Fe3+ Coordination and Redox Properties of a Bacterial
Transferrin*
Céline H.
Taboy
,
Kevin G.
Vaughan§,
Timothy A.
Mietzner§,
Philip
Aisen¶, and
Alvin L.
Crumbliss
From the
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
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ABSTRACT |
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.
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INTRODUCTION |
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 |
Isolation and Purification of Ferric-binding
Protein--
Recombinant nFbp was prepared by a variation
of the method described previously (15). Briefly, Escherichia
coli strain DH5
MCR (Life Technologies, Inc.) containing the
plasmid pSBGL (16), which encodes nFbp as a fusion protein
with lacZ
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 (
= 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;
= 392 and 598 nm (mediator signal) and
= 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 |
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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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.
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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.
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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 (
= 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.
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(A)
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(B)
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The overall spectroelectrochemical system has been summarized in
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.
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DISCUSSION |
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).
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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
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.
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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.
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(Eq. 1)
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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.
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(Eq. 2)
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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.
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.
 |
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