(Received for publication, May 11, 1995; and in revised form, June 21, 1995)
From the
The bacterioferritin (BFR) of Escherichia coli is a
heme-containing iron storage molecule. It is composed of 24 identical
subunits, which form a roughly spherical protein shell surrounding a
central iron storage cavity. Each of the 12 heme moieties of BFR
possesses bis-methionine axial ligation, a heme coordination
scheme so far only found in bacterioferritins. Members of the BFR
family contain three partially conserved methionine residues (excluding
the initiating methionine) and in this study each was substituted by
leucine and/or histidine. The Met variants were devoid of
heme, whereas the Met
and Met
variants
possessed full heme complements and were spectroscopically
indistinguishable from wild-type BFR. The heme-free Met
variants appeared to be correctly assembled and were capable of
accumulating iron both in vivo and in vitro. No major
differences were observed in the overall rate of iron accumulation for
BFR-M52H, BFR-M52L, and the wild-type protein. The iron contents of the
Met
variants, as isolated, were at least 4 times greater
than for wild-type BFR. This study is consistent with the reported
location of the BFR heme site at the 2-fold axis and shows that heme is
unnecessary for BFR assembly and iron uptake.
Iron storage proteins are found in both eukaryotes and
prokaryotes where they are thought to store excess iron in a non-toxic
form and provide a reserve of iron for utilization in cellular
metabolism (Theil, 1987). They are composed of 24 structurally
identical subunits of M approximately 19,000,
which assemble to form a spherical shell surrounding a central cavity
where up to 4500 iron atoms can be sequestered as a ferric
oxide-hydroxide-phosphate core (Ford et al., 1984). The
deposition of ferric iron in the storage cavity is preceded by the
oxidation of ferrous iron, which is mediated by the ferroxidase
activity of the storage protein. Two types of iron storage protein are
known: the ferritins of animals, plants, fungi, and bacteria; and the
bacterioferritins, so far only found in bacteria. Similar
three-dimensional structures have been deduced for human H-chain, horse
spleen, rat liver, Escherichia coli, and bullfrog ferritins,
and for the bacterioferritin (BFR) (
)of E. coli (Lawson et al., 1991; Ford et al., 1984; Thomas et al., 1988; Hempstead et al., 1994; Trikha et
al., 1994; Frolow et al., 1994). Each subunit comprises a
bundle of four long
helices (A to D) and a short helix (E), which
together account for approximately 75% of the total secondary
structure. The ferroxidase activity of mammalian ferritins is due to an
active site (the ferroxidase center) in the middle of the four-helix
bundle of each H-chain subunit where a dinuclear iron species is
thought to form (Lawson et al., 1989, 1991). Key amino acid
residues in the ferroxidase centers of ferritins are conserved in
bacterioferritins, suggesting that BFR possesses a ferroxidase center
similar to that of H-chain ferritins (Andrews et al., 1991;
Grossman et al., 1992). This suggestion has been confirmed by
studies on iron uptake by wild-type BFR and site-directed variants of
BFR (Le Brun et al., 1993, 1995), and is supported by the
presence of a dinuclear metal-binding site at the ferroxidase center in
the BFR crystal structure (Frolow et al., 1994).
Although
ferritins and bacterioferritins have similar structures and functions,
they possess only 17% amino acid sequence identity and are therefore
distantly related in evolution (Andrews et al., 1991).
Furthermore, bacterioferritins differ from ferritins in possessing heme
b prosthetic groups (up to 12 groups/24 subunits), which have a very
low redox potential that depends on the presence (-475 mV) or
absence (-225 mV) of an iron core (Watt et al., 1986).
Magnetic circular dichroism (MCD), electron-paramagnetic resonance
(EPR) spectroscopy, extended x-ray absorption fine structure, and x-ray
crystallographic studies have shown that the heme iron of the
bacterioferritins has a unique bis-methionine ligation
(Cheesman et al., 1990, 1992; George et al., 1993;
Frolow et al., 1994). Modeling studies suggested that the E. coli BFR possesses two potential heme-binding sites (site I
and site II), each containing a pair of methionine residues correctly
disposed to bind heme (Cheesman et al., 1993). Site I is an
intrasubunit site near the outer molecular surface with heme iron
ligands Met and Met
(24 heme sites/24
subunits), and site II is an intersubunit site near the inner surface
with ligands Met
and Met
from diad-related
neighboring subunits (12 sites/24 subunits). The validity of site II
has recently been confirmed by x-ray crystallography (Frolow et
al., 1994). However, neither Met
, Met
,
nor Met
are absolutely conserved in the BFR family.
Met
is replaced by Leu in the BFR of Nitrobacter
winogradskyi, Met
is substituted by Thr in both the
BFR of Synechocystis PCC 6803 and the BFR
-subunit of Pseudomonas aeruginosa, and Met
is replaced by
Gln in the BFR of both Mycobacterium leprae and M. avium (Kurokawa et al., 1989; Laulhère et al., 1991; Moore et al., 1994; Pessolani et
al., 1994; Inglis et al., 1994).
This paper describes
the effects of substituting the three potential heme-ligating
methionine residues of E. coli BFR and demonstrates that
whereas Met and Met
are not involved in heme
binding, Met
substitution generates a heme-free protein.
Iron uptake studies of two heme-free variants show that the heme group
is not essential for the ferroxidase activity of BFR.
The nucleotide sequences of the primers are identical to the corresponding bfr sequence except for the mismatches (bold uppercase letters: C(A/T)C) in the Met codon (ATG) designed to direct amino acid substitutions. The complete bfr nucleotide sequence of each identified mutant was determined and verified. Double-stranded replicative form DNA was prepared for each verified mutant in order to subclone the 1.2-kilobase pair EcoRI-PstI bfr fragment into the corresponding sites of pUC119. The mutated bfr genes were expressed from the natural bfr promoter (Andrews et al., 1993).
EcoRI-PstI
fragments containing mutated bfr genes were subcloned into
pUC119 generating plasmids pGS725-729, as listed in Table 1. The
mutated bfr genes were then overexpressed in transformants of
the BFR-free strain, JRG2157, so that the overproduced BFR variants
were not contaminated with wild-type protein. The overproducing strains
were grown to late stationary phase and harvested. Bacterial pellets
containing BFR-M31H, -M31L, and -M86L were pink due to their high
content of heme-containing BFR, whereas those containing BFR-M52H and
-M52L were beige (the normal color for E. coli), indicating
that the Met variants have a low heme content.
Densitometric analysis of Coomassie Blue-stained SDS-polyacrylamide
gels showed that the BFR variants represent 6-12% of total cell
protein, which is somewhat lower than for wild-type BFR (18%; Table 1). The different degrees of overproduction may be due to
the codon changes associated with the missense mutations. The iron
contents of the overproducing strains were higher than that of the
control strain by between 0.011 and 0.031%, dry cell weight (Table 1), presumably due to relatively high amounts of
BFR-associated iron in the overproducers. The increase in cellular iron
contents (extra whole cell iron; Table 1) was greater for the
strains containing BFR-M52H and BFR-M52L (0.031 and 0.021%, dry cell
weight, respectively) than for the strains containing the other BFR
variants (approximately 0.012%) (Table 1). The
BFR-M52-overproducing strains had much higher extra whole cell iron:BFR
ratios than the other overproducing strains (Table 1), suggesting
a higher iron content for the BFR-M52 variants relative to the other
variants and wild-type BFR (see below).
In order to isolate BFR-M52L in its fully assembled state, a modified purification protocol was employed (Method 2) in which 20 mM Hepes (pH 7.8) was used in place of 20 mM histidine (pH 5.5) for anion-exchange chromatography. Under the new conditions, both BFR-M52L and the wild-type protein eluted from the anion-exchange column in single peaks (at 25-50 mM ammonium sulfate) as fully assembled proteins, as indicated by analytical gel permeation chromatography (results not shown). The analyses of BFR-M52L, described below, were performed using the preparation purified via Method 2.
The ultraviolet-visible spectra of the wild-type and variant apo-
(non-heme iron-free) proteins confirmed that the Met variants lack heme, whereas the Met
and Met
variants possess high heme contents (Fig. 1A and Table 2). The spectra of the Met
and Met
variants were very similar to that of the wild-type protein (Fig. 1A) with absorption peaks at 418, 525, and 560 nm
associated with the Soret,
, and
bands of the oxidized heme
moiety. No heme-associated absorption peaks were detected in the
spectra of the Met
variants, even at high sensitivity,
indicating that they are completely free of heme. The spectra of the
iron-containing proteins were similar to those of the apoproteins,
except for higher absorptions at 280 nm (Fig. 1B),
which were directly related to the iron contents of the proteins. The
spectra of the Met
variants possessed a minor peak at
approximately 475 nm that has not previously been reported for
bacterioferritins or ferritins (Fig. 1C). The 475 nm
absorbance peak disappeared from the spectra of the BFR-M52 variants
upon reduction with sodium dithionite or upon removal of associated
iron, suggesting that it arises from a non-heme ferric iron species.
This was confirmed by the reappearance of the 475 nm absorbance band
following addition of ferrous iron (50 iron atoms/BFR molecule) to
aerobic solutions of apoBFR-M52H (Fig. 1D). Initially,
it was not possible to determine whether a similar component is present
in the spectra of native (iron-containing) heme-containing
bacterioferritins because of the high intensity heme absorption band
that obscures the relevant region of the spectrum (Fig. 1C). However, the difference spectrum of the
iron-loaded (50 iron atoms/molecule) and apo forms of BFR revealed
minor absorption bands at 427 and 475 nm, showing that the 475
nm-absorbing component forms in both the heme-containing and
non-heme-containing protein upon iron uptake (Fig. 1D).
A similar weak absorption band (at 470 nm) has been reported in the
absorption spectrum of castor stearoyl-ACP
desaturase
and was assigned to the Fe-O-Fe center of the enzyme (Fox et
al., 1994). It is possible that the 475 nm band of BFR also
derives from a dinuclear iron center. The 427 nm peak observed in the
BFR-difference spectrum (Fig. 1D) is consistent with a
slight red shift in the position of the Soret band, suggesting that
iron uptake results in a structural perturbation of the heme-binding
region. A similar effect has been observed previously following an
initial, and very rapid, 1 nm blue shift in the Soret band (Le Brun,
1993). The minor absorption peaks at 650 and 737 nm, observed
previously with wild-type BFR (Andrews et al., 1993), were
absent from the spectra of the Met
variants (Fig. 1E), indicating that these bands are heme
associated. This is consistent with the assignment of the MCD spectrum
of bis-methionine low spin ferric heme (Cheesman et
al., 1993).
Figure 1:
Ultraviolet-visible spectra of the
oxidized forms of BFR and BFR variants. The proteins were in 200 mM Mes, pH 6.5, at 0.1 mg/ml (A) or at 0.7 mg/ml (D) or in buffer C at 0.5 mg/ml (B, C, and E). A, spectra (250-600 nm) of the apoproteins; B, spectra (250-600 nm) of the native proteins; C, comparison of the spectra of BFR and the BFR-M52 variants
at 400-600 nm; D, difference spectra (300-600 nm)
of iron-containing (50 iron atoms/molecule) BFR or BFR-M52H, and apoBFR
or apoBFR-M52H, respectively (the reference cuvette contained
apoprotein and the sample cuvette contained an identical solution to
which Fe had been added at least 20 min prior to
measurement of the difference spectra); E, comparison of the
spectra of BFR and the BFR-M52 variants at 600-800
nm.
The 10 K EPR spectrum of apoBFR (Fig. 2A) contains signals at g = 2.88,
2.31, and 1.45, which have previously been assigned to the S =
1/2 low spin ferric heme groups of the protein (Cheesman et
al., 1991). The EPR spectra of apoBFR-M31H, -M31L, and -M86L (Fig. 2, B-D) each contain low spin ferric heme
signals at g values equal to, or very close to, those of the
wild-type protein. Hence, substitution of either Met or
Met
fails to produce any significant changes in the heme
group g values. The low spin heme signals are absent in the
spectra of the Met
variants (Fig. 2, E and F), confirming the lack of heme in these proteins. The
Met
variant spectra contain a large feature at g = 4.28, which has previously been assigned to S
= 5/2 high spin mononuclear Fe(III) bound at specific sites on
the protein (Cheesman et al., 1992). Such signals are absent
from the spectra of Fig. 2(A-D) because the
non-heme iron has been removed.
Figure 2: EPR spectra of BFR and BFR variants. ApoBFR (A) was in 100 mM Mes, pH 6.5, at a concentration of 8.1 mg/ml. ApoBFR-M31H (B), apoBFR-M31L (C), and apoBFR-M86L (D) were in phosphate-buffered saline, pH 7.2, at concentrations of 7.3, 4.2, and 3.5 mg/ml, respectively. BFR-M52H (E) and BFR-M52L (F) were in 100 mM Hepes, pH 7.8, at concentrations of 9.5 and 14 mg/ml, respectively. Spectra were recorded at a microwave frequency of 9.39 GHz, a microwave power of 2.01 mW, a modulation amplitude of 10 gauss and a temperature of 10 K. The spectra in A-D were subjected to a base-line subtraction procedure.
To ensure
that BFR-M52L remained fully assembled, the iron uptake properties of
the Met variants and wild-type BFR were compared at pH 7.1
rather than pH 6.5. Adding 1000 iron (II) ions/apoBFR molecule at pH
7.1 gave similar overall rates of oxidation with all three proteins,
when monitored at 340 nm (Fig. 3). Hence, loss of heme from the
protein causes no major effect on catalytic activity. Indeed, a slight
enhancement of catalytic activity was observed with BFR-M52H, although
there was a slight decrease with BFR-M52L (Fig. 3).
Figure 3: Absorbance changes measured at 340 nm after the addition of 1000 iron (II) ions/molecule to apoBFR and apoBFR-M52 variants. Proteins were 0.5 µM (after mixing) in 100 mM Hepes buffer, pH 7.1; the temperature was 30 °C; and the pathlength was 1 cm.
In order
to resolve phases 2 and 3, it was necessary to measure iron uptake at
lower iron (II):apoBFR ratios (between 10 and 133 Fe:BFR). For
wild-type BFR, a single phase (phase 2) was observed at iron (II):BFR
ratios of <50, whereas two distinct phases (corresponding to phases
2 and 3) were observed at ratios >50 (Fig. 4A).
Phase 2 is shown more clearly in the plot of the initial 5 s of the
reaction (Fig. 4B). The amplitude of phase 2 was
obtained by fitting each trace to a first order process. A plot of
phase 2 amplitude as a function of added iron (II) indicates that
saturation occurs at a level of approximately 48 iron (II) ions/BFR
molecule (Fig. 4C). These data are consistent with
previous measurements at pH 6.5 (Le Brun et al., 1993).
However, at pH 7.1 the observed rate of phase 2, measured at a ratio of
50 iron (II) ions:BFR (0.5 µM BFR concentration, 30
°C), was 0.55 ± 0.05 s, while at pH 6.5
the rate was slower, 0.17 ± 0.05 s
.
Figure 4: Iron uptake properties of apoBFR and the apoBFR-M52 variants at pH 7.1. A, D, and G show absorbance changes measured at 340 nm over the first 20 s after the addition of variable amounts of iron (II) ions/apoBFR molecule for wild-type BFR (0.50 µM), BFR-M52H (0.35 µM), and BFR-M52L (0.50 µM), respectively. The proteins were in 100 mM Hepes buffer (pH 7.1), the temperature was 30 °C, and the pathlength was 1 cm. The ratio of iron (II):BFR is shown against each trace. B, E, and H are as for A, D, and G, respectively, except that the measurements were over the first 5 s of the reaction. C, F, and I are plots of absorbance increases at 340 nm versus the ratio of iron (II):BFR. The absorbance changes at 5 s were obtained from deconvolution of the rates of phases 2 and 3. To enable easy comparison between the plots, the amplitudes for BFR-M52H were adjusted to those expected for a 0.5 µM protein solution. The plots include some additional points derived from data not shown in the traces of absorbance versus time.
The
data for BFR-M52H also show a single phase at iron (II):BFR ratios of
<50, whereas two distinct phases can be distinguished at ratios of
>50 (Fig. 4D). The kinetic parameters of the latter
differ from those of wild-type BFR, but they clearly correspond to
phases 2 and 3. This is more apparent in Fig. 4(E and F), where phase 2 is saturated at approximately 48 iron (II)
ions/BFR-M52H, as for the wild-type protein. The observed rate of phase
2 for BFR-M52H, measured at a ratio of 50 iron (II) ions:BFR-M52H was
1.9 ± 0.1 s, which is 3.5-fold higher than
that measured for wild-type BFR under similar conditions. Despite this,
the two phases for BFR-M52H are less well resolved than in wild-type
BFR, indicating that phase 3 also proceeds at an enhanced rate relative
to wild-type, as shown in Fig. 3.
Results for BFR-M52L again
show a single fast phase at iron (II):BFR-M52L ratios of <50, while
at higher ratios, two distinct phases are observed (Fig. 4, G and H). However, the two phases appear to be less
well resolved than for wild-type BFR and BFR-M52H, such that saturation
of phase 2 is not obvious. The initial 1.3-s traces were fitted to a
first order process and the resultant amplitude plotted against iron
(II):BFR-M52L ratio. This plot shows that phase 2 saturation occurs at
approximately 48 iron (II) ions/BFR-M52L (Fig. 4I). The
rate of phase 2 for the addition of 50 iron (II) ions/BFR-M52L was 1.2
± 0.1 s, approximately 2-fold greater than
that observed in wild-type BFR, but slightly lower than that for
BFR-M52H.
The iron uptake properties of wild-type BFR measured at pH
7.1 are qualitatively identical to those observed at pH 6.5 (Le Brun et al., 1993), but a considerable enhancement of the rate of
phases 1 (data not shown) and 2 is observed at the higher pH. This pH
effect could be due to the need to deprotonate a residue (or residues)
at the ferroxidase center during the binding of iron (II) in phase 1.
The iron uptake properties of the two Met variants show
that there is no significant effect on the overall catalytic activity
of the protein as a result of the lack of heme. Differences between
wild-type BFR and the Met
variants are observed in the
relative rates of the two oxidation phases; these are clearly resolved
in wild-type, but are increasingly less well resolved in BFR-M52H and
BFR-M52L. Hence, saturation of the first oxidation phase, phase 2, was
more difficult to detect in the Met
variants.
The complete lack of heme in the BFR-M52 variants confirms
that Met serves as the heme iron ligand (Cheesman et
al., 1990; George et al., 1993; Frolow et al.,
1994). It further shows that heme binds within a pocket at the
intersubunit two-fold axis as previously proposed by model building
(Andrews et al., 1991; Cheesman et al., 1993;
Grossman et al., 1992) and subsequently shown by x-ray
crystallography (Frolow et al., 1994). The present studies
also show that Met
and Met
do not participate
in heme iron ligation in BFR. This excludes the existence of the
intrasubunit heme sites discussed by Grossman et al. (1992)
and Cheesman et al.(1993) as alternative possibilities to the
Met
site. The location of the BFR heme site between
two-fold related subunits is novel for a hemoprotein, and its proximity
to both the dinuclear iron site and the iron core in the central cavity
raises the possibility of heme-mediated redox interactions between
these iron centers. The Met
His replacement in BFR
resulted in a heme-free protein, despite the fact that histidine
residues act as coaxial heme iron ligands in many other hemoproteins.
The failure of His
to replace Met as a functional heme
ligand probably stems from the importance of Met
in
mediating the tight fit of the heme at its binding site in BFR
(Met
contributes 58 of the 108 reported van der Waals
contacts between heme and the protein) (Frolow et al., 1994).
Met is conserved in the bacterioferritins of E.
coli, Azotobacter vinelandii, M. leprae, P.
aeruginosa (subunit
), and M. avium, but not Synechocystis PCC 6803 or the
subunit of P.
aeruginosa BFR where the corresponding residue in both cases is
Thr (Andrews et al., 1989; Grossman et al., 1992;
Inglis et al., 1994; Laulhère et
al., 1991; Pessolani et al., 1994; Moore et al.,
1994). However, despite the absence of Met
, the purified Synechocystis and P. aeruginosa bacterioferritins
contain heme (6 and 3-9 hemes/24 subunits, respectively). The
reason for the apparent discrepancy is uncertain. A possible
explanation arises from the observation that Synechocystis and P. aeruginosa bacterioferritins, unlike the other
bacterioferritins so far sequenced, possess a methionine at position
48. This has led to the suggestion that Met
could function
as a heme ligand in the absence of Met
(Moore et
al., 1994).
The assembly of E. coli BFR at
physiological pH was not affected by the lack of heme and the
Met substitutions. Furthermore, the overproduced heme-free
variants acquired more iron in vivo than the overproduced
heme-containing bacterioferritins, showing that heme is unnecessary for
the intracellular uptake of iron by BFR. The relatively high in
vivo iron content of the Met
variants indicates that
they either have higher iron uptake activities or reduced rates of iron
release in vivo. The results show that heme is also
unnecessary for the uptake of iron by BFR in vitro. This is
not surprising, since the heme iron does not undergo redox cycling when
iron (II) and dioxygen are the substrates for the iron loading of BFR
(Le Brun et al., 1993).
The Met substitutions
and absence of heme resulted in a more rapid (2-3.5-fold)
oxidation of Fe(II) at the ferroxidase centers during phase 2 of iron
loading. The differences in the phase 2 and 3 rates of iron loading of
the two Met
variants presumably relates to the nature of
the replacement amino acid residues (His or Leu). The effects of the
Met
substitutions on phases 2 and 3 may be explained by
the close proximity of residue 52 to the ferroxidase center. The heme
group lies at the interface between a pair of two-fold related
subunits. A pair of methionine residues, Met
and
Met
, from two-fold related subunits act as the coaxial
heme ligands, as shown by the x-ray structure (Frolow et al.,
1994) and confirmed by the mutagenesis results reported here. The
adjacent residue, Glu
, provides a carboxylate side chain
that can bridge two divalent metal ions, such as iron (II), bound at
the ferroxidase center of BFR.
Therefore, the substitution
of residue 52 might be expected to have an affect on the coordinating
ability of Glu
. The binding of iron (II) ions at the
ferroxidase center of wild-type BFR causes a perturbation of the heme
absorption spectrum (Le Brun et al., 1993), suggesting that
there is interaction between the heme-binding site (which includes
Met
) and the dinuclear metal binding site (which includes
Glu
). The present work provides further support for
interaction between these two metal centers.
Previous studies have
shown that the incorporation of heme into mammalian ferritins increases
the rate at which iron can be reduced and released from the core by
enhancing the delivery of electrons from electron donors through the
protein shell to the core (Kadir et al., 1992). It is
therefore possible that the high in vivo iron contents of the
Met variants are due to inefficient iron release from the
proteins resulting from their lack of heme. However, the true function
of the hemes within BFR is still uncertain.. Although the hemes appear
not to be required for iron oxidation or for assembly of the protein,
the replacement of Met
and absence of heme do influence
the amount of iron accumulated in vivo. Whether or not the in vivo rate of iron accumulation is significantly affected by
the Met
substitutions remains to be determined, as does
the possibility that the hemes are involved in iron core reduction and
iron release. The availability of the BFR-Met
variants
should now allow the initiation of further studies aimed at defining
the function of heme in BFR and providing a deeper insight into the
physiological role of BFR in E. coli and other bacteria.