFbp, indicating that the bicarbonate-induced alteration represents a specific effect of the anion rather than a simple pH change. A likely interpretation, therefore, is that bicarbonate is capable of fulfilling the anion requirement of Fbp as it is for eukaryotic transferrins.



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Fig. 1.   X-band EPR spectrum of 370 mM recombinant nFbp in 50 mM HEPES, 100 mM KCl, pH 7.4 buffer before (A) and after (B) addition of bicarbonate to a concentration of 17 mM. Microwave frequency, 9.297 GHz; microwave power, 10 milliwatts; temperature, 77 K; receiver gain, 6.3 × 105; conversion time and time constant, 655 ms; sweep time, 671 s.

31P NMR Characterization-- 31P NMR experiments were performed to determine the presence of phosphate anion bound to apo and holo nFbp. Spectra for freshly prepared apo-nFbp and reconstituted holo-nFbp were obtained in the presence and absence of competing Fe3+-chelating agents, deferriferrioxamine B, and EDTA.

A sample of recombinant apo-nFbp in 0.05 M phosphate buffer at pH 7.0 in the presence of Feaq3+ was allowed to come to equilibrium overnight to produce a pink solution (consistent with reconstituted holo-nFbp) and was then dialyzed against 0.05 M MES, 0.2 M KCl at pH 6.5 for 3 h. The 31P NMR spectrum of the resulting solution (Fig. 2A) shows that the normally sharp phosphate signal is significantly broadened and shifted downfield from 1.2 to ~2.0 ppm. This is consistent with phosphate bound to a paramagnetic metal ion with a long electron relaxation time. We attribute this signal to the Fe3+-phosphate complex, present in the holo form of nFbp, as has been established in the solid state by x-ray crystallography (11). This is also consistent with a report of a 1:1 iron/phosphate stoichiometry for nFbp purified directly from Neisseria (21).



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Fig. 2.   31P NMR spectra. A, reconstituted holo Fe3+nFbp (~1 mM) (broad peak, 2 ppm) in the presence of 20 mM (CH3)4PCl (sharp peak, 24 ppm) in 0.05 M MES, 0.2 M KCl, pH 6.5. B, apo-nFbp (~1 mM) (sharp peak, 1.2 ppm) in the presence of 4 mg EDTA and 20 mM (CH3)4PCl (sharp peak, 24 ppm) in 0.05 M MES, 0.2 M KCl, pH 6.5.

To further confirm this assignment, the holo protein complex was exposed to a competing Fe3+ chelator. Deferriferrioxamine B (Desferal®), despite its high thermodynamic affinity for Fe3+ (22), was kinetically ineffective in removing iron from the holoprotein over a 72-h period. In contrast, EDTA was successful in removing iron from Fe3+-nFbp within 3 h at 25 °C. The 31P NMR spectrum of the protein solution after dialysis against EDTA is presented in Fig. 2B and shows the presence of a sharp peak at 1.2 ppm, consistent with free phosphate in solution. The sample was then dialyzed further against 0.05 M MES, 0.2 M KCl at pH 6.5 for 3 h to determine whether the free phosphate was bound to the apo form of the protein. No detectable 31P signal was observed, confirming the absence of phosphate bound to the protein after the removal of Fe3+. Holo-nFbp was then successfully reconstituted in the presence of both phosphate and Fe3+ to confirm that the protein had not been denatured by repeated dialysis and manipulation.

Our experiments illustrate that in solution phosphate is bound to the holo protein in proximity to Fe3+ and importantly that removal of the bound Fe3+ by dialysis against EDTA results in release of the bound phosphate. This is consistent with extremely weak binding of the phosphate to the apo-nFbp structure, if it is bound at all.

Fe3+/2+nFbp Redox Potential-- The spectroelectrochemical technique involves equilibrium spectral measurements at incremental solution potentials. In our case, an increasingly negative (reducing) potential was applied to an anaerobic cell containing an optically transparent electrode, the oxidized protein, and a mediator. Spectral changes associated with a change in the oxidation state of Fe3+nFbp and methyl viologen are reported in Fig. 3. The broad band, associated with Fe3+nFbp, centered around 481 nm (epsilon  = 2430 M-1 cm-1) decreased in intensity with the application of increasingly negative potentials (Eapp) over the range -200 to -400 mV (NHE). In addition, the characteristic peaks for methyl viologen mediator (MV·+; 396 and 602 nm) started to be apparent at Eapp = -340 mV and increased throughout the end of the experiment (-420 mV). An equilibrium position was reached at each applied potential within 30 min. As the Fe3+ was reduced to Fe2+, its low affinity for apo-nFbp, as well as its rather high lability, led to the following two-step process shown in Reaction 2 (19).



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Fig. 3.   Nernst plot corresponding to the data shown in the inset for recombinant nFbp (~0.4 mM iron) with a 1:7 iron/mediator ratio at pH 6.5, 20 °C. The potentials are reported versus NHE. Inset, collection of spectra at different applied potentials (-200, -240, -280, -300, -320, -340, and -360 mV versus NHE, top to bottom) for an OTTLE cell containing a fresh sample of recombinant nFbp (~0.4 mM Fe) with a 1:7 iron/mediator ratio at pH 6.5, 20 °C.


<UP>Fe<SUP>3+</SUP></UP>n<UP>Fbp</UP>+<UP>e<SUP>−</SUP>⇌Fe<SUP>2+</SUP></UP>n<UP>Fbp</UP> (A)

<UP>Fe<SUP>2+</SUP></UP>n<UP>Fbp</UP>⇌<UP>Fe</UP><SUP><UP>2+</UP></SUP><SUB><UP>aq</UP></SUB>+<UP>apo</UP>-n<UP>Fbp</UP> (B)

<UP><SC>Reaction</SC> 2</UP>
The overall spectroelectrochemical system has been summarized in Scheme 1.



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Scheme 1.  

The relationship between the concentration ratio and the absorbance readings has previously been demonstrated (19, 23-25) and can be used to create a Nernst plot (Fig. 3). From the intercept of the Nernst plot, Erxn was found to be -290 mV (NHE), with a slope of 1.0 corresponding to a 1-electron reduction. The measured Erxn corresponds to the sum of two steps: the reduction step followed by Fe2+ dissociation, as illustrated in Reaction 2.

A value for the formal potential (Eo') associated with the reversible reduction reaction (Reaction 2A) requires correcting the observed Erxn for the dissociation equilibrium in Reaction 2B (19). The correction requires a value for Fe2+ binding to apo-nFbp, which is not experimentally available because of the sensitivity to oxidation by O2, the lability of Fe2+, and its low affinity for apo-nFbp. However, we estimate K(Fe2+) for Fe2+ sequestration by apo-Fbp at our conditions to be ~103 M-1, based on a linear free energy relationship established for Fe2+ binding by apo-hTf (26). Consequently, assuming the equilibrium constant for Reaction 2B to be 10-3 M at our conditions, we estimate the formal reduction potential (Eo') for the Fe3+/2+nFbp Reaction 2A to be ~-305 mV. This correction shows little sensitivity to the assumed equilibrium constant for Reaction 2B over the range 10-3 to 10-6 M.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

EPR spectra confirm the Fe3+ bound to nFbp to be high-spin in a strong field ligand site with low symmetry. Comparison of EPR spectra for Fe3+nFbp with those for mammalian transferrin (6, 20, 27-29) shows that the synergistic anions are different for each protein, which is consistent with the presence of PO43- (in monodentate coordination) and H2O exogenous ligands in the former case and CO32- (in bidentate coordination) in the latter, as shown in their respective crystal structures (10, 11, 30). A comparison of the inner coordination shell of Fe3+ for the two proteins is illustrated in Fig. 4. This similarity in binding site further manifests itself in the Fe3+ binding constant for nFbp at pH 6.5, 2.4 × 1018 M-1, which is within a factor of 13 of the effective binding constant of the isolated N-lobe of transferrin measured in air at pH 6.7 (1.8 × 1017 M-1) (27).



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Fig. 4.   Illustration of the iron inner coordination shell for nFbp (top, Ref. 11) and the N-terminal lobe of human transferrin (bottom, Ref. 35).

A defining structural feature for all transferrins is the dependence of Fe binding on the concomitant binding of an exogenous anion. This requirement is evident in nFbp, in which the anion binding site can accept a phosphate or apparently, a bi(carbonate). However, the change in binding site symmetry brought about by the change in exogenous ligands (H2O and PO43- for nFbp and CO32- for hTf), and the increased exposure of the nFbp binding site to solvent (Fig. 5) have resulted in a significant (~200 mV) positive shift in Fe3+/2+ redox potential for Fe3+nFbp relative to Fe3+hTf (19, 31, 32). The lower affinity of HPO42- for Fe3+ relative to that of CO32- (based on Keq for the equilibria Feaq3+ + L2- right-arrow FeL+ where L2- = HPO42-, CO32-) (33, 34) is also consistent with a more positive redox potential.



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Fig. 5.   Two-dimensional ribbon diagrams of N-terminal lobe of human transferrin (left, Ref. 35) and nFbp (right, Ref. 11) crystal structures showing the iron center exposure to solvent. Crystallographic data were obtained from the Cambridge Protein Data Bank. The graphics were produced using kinemage.

Consideration of the following thermodynamic cycle shown in Scheme 2, allows us to compare the ratios of Fe3+/Fe2+ binding affinities for hTf and nFbp. The following relationship shown in Equation 1 can be derived from this cycle at 20 °C.
<UP>E</UP><SUP><UP>0</UP></SUP><SUB><UP>complex</UP></SUB>=<UP>E</UP><SUP><UP>0</UP></SUP><SUB><UP>aq</UP></SUB>−58.16 <UP>log</UP>(K<SUP><UP>III</UP></SUP>/K<SUP><UP>II</UP></SUP>) (Eq. 1)
Using this relationship and either the corrected or uncorrected redox potentials determined here for Fe3+nFbp and previously for Fe3+ hTf (19), and 770 mV for Eaq0 yields the following approximation.
<UP>log</UP>(K<SUP><UP>III</UP></SUP>/K<SUP><UP>II</UP></SUP>)<SUB>h<UP>Tf</UP></SUB>−<UP>log</UP>(K<SUP><UP>III</UP></SUP>/K<SUP><UP>II</UP></SUP>)<SUB>n<UP>Fbp</UP></SUB>∼3.5 (Eq. 2)
Acknowledging that we must use effective equilibrium constants rather than true thermodynamic constants, we can use Eq. 2 to estimate the affinity of nFbp for Fe2+ in the periplasmic space. Using either 1.8 × 1017 M-1 (isolated N-lobe hTf, Ref. 27)) or 1.4 × 1018 M-1 (upper limit for the N-lobe site in holo-hTf, Ref. 28)) for KIII(hTf) at pH 6.7 and our value of 2.4 × 1018 M-1 for KIII(nFbp) at pH 6.5 (the 0.2 pH unit difference is not likely to have a substantial effect on the stability constants), we estimate that KII(nFbp) in the presence of phosphate is 103.5-104.5 greater than KII(hTf) in the presence of carbonate. A direct determination of Fe2+ binding by either protein has not been made, largely because of the extreme air sensitivity of the systems. However, an indirect estimate of 103 M-1 based on a LFER at pH 7.4 has been made for Fe2+ binding to the N-terminal lobe of hTf (26). KII(hTf) at pH 6.5 is expected to be lower than this value because of the proton dependence of the Fe2+ binding constant. Based on these considerations we use Eq. 2 to estimate the Fe2+ binding constant for nFbp in the presence of phosphate to be within an order of magnitude of 106 M-1. We acknowledge that the binding constants used in Eqs. 1 and 2 for this estimate are effective and not thermodynamic constants as would be required in the strict application of the thermodynamic cycle shown above. However, our calculations are internally consistent and the expressed uncertainty adequately reflects the uncertainty in the parameters used to make this estimate.



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Scheme 2.  

An implication of the positive shift in redox potential for nFbp relative to hTf is that the redox potential for Fe3+nFbp is in the range for NADH- or NADPH-driven reduction. Therefore Fe3+/2+ reduction emerges as a viable chemical mechanism for iron release from Fbp, given our estimate that Fe2+ binding to Fbp is about 12 orders of magnitude weaker than Fe3+ binding. This is in contrast to Fe3+hTf, which as a result of a more negative redox potential requires the presence of a Fe2+ chelator with an affinity constant in the range 103 M-1 or greater for NAD+ or NADP+ driven Fe3+/2+ reduction to play a role in iron release from the protein (19).

In conclusion, we have shown that the effective stability constant for Fe3+nFbp in the presence of phosphate at the periplasmic pH 6.5 is 2.4 × 1018 M-1. This is within an order of magnitude of the Fe3+ binding to N-terminal site for hTf in the presence of carbonate. EPR spectra of Fe3+nFbp in the presence of phosphate and carbonate are consistent with a displaceable phosphate ligand in the wild-type protein. 31P NMR results show that phosphate is present in the holo form of reconstituted nFbp, consistent with x-ray crystallographic results that show monodentate binding of phosphate to iron (10, 11) and that phosphate is released when Fe3+ is removed by dialysis against EDTA. As in transferrin, the synergistic anion in nFbp is bound weakly if at all in the absence of bound metal ion. The similarity in metal and anion binding properties of the bacterial and eukaryotic transferrins, despite the absence of sequence homology, is a striking example of convergent evolution. However, a significant finding in our study is that nFbp has a very different redox potential from mammalian transferrin. Our spectroelectrochemical measurements imply that Fe3+nFbp may be reduced in vivo by pyridine nucleotides making a reductive iron release feasible for iron transport from outer membrane to cytoplasmic membrane across the periplasmic space in these Gram-negative bacterial pathogens.


    ACKNOWLEDGEMENTS

We thank Dr. H. Boukhalfa (Duke University) for fruitful discussions and preliminary experiments associated with the 31P NMR results, and B. Weiner (Duke University) for generating the two-dimensional ribbon diagrams shown in Fig. 5.


    FOOTNOTES

* This work was supported by National Institutes of Health Grants 2 RO1 DK15056 (to P. A.), 1 R29 AI32226 (to T. A. M.), and RO1 HL58248 (to A. L. C.) and the American Chemical Society Petroleum Research Fund (to A. L. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed. Tel.: 919-660-1540; Fax: 919-660-1605; E-mail: alc@chem.duke.edu.

Published, JBC Papers in Press, October 11, 2000, DOI 10.1074/jbc.M004763200


    ABBREVIATIONS

The abbreviations used are: Fbp, ferric-binding protein; OM, outer membrane; CM, cytoplasmic membrane; MES, 4-morpholineethanesulfonic acid; OTTLE, optically transparent thin layer electrode.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


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