The x-ray crystal structure of the N-lobe of
human serum transferrin has shown that there is a hydrogen bond
network, the so-called "second shell," around the transferrin iron
binding site. Tyrosine at position 85 and glutamic acid at position 83 are two nonliganding residues in this network in the human serum transferrin N-lobe (hTF/2N). Mutation of each of these two amino acids
has a profound effect on the metal binding properties of hTF/2N. When
Tyr-85 is mutated to phenylalanine, iron release from the resulting
mutant Y85F is much more facile than from the parent protein.
Elimination of the hydrogen bond between Tyr-85 and Lys-296 appears to
interfere with the "di-lysine (Lys-206-Lys-296) trigger," which
affects the iron binding stability of the protein. Surprisingly,
mutation of Glu-83 to alanine leads to the absence of one of the normal
iron binding ligands; introduction of a monovalent anion is able to
restore the normal first coordination sphere. The missing ligand
appears to be His-249, as revealed by comparison of the metal binding
behaviors of mutants H249Q and E83A and structural analysis. Glu-83 has
a strong H bond linkage with His-249 in apo-hTF/2N, which helps to hold
the His-249 in the proper position for iron binding. Disabling Glu-83
by mutation to an alanine seriously disturbs the H bond network,
allowing His-249 to move away. A monovalent anion can help reestablish
the normal network by providing a negative charge near the position of
Glu-83 to reach charge balance, so that ligand His-249 is available
again for iron binding.
 |
INTRODUCTION |
Transferrins are a family of iron-binding proteins that provide
transport of iron from plasma to cells and buffer the iron level in
plasma (1-3). The full-length transferrin molecule (~80 kDa) has two
structurally similar iron binding sites distributed into two halves
termed the N- and C-lobes, linked by a short peptide. Each lobe is
subdivided into two domains, the NI and NII domain for the N-lobe and
the CI and CII for the C-lobe. X-ray crystallographic studies of four
homologous proteins, human lactoferrin (4), human serum transferrin
(hTF)1 (5, 6), rabbit serum
transferrin (7), and chicken ovotransferrin (8) have shown that the
iron coordination in the binding sites is the same, with the ferric ion
bound octahedrally to four amino acid ligands (two tyrosines, one
aspartate, and one histidine) and two oxygens of the obligate
synergistic anion, carbonate. When iron is released, the two domains of
the N-lobe rotate 63° with respect to the hinge to form an "open"
conformation (6).2 Based on
the structural analysis of apo versus iron-containing human
lactoferrin, rotation of the domains in the C-lobe is only about 15°
(10). Besides iron, other metal ions having similar ionic radius and
charge can be accommodated in the binding sites (10-12), and some
anions bearing a carboxylate group can take the place of the
"synergistic" anion, carbonate (10, 13, 14).
We have employed recombinant DNA techniques to address the specific
role a particular residue plays in the metal binding properties of
transferrin. To date all of the mutations have been to residues in the
N-lobe of human transferrin (hTF/2N), because the N-lobe and the
numerous single point mutants are produced in high yield in our
mammalian expression system. In contrast the recombinant C-lobe has
proved to be difficult to produce, and the yield has been very low
(15). The N- and C-lobes of hTF differ rather markedly in their iron
binding and release properties despite having identical ligands to
iron, but the hTF/2N very effectively mimics the behavior of the N-lobe
of the complete protein (16, 17). It is more straightforward to
characterize the iron binding behavior of an isolated lobe than to
dissect it from studies with full-length transferrin in which both
lobes are contributing to the results.
Numerous reports have demonstrated that the metal binding behavior of
transferrin is altered by introduction of single point mutations
(16-22). Not surprisingly, mutation of the binding ligands severely
affects the metal binding properties of the protein. Mutations at other
residues may also have an effect, more or less, depending on the
environment of these residues. As pointed out by Baker et
al. (23), so-called second shell effects can influence the metal
binding properties of the protein and may account for the differences
between the N- and C-lobe noted above. The second shell residues in
hTF/2N include Gly-65, Glu-83, Tyr-85, Arg-124, Ser-248, Lys-206, and
Lys-296, which form a hydrogen bond network around the metal binding
site (see Fig. 1) (6).2 This network is undoubtedly
important for the stabilization of the binding site. A structural
feature termed the di-lysine trigger (24), which involves a hydrogen
bond between Lys-206 and Lys-296 in human hTF/2N, is located in the
network. It is proposed that the "open-closed" conformation of the
protein may be modulated by the di-lysine linkage under the different
pH conditions encountered in the uptake and release of transferrin by
cells. Mutation of any of the amino acids in the second shell might be
predicted to have an influence on iron binding and has been
demonstrated previously. For example, compared with those for wild-type
hTF/2N, the thermodynamic stability of mutant G65R (19) and the kinetic stability of mutants R124S and R124K (21) significantly decrease, whereas the EPR spectra of mutants G65R, K296E, and K296Q show pronounced variation (20). Although the rate of iron release from
mutant K206R is not very different from that of wild-type hTF/2N (21),
the binding of iron to mutant K206Q is considerably stronger than that
to the normal N-lobe (18, 19).
Tyr-85 and Glu-83 are highly conserved residues in the N-lobe of
transferrins and in equivalent positions in the C-lobe (25). They are
linked together by hydrogen bonds, and both are part of the second
shell network in hTF/2N (Fig. 1). Because
of the structural importance of Tyr-85 and Glu-83, we undertook a study of the effect these amino acid residues exert on the metal binding site
and therefore produced the mutants, Y85F and E83A. Although neither of
these side chains is ligated directly to the metal center, these
mutants show considerably weaker metal binding ability. In the present
report, the unique properties of these two mutant proteins are
described, and the roles of these particular residues, Tyr-85 and
Glu-83, in metal binding and release are examined.

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Fig. 1.
The so called second shell hydrogen bond
network, around the metal binding site in the wild-type hTF/2N
(6). The four iron binding ligands are Asp-63, Tyr-95, Tyr-188,
and His-249. The corresponding ligands in C-lobe of hTF are Asp-392,
Tyr-426, Tyr-517, and His-585.
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EXPERIMENTAL PROCEDURES |
Materials--
Chemicals were reagent grade. Stock solutions of
HEPES, Bis-Tris propane, and other buffers were prepared by dissolving
the anhydrous salts in Milli-Q (Millipore) purified water and adjusting the pH to desired values with 1 N NaOH or HCl. Standard
solutions of iron(II) (1,000 µg/ml) in 5% HNO3 were
obtained from Johnson Matthey. Cupric chloride came from J. T. Baker Inc., 4,5-dihydroxy-1,3-benzenedisulfonate (Tiron) came from
Fisher, EDTA came from Mann, ethylenediaminetetrapropionate (EDTP)
(20% solution in water, ~ 0.573 M) came from Aldrich,
and nitrilotriacetate (NTA) came from Sigma. Centricon 10 microconcentrators were from Amicon. Milli-Q (Millipore) purified water
was used for all solutions and dilutions.
Molecular Biology--
The E83A N-lobe mutant (in which the
glutamic acid at position 83 was mutated to an alanine residue) and the
mutant, Y85F (in which the tyrosine at position 85 was mutated to a
phenylalanine), were constructed in the hTF/2N cDNA using
polymerase chain reaction-based mutagenesis as described in detail
previously (22). In vitro mutagenesis was performed
utilizing Bluescript flanking oligonucleotides and the following
mutagenic primers: E83A, 5'-GTGGTGGCAGCGTTCTATGGG-3'; Y85F,
5'-TGGCAGAGTTCTTTGGGTCAAAA3'.
Nucleotide sequences of each insert were determined to confirm the
introduction of the specific mutation and the absence of any polymerase
chain reaction-induced spurious mutations. As per our standard
protocol, the mutated hTF/2N cDNAs were excised from the Bluescript
vector, the ends were made blunt by treatment with the Klenow fragment
of Escherichia coli. DNA polymerase I and the blunt-ended
fragments were ligated into the SmaI site of the expression
vector pNUT. The correct orientation of the hTF/2N cDNA in the pNUT
vector was confirmed by restriction endonuclease mapping.
Expression, Purification, and Preparation of Proteins--
The
N-lobe of hTF and the single-point mutants of hTF/2N were expressed
into the medium of baby hamster kidney cells containing the relevant
cDNA in the pNUT vector and were purified as described in detail
previously with modifications (18, 26, 27). After chromatography on a
Poros 50 HQ column (PerSeptive Biosystems), the sample was applied to a
Sephacyl S-100 gel filtration column (Amersham Pharmacia Biotech). At
this stage, the E83A mutant was ~95% pure, whereas the Y85F mutant
required further purification on a Sephadex G-75 gel filtration column
(Amersham). The preparation of apo-protein samples followed the
procedure described previously (16). Fe-loaded samples were prepared by
adding a slight excess of ferric-NTA to the apo-proteins in the
presence of bicarbonate (25 mM). Two more exchanges with
the bicarbonate solution (25 mM) were performed to
eliminate the possibility of NTA taking part in the metal binding of
the proteins. The resulting samples were finally exchanged into the
desired buffer (usually 50 mM HEPES, pH 7.4). The
63Cu protein samples were prepared as described
previously (22).
Electronic Spectra--
UV-visible spectra were recorded on a
Cary 219 spectrophotometer under the control of the computer program
Olis-219s (On-line Instrument Systems, Inc., Bogart, GA). The
appropriate buffer (50 mM HEPES, pH 7.4) served as the
reference for full-range spectra from 236 to 650 nm. Difference spectra
were generated by storing the spectrum of the apo-protein as the base
line and subtracting it from the sample spectra. Anion exchange
experiments between NTA and carbonate were performed as reported
previously (16).
Kinetics of Metal Release--
As described in detail (16,
17), kinetics of metal removal from transferrin were measured by
monitoring the UV-visible absorbance change at 480 nm (for the iron
release with Tiron) or at 295 nm (for the iron release with EDTP).
Tiron (12 mM) was used as a chelator for iron release from
mutant Y85F and wild-type hTF/2N, and EDTP (10 mM) was used
as a chelator for mutants E83A and H249Q. All data were analyzed with
single-exponential functions, giving R2 values
(coefficients of determination) greater than 0.99 in every case.
Effect of Anions--
To a HEPES (50 mM) buffer
solution (pH 7.4) containing bicarbonate (1 mM) and the
target protein, Fe-E83A-CO3 (~ 40 µM), an aliquot of anion (X
) stock solution (pH 7.4)
was added. After each addition, UV-visible spectra were taken until the
max no long changed. At this point, the original protein
(yellow) was completely converted to a new species,
Fe-E83A-CO3(X) (orange or pink).
About 2 min was required to reach equilibrium after each addition.
After conversion, the mixture was concentrated in a Centricon 10 microconcentrator and then exchanged into the desired buffer (50 mM HEPES, pH 7.4, 3 × 2.5 ml). The resulting samples
were used for iron release by EDTP.
EPR Spectroscopy--
Frozen solution EPR spectra of copper and
iron transferrins were obtained as described in detail elsewhere
(22).
 |
RESULTS |
Iron Binding and Release--
As demonstrated in our previous
studies, electronic spectroscopy is a simple and effective method to
study the metal binding behaviors of transferrin (16, 22). Specific
binding in transferrin with a transition metal such as iron or copper
generates an absorption band in the visible region and two or three
absorption peaks in the UV region (observable in difference spectra),
which can be used to monitor the interaction between the metals and the
protein. The UV-visible spectra for the hTF/2N mutants, Y85F and E83A, indicated that these proteins are able to bind iron specifically. Some
of the spectral parameters including those for mutants H249Q, D63A,
Y95F, and wild-type hTF/2N are listed in Table
I. Compared with the wild-type hTF/2N
(
max = 472 nm, pink), mutants Y85F and H249Q
have slightly blue-shifted
maxs, whereas mutants E83A, D63A, and Y95F have significant blue shifts of 52, 58, and 62 nm
respectively and are yellow in color. The
max
derives from the charge transfer between phenolate (
) and the metal
ions (d
*) and therefore reflects the interaction between tyrosine
ligands and the iron center (28-30). The blue shift of the
max suggests that the iron coordination sphere in mutant
E83A has been seriously perturbed by the mutation, resulting in a
strengthened Fe-O(Tyr) interaction, similar to that found for mutants
D63A and Y95F (16, 22).
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Table I
A comparison of the spectral characteristics for recombinant wild type
(WT) and mutants of hTF/2N (iron saturated)
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An inference for the unusual impact of changing the glutamic acid at
position 83 to an alanine is that a binding ligand is lost due to the
mutation. This implies that mutant E83A might display
NTA2
preference upon binding with iron, as
seen with mutants D63A and Y95F, which are both missing a ligand (16,
22). An
NTA2
-CO32
anion exchange experiment was therefore performed to test this possibility. The resulting exchange spectra featured an almost identical spectral pattern to those produced with the Asp-63 mutants (16). This titration showed that one equivalent of
NTA2
was able to replace
CO32
instantly, but the reverse
required more than a 200-fold molar excess of
HCO3
, and that the anion exchange occurs
via an intermediate species.
EPR measurements were carried out to provide additional evidence as to
the nature of the iron binding in these mutants. The EPR spectrum for
ferric Y85F (Fig. 2) is quite similar to
that for wild-type hTF/2N, showing three peaks near g' = 4.3 corresponding to a slightly axial system (20). This result indicates
that the mutation of the tyrosine at position 85 to a phenylalanine has
little effect on the coordination of iron in the mutant. In contrast,
the spectrum for the ferric E83A mutant displays a pronounced change to
a purely rhombic spectrum (Fig. 2), suggesting that a very different
iron binding mode has been adopted. In all cases where a known protein
ligand had been mutated to a noncoordinating amino acid residue,
i.e. D63A, Y95F, and H249Q, a purely rhombic iron(III) EPR
signal was obtained (Fig. 2). In contrast, mutation of His-249 to
glutamate (i.e. H249E), which can likewise coordinate the
iron, resulted in a variant that showed axial character in its EPR
signal (20). These observations support the hypothesis that one amino
acid ligand is lost in the mutant E83A, namely His-249 (see below),
perhaps being replaced by H2O or OH
.

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Fig. 2.
X-band (9.38 GHz) EPR spectra of frozen
samples of 90% iron saturated hTF/2N and various mutants. Protein
samples (~0.3 mM), HEPES (~0.1 M),
NaHCO3 (~10 mM), pH 7.5, temperature 90 K. The instrument settings are similar to those given elsewhere
(20).
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The kinetics of iron release were measured to determine the labilities
of iron binding in the mutant proteins. A comparison of rate constants
for iron release from mutant Y85F and wild-type hTF/2N by Tiron is
presented in Table II. In general, iron
release from mutant Y85F is unexpectedly facile. In the absence of
chloride, iron release from mutant Y85F was 7-fold faster than that
from the wild-type hTF/2N, despite the fact that electronic and EPR spectra did not show much difference between these two proteins. Interestingly, chloride had a negative effect on the iron release from
wild-type hTF/2N but a sharp positive effect for the mutant Y85F. In
the presence of [Cl
] at 0.14 and 0.50 M,
iron release from Fe-Y85F was accelerated ~2.5 and 5 times,
respectively. Iron release from mutant E83A was even faster. Tiron
removes iron from mutant E83A instantly. To measure iron release from
this mutant, it was necessary to use a much weaker chelator, EDTP,
having a logK = 14.4 (31) for iron(III) (see below).
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Table II
A comparison of rate constant (min 1) for iron release
from mutant Y85F and wild-type hTF/2N by Tiron (12 mM)
HEPES buffer (50 mM, no NaHCO3), pH 7.4, 25 °C.
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Effect of Monovalent Anions on Mutant E83A--
Another unexpected
phenomenon is that monovalent anions (Ac
,
F
, Cl
, NO3
, and
ClO4
, referred to as
X
) are able to affect the iron binding
properties of mutant E83A. When one of these anions was added to a
solution containing the protein Fe-E83A-CO3, the color was
converted from yellow to orange or pink. After conversion, the
maxs shifted 36, 36, 46, 46, and 50 nm, respectively,
suggesting a new species, namely Fe-E83A-CO3(X) (see below), was generated upon conversion. Fig.
3A shows typical spectral
changes when aliquots of KClO4 were added into a HEPES solution, pH 7.4, containing Fe-E83A-CO3. Fig.
3B is the difference spectra produced by subtracting the
original Fe-E83A-CO3 spectrum from the whole family of
spectra. Plotting the difference absorbance (
A) of the
positive peak against the concentration of perchlorate yielded Fig.
3C. The saturation plot in Fig. 3C provided the
absorbance maximum (
Amax) of saturated anion
binding and the anion concentration for 90% conversion. Hill plots:
log(
/(1
)) = n × log X
log Kd, where
=
A/
Amax, X = anion
concentration, and n = slope (Fig. 3D),
yielded the dissociation constant for anion binding
(Kd) and the number of anions bound (n).
Table III lists the Kd
and n values, including the data of the kinetic stability
for all the cases. The amount of anion required for 90% conversion
decreases in the order Ac
> F
> Cl
> NO3
> ClO4
, corresponding to the dissociation
constant Kd values. Iron release from the original
mutant and the new species by EDTP follows the same order in terms of
lability, Fe-E83A-CO3 > Fe-E83A-CO3(Ac
) > Fe-E83A-CO3(F
) > Fe-E83A-CO3(Cl
) > Fe-E83A-CO3(NO3
) > Fe-E83A-CO3(ClO4
). Of greater
interest, the n values for all cases are very close to 1, suggesting that a single molecule of anion is involved in the
conversion. Identical experiments with the divalent anions SO42
and
HPO42
did not give similar
results. These divalent anions slowly removed iron from mutant E83A
rather than stabilizing the protein. No such behavior with regard to
the anions is observed in the wild-type hTF/2N or in the Y85F
mutant.

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Fig. 3.
Typical UV-visible titration spectra and Hill
plots of anion binding to mutant E83A. A, the spectral
change of anion effect. Upon the addition of aliquots of
ClO4 into a HEPES solution containing
Fe-E83A-CO3, the spectrum switched gradually from pattern
a to b. HEPES (50 mM),
NaHCO3 (1 mM), pH 7.4. B, the
difference spectra produced by subtracting spectrum a from
the whole family of spectra. Both positive and negative peaks can be
used for the following Hill plots. C, saturation plot of
A, taken from the positive peak in B, against the anion
concentration used. D, Hill plot using log ( /(1 )) = n × log X log Kd,
where = A/ Amax,
X = anion concentration and Kd = dissociation constant.
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Fig. 4 shows the influence of KCl on the
iron(III) EPR spectrum of mutant E83A. Chloride converts the rhombic
signal of Fe-E83A-CO3 to the spectrum of
Fe-E83A-CO3(Cl
), which has the same features
as that of the native protein, Fe-hTF/2N-CO3
(cf. Figs. 2 and 4). This result suggests that, in the
presence of chloride, a normal first coordination sphere has been
reestablished in the mutant, a finding consistent with the visible
spectral data. A similar spectral change is observed with KF; however,
unlike chloride, fluoride slowly strips iron from the protein (data not
shown). Furthermore, no 19F superhyperfine splitting is
seen in the EPR spectrum of Fe-E83A-CO3(F
),
arguing against direct coordination of fluoride to the iron in this
complex.

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Fig. 4.
Iron EPR spectra of Fe-E83A showing the anion
(Cl ) effect. Protein samples (~0.3
mM), HEPES (~0.1 M), NaHCO3
(~10 mM), pH 7.5, temperature 90 K.
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Copper(II) Binding--
Difference UV-visible spectra for the
binding of copper(II) to the mutants Y85F and E83A, together with those
for mutant H249Q and wild-type hTF/2N, are shown in Fig.
5. These spectra were taken after the
addition of 1 equivalent of copper(II) to the corresponding
apo-proteins. Mutant Y85F shows specific copper binding, even though
there are two absorption bands in its difference spectrum compared with
three absorption peaks in the spectrum of wild-type Cu-hTF/2N. In
contrast, the spectra for mutants E83A and H249Q reveal that copper is
not specifically bound under the identical conditions; the small
positive absorption in the visible region appears to be from
nonspecific binding. These negative absorptions tailing into the UV
region, observed in all cases, signify the binding of anion to the
apo-proteins (32).

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Fig. 5.
Difference spectra for copper binding to
hTF/2N proteins with carbonate at pH 7.4. HEPES (25 mM), NaHCO3 (1 mM). B is
for base line, and WT is wild type.
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63Cu EPR spectra given in Fig.
6 confirmed the results of the electronic
spectra above. Compared with the spectrum for wild-type hTF/2N, similar
superhyperfine splitting and resonances in both g
and g
regions are
observed in the spectrum of 63Cu-Y85F. In contrast, the
spectra for 63Cu-E83A and 63Cu-H249Q do not
show characteristic resonances of specific copper binding. The
relatively broad featureless spectra are similar to that of
63Cu in buffer, suggesting nonspecific binding in the both
cases.

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Fig. 6.
X-band (9.38 GHz) EPR spectra of frozen
samples of 90% 63Cu-saturated hTF, hTF/2N, and various
mutants. Protein samples (~0.5 mM), HEPES (~0.1
M), NaHCO3 (~20 mM) in
D2O, pD 7.9. The temperature is 90 K. The instrument
settings are similar to those given elsewhere (22).
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 |
DISCUSSION |
The Impact of Mutations at Tyr-85 and Glu-83--
The profound
impact of mutations at Tyr-85 and Glu-83 clearly demonstrated that a
functional second shell effect exists in iron binding by the human
transferrin N-lobe. The significant role of Tyr-85 in the second shell
effect is indicated by the fact that iron is removed from the mutant
Y85F much more readily than from wild-type hTF/2N. Since the mutation
of Tyr-85 to phenylalanine does not alter the binding of His-249 (see
below), we speculate that the mutation of Tyr-85 may weaken the
di-lysine trigger in the Fe-hTF/2N. The NZ of Lys 206 from the NI
domain is bound to the NZ of Lys-296 from the NII domain in the
iron-loaded protein (Fig. 1). It has been postulated that in the acidic
endosome (pH ~ 5.5), both lysine residues become protonated to
provide the driving force that opens the two domains of the protein,
thus facilitating iron release (24, 33). Facile release of iron from
the Y85F mutant even at pH 7.4 is attributed to the weakening of the
di-lysine linkage because of the disruption of the hydrogen bond
between Tyr-85 and Lys-296 (Fig. 1). The destabilization of the
lysine-lysine interaction then makes it easier for chloride to enter
the binding cleft, thereby aiding Tiron, the chelator, in facile iron
removal.
One unexpected finding was that mutation of the glutamic acid at
position 83 to an alanine residue had a dramatic effect on the
properties of hTF/2N. Both UV and EPR spectra revealed that in mutant
E83A, the elimination of the hydrogen-bonding ability of Glu-83 causes
the loss of a normal iron binding ligand. This missing ligand appears
to be His-249, as demonstrated by the fact that mutant E83A displayed
similar iron and copper binding behavior to mutant H249Q, in which the
His-249 ligand was changed to glutamine. In apo-hTF/2N, His-249 is
hydrogen-bonded to Glu-83 at imidazole ND1 and to Lys-296 at imidazole
NE2 (Fig. 1). This network serves to hold His-249 at the proper
position, in preparation for metal binding. The glutamic acid at
position 83 appears to be a key component in the hydrogen-bonding
network; without it, His-249 may move away and not be available for
iron binding.
Effect of Monovalent Anions--
A unique finding of the present
study is that monovalent anions are able to return normal iron binding
to the mutant protein E83A but not Y85F. As indicated by the anion
conversion titrations and iron release kinetics (Table III), monovalent
anions exert their influence according to the lyotropic sequence
Ac
< F
< Cl
< NO3
< ClO4
. The
effect of these anions is to convert the iron complexes to a form of
protein with typical absorption and EPR spectra, i.e.
maxs in UV-visible spectra significantly red-shift
(Table III), and EPR spectra change from a single rhombic feature to
one more typical of wild-type protein (Fig. 4). It seems clear that the
normal six-coordinate species is restored in the presence of the
monovalent anions. Hill plot calculations from the UV-visible titrations suggest that a single anion participates in this conversion (Table III). The lyotropic sequence is inconsistent with coordination of the anion directly to the metal. Furthermore, no 19F
coupling is seen in the EPR spectrum of mutant E83A in the presence of
KF. The present data therefore suggest an interaction of these anions
with an anion binding site near the position of Glu-83. The fact that
only monovalent rather than divalent anions show the stabilization
effect further suggests that the charge balance is restored by a
monovalent anion supplying a negative charge missing at position 83 in
the E83A mutant, an argument consistent with the fitting results of the
Hill plots. The presence of such an anion around the Glu-83 position
therefore helps to rebuild the original hydrogen bond network, allowing
His-249 to serve as a binding ligand again.
Concluding Remarks--
Complementary spectral data provide
evidence that mutant E83A has only five of the ligands normally found
in hTF/2N, and the missing ligand appears to be His-249. Interestingly,
monovalent anions are able to convert the ferric E83A back to the
normal six-coordinate structure. A single anion takes part in the
conversion but does not exert its effect by directly binding to the
iron center. Structural analysis shows that residue Glu-83 may be the key component of the network, holding His-249 in the proper position for iron binding. The anion may stabilize mutant E83A by replacing the
negative charge around the position 83 lost in the mutation and
rebuilding the H-bonding network thus allowing His-249 to again bind
the iron. In summary, mutations at nonliganding residues Tyr-85 and
Glu-83 produce profound effects on the metal binding properties of the
hTF/2N protein, since these two residues make up part of the
hydrogen-bonding network around the binding cleft, the so called second
shell. This second shell effect may well exist in the C-lobe of hTF but
has not yet been studied. Similar second shell effects have also been
reported in other metal-containing proteins such as carbonic anhydrase
(34) and metallophosphatases calcineurin (9). Comprehensive
characterization of the role of second shell residues in metal binding
stability may lead to a better understanding of the structure-function
relationship in metalloproteins in general.