©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Site-directed Replacement of the Coaxial Heme Ligands of Bacterioferritin Generates Heme-free Variants (*)

(Received for publication, May 11, 1995; and in revised form, June 21, 1995)

Simon C. Andrews (1)(§) Nick E. Le Brun (¶) Vladimir Barynin (1)(**) Andrew J. Thomson Geoffrey R. Moore John R. Guest (1) Pauline M. Harrison (1)

From the Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, S10 2TN and the Centre for Metalloprotein Spectroscopy and Biology, School of Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

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(r) 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) (^1)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 alpha 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 alpha-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.


EXPERIMENTAL PROCEDURES

Materials and Strains

Oligonucleotides were made on an Applied Biosystems DNA synthesizer. The alpha-[S]thio-dATP was supplied by Amersham International; restriction enzymes, T4 polynucleotide kinase, and DNA ligase were from Northumbria Biologicals Limited; and DNA polymerase (Klenow fragment) was from Pharmacia Biotech Inc. The strains of E. coli were: JM101 (thi, supE, DeltaproAB-lac/F` traD36, proAB, lacI^qZDeltaM15) (Messing, 1983) for propagating bacteriophage-M13mp18 derivatives for nucleotide sequence analysis and as the host for pUC119-derived phagemids, JRG2157 (JM101 Deltabfr::kan) (Hudson et al., 1993) as the host for overproducing BFR variants, and BW313 (dut, ung, thi, relA, spoT/F` lysA) (Kunkel et al., 1985) for preparing uracil-incorporated single-stranded DNA templates for site-directed mutagenesis.

Site-directed Mutagenesis

Site-directed mutagenesis was performed by the single-primer method (Kunkel et al., 1985). The target for mutagenesis was a single-stranded M13mp18 derivative containing the 4.9- kilobase pair EcoRI-HindIII bfr fragment from the plasmid pGS280 (Andrews et al., 1989). Single-stranded DNA was prepared according to Sanger et al.(1980) using E. coli strain BW313 as the host and uridine (0.25 µg/ml) in the growth medium. The mutagenic oligonucleotide primers were phosphorylated and annealed to the template by heating to 70 °C for 10 min and cooling at room temperature before use in the primer-extension ligation reaction. The extension-ligation mixture was transfected into JM101(ung), and progeny phage were screened by single-channel tracking and nucleotide sequence analysis using the Sequenase DNA sequencing kit (U. S. Biochemical Corp.) and buffer-gradient gels (Biggin et al., 1983). Three mixed-site mutagenic primers were used to direct the following substitutions (binding coordinates from Andrews et al., 1989): S268, Met His or Leu (5`d(CAT GCC CGA C(A/T)C TTT AAA AAC TGG)-3`; 592-615); S269, Met His or Leu (5`d(ATT GAT GAG C(A/T)C AAA CAC GCC)-3`; 655-675); and S270, Met His or Leu (5`d(TT GAG GAA C(A/T)C CTG CGT TCT GAT C)-3`; 758-781).

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).

Overproduction and Purification of BFR and Variants

Derivatives of the high copy phagemid, pUC119, carrying the mutated bfr genes were used to transform a BFR-free derivative of JM101, JRG2157 (Deltabfr::kan). The resultant overproducing strains were grown aerobically for 16 h at 37 °C in L-broth containing 150 µg/ml ampicillin. The BFR-variants were purified using either the method described previously (Method 1; Andrews et al., 1993) or a variation of this method (Method 2) with the following modifications; buffer C (20 mM Hepes, pH 7.8, 100 mM KCl, 0.1 mM EDTA, 10% glycerol) was used in place of phosphate-buffered saline (buffer A; pH 7.2), and anion-exchange chromatography was performed with a Protein-Pak Q (20 times 100 mm) column from Waters (in place of the Q-Sepharose column) equilibrated with buffer C (instead of 20 mM histidine, pH 5.5) and eluted at 2.5 ml/min with a 300-ml linear gradient of 0.0-0.1 M (NH(4))(2)SO(4) in buffer C. Purity was assessed by polyacrylamide gel electrophoresis (PAGE) (Laemmli, 1970).

Iron and Protein Assay

Iron was assayed by the method of Drysdale and Munro(1965) with 1% ferrozine instead of 0.5% 2,2`-bipyridine. Protein was measured as described by Lowry et al.(1951) with bovine serum albumin as standard. The absolute concentration of pure BFR was determined using a Waters Pico-Tac amino acid analysis system. Heme was assayed using the pyridine hemeochromogen method (Falk, 1964).

Iron Removal and Incorporation

Iron was removed from BFR by reduction with sodium dithionite (Bauminger et al., 1991). Iron was incorporated by the aerobic addition of 10 mM ferrous ammonium sulfate (freshly prepared in deoxygenated ultrapure water) to wild-type and variant apobacterioferritins (0.5 µM final concentration) in 100 mM Hepes (pH 7.1). Absorbance changes were measured using an Applied Photo Physics stopped flow apparatus. Iron uptake experiments were performed at 30 °C.

Spectroscopy

EPR spectra were measured with an X-band spectrometer (Bruker ER200D with an ESP 1600 computer system) fitted with a liquid helium flow cryostat (Oxford Instruments plc; ESR9). Ultraviolet-visible spectra were obtained using a Carey 3E or a Unicam UV4 spectrophotometer. Difference spectra between iron-containing and iron-free BFR were obtained following the addition of 11.2 µl of 10 mM ferrous ammonium sulfate to the sample cuvette containing 0.7 mg of apoprotein in 1 ml and the addition of an equal volume of H(2)O to an identical sample in the reference cuvette. A zero base line was obtained prior to addition of iron.

Other Methods

Electrospray mass spectrometry was performed with BFR (20 pg/ml in 5% formic acid, 50% acetonitrile) by David Towers (Department of Chemistry, Sheffield University) using a VG Platform spectrometer. Analytical gel permeation chromatography was performed using a Waters 625 HPLC and a Protein Pak 300SW (0.8 times 300 mm) column at a flow rate of 0.8 ml/min in 200 mM Mes buffer (pH 6.5).


RESULTS

Design, Construction, and Overproduction of BFR Variants

The three putative heme iron-ligating methionine residues were changed, individually, to leucine or histidine. The Met Leu substitutions should minimize structural perturbation in the altered proteins, whereas the Met His substitutions could provide alternative heme iron ligands that should be detectable by EPR and MCD spectroscopy, thus allowing the unambiguous assignment of the heme ligands. The strategy for mutagenesis involved the use of mutagenic primers containing mixed sites each designed to generate two missense mutations (ATG CAC or CTC; Met His or Leu). Nucleotide sequence analysis of 150 potential mutants identified 22 valid mutants representing five of the six desired classes. The Met His class was not recovered, nor was it further sought since studies with the BFR-M86L variant showed that Met is not involved in heme ligation.

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).



Purification of Variant BFR Proteins

The BFR variants and wild-type BFR were initially purified using Method 1. The BFR variants resembled the wild-type protein in being heat stable (65 °C for 15 min) and in eluting as 500-kDa proteins from a gel permeation column. However, during anion-exchange chromatography at pH 5.5 in 20 mM histidine buffer, the elution pattern of BFR-M52L was markedly different from those of the other variants and the wild-type protein. Approximately 80% of the BFR-M52L sample (A) eluted in peak 1 (at 75-110 mM NaCl) and only 20% in peak 2 (at 130 to 200 mM NaCl), whereas wild-type BFR (and the other variants) eluted mainly (88%) in peak 2 with a smaller proportion (12%) in peak 1 (results not shown). SDS-PAGE analysis confirmed that peaks 1 and 2 correspond to BFR and absorbance measurements at 418 nm showed that the main component of peak 1 is heme-free BFR for both the wild-type and variant preparations, whereas peak 2 possessed heme for the wild-type, BFR-M31, and BFR-M86 samples but not for the BFR-M52 samples. Non-denaturing PAGE indicated that the heme-free BFR in peak 1 is in a disassembled form because it only contained the high mobility subunit monomer (Andrews et al., 1993), and that BFR in peak 2 is mostly in the 24-meric state. These findings suggest that, during anion-exchange chromatography, a small proportion of BFR dissociates into individual subunits with the concomitant loss of any associated heme, and that BFR-M52L is more susceptible to this dissociation. Since Met projects into the interface between symmetry-related subunits (Frolow et al., 1994), it is possible that the Met Leu substitution causes a destabilization of interactions along the two-fold axis resulting in the increased susceptibility of BFR-M52L to dissociation.

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.

Properties of BFR Variants

The subunit masses of the purified BFR variants estimated by electrospray-mass spectroscopy were within +3 and -6 daltons of the predicted masses (Table 2) and are therefore consistent with the genetically engineered substitutions. The iron contents of BFR-M31H, BFR-M31L, and BFR-M86L were similar to that of wild-type BFR (Table 2). However, the Met variants possessed 4-11-fold higher iron contents than the other bacterioferritins. These high iron contents resulted in the high extra whole cell iron:BFR ratios for the BFR-M52-overproducing strains (Table 1), as described above.



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, beta, and alpha 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 Delta^9 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.



Iron Uptake

Previous studies of wild-type apoBFR at pH 6.5 revealed that the iron uptake process involves at least three distinct kinetic phases (Le Brun et al., 1993), and this led to the following hypothesis for the mechanism of iron uptake. In phase 1 (t 50 ms), two iron (II) ions bind at the ferroxidase center to form a dinuclear iron site. In phase 2 (t 3 s), the two iron (II) ions are oxidized to iron (III) ions. If sufficient iron (II) is present, phase 3 (t 5 min) occurs, in which there is a slow formation of iron core.

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.


DISCUSSION

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 beta), and M. avium, but not Synechocystis PCC 6803 or the alpha 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.^2 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.


FOOTNOTES

*
This work was supported in part by the BBSRC through funding of an Electrospray Mass Spectrometer under the Molecular Recognition Initiative and by providing funds for the UEA Centre for Metalloprotein Spectroscopy and Biology, and was also supported by the Wellcome Trust by the provision of a project grant (to J. R. G. and P. M. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of an Advanced Fellowship from the BBSRC. To whom correspondence should be addressed: Dept. of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Firth Court, Sheffield S10 2TN, United Kingdom. Tel.: 114-282-6194; Fax: 114-273-9826; s.andrews{at}sheffield.ac.uk.

Recipient of a Prize Fellowship from the Wellcome Trust.

**
Supported by an EC Bridge Programme Grant (BIOT-CT91-0262) and the SERC.

(^1)
The abbreviations used are: BFR, bacterioferritin; apoBFR, BFR lacking non-heme iron; PAGE, polyacrylamide gel electrophoresis; Mes, 2-(N-morpholino)ethanesulfonic acid; MCD, magnetic circular dichroism; EPR, electron paramagnetic resonance.


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