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J. Biol. Chem., Vol. 281, Issue 38, 27827-27835, September 22, 2006

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Paired Bacillus anthracis Dps (Mini-ferritin) Have Different Reactivities with Peroxide^*

Xiaofeng Liu, Kijeong Kim^¶, Terrance Leighton, and Elizabeth C. Theil¹

From the Children's Hospital Oakland Research Institute, Oakland, California 94609, Department of Nutritional Science and Toxicology, University of California, Berkeley, California 94720, and ^¶Department of Microbiology, Chung-Ang University College of Medicine, Seoul 156-756, Korea

Received for publication, February 14, 2006 , and in revised form, June 26, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Dps (DNA protection during starvation) proteins, mini-ferritins in the ferritin superfamily, catalyze Fe²⁺/H₂O₂/O₂ reactions and make minerals inside protein nanocages to minimize radical oxygen-chemistry (metal/osmotic/temperature/nutrient/oxidant) and sometimes to confer virulence. Paired Dps proteins in Bacillus, rare in other bacteria, have 60% sequence identity. To explore functional differences in paired Bacilli Dps protein, we measured ferroxidase activity and DNA protection (hydroxyl radical) for Dps protein dodecamers from Bacillus anthracis (Ba) since crystal structures and iron mineralization (iron-stain) were known. The self-assembled (200 kDa) Ba Dps1 (Dlp-1) and Ba Dps2 (Dlp-2) proteins had similar Fe²⁺/O₂ kinetics, with space for minerals of 500 iron atoms/protein, and protected DNA. The reactions with Fe²⁺ were novel in several ways: 1) Ba Dps2 reactions (Fe²⁺/H₂O₂) proceeded via an A_{650 nm} intermediate, with similar rates to maxi-ferritins (Fe²⁺/O₂), indicating a new Dps protein reaction pathway, 2) Ba Dps2 reactions (Fe²⁺/O₂ versus Fe²⁺/O₂ + H₂O₂) differed 3-fold contrasting with Escherichia coli Dps reactions, with 100-fold differences, and 3) Ba Dps1, inert in Fe²⁺/H₂O₂ catalysis, inhibited protein-independent Fe²⁺/H₂O₂ reactions. Sequence similarities between Ba Dps1 and Bacillus subtilis DpsA (Dps1), which is regulated by general stress factor (SigmaB) and Fur, and between Ba Dps2 and B. subtilis MrgA, which is regulated by H₂O₂ (PerR), suggest the function of Ba Dps1 is iron sequestration and the function of Ba Dps2 is H₂O₂ destruction, important in host/pathogen interactions. Destruction of H₂O₂ by Ba Dps2 proceeds via an unknown mechanism with an intermediate similar spectrally (A_{650 nm}) and kinetically to the maxi-ferritin diferric peroxo complex.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Dps (DNA protection during starvation) proteins are a family of bacterial proteins in the ferritin superfamily, first identified in Escherichia coli as dodecameric DNA-binding proteins in cells stressed by starvation (1). The proteins are stable nanocages with large (5-nm diameter) central cavities (2, 3) and have been observed in vivo as part of biocrystalline complexes with DNA (e.g. see Ref. 4). Protection of DNA from damage by free radicals, the common property of Dps proteins, is accomplished by converting oxidants and iron released during stress to benign, hydrated, ferric oxide minerals inside the Dps protein cage (2, 3). Because only some Dps protein dodecamers bind DNA but all protect DNA from hydroxyl radical damage in vitro, the mechanisms of Dps protein protection of DNA from oxidant damage and other stresses such as nucleases, radiation, and heat are subjects of active study (e.g. Refs. 2–9). When DNA-Dps interactions occur, N- or C-terminal protein extensions that protrude from the protein nanocages are thought to be involved (10, 11). The ability of Helicobacter pylori, Borrelia burgdorferi, and other pathogens to survive oxidative burst killing within macrophages and neutrophils has been attributed to Dps protein dodecamer protection of DNA based on the increased oxidant sensitivity of dps deletion mutants (e.g. Refs. 12–18). Bacteria with two dps genes are rare although present in many Bacilli (see Fig. 1). No comparative studies of the reaction properties of pairs of Dps proteins have been made to our knowledge.

The ferritin superfamily, of which Dps protein dodecamers are members, are protein nanocages that form protein-encased minerals of hydrated ferric oxide through a series of steps. First, oxidation of two ferrous ions occurs at catalytic sites in the protein cage that are related to catalytic sites in di-iron oxygenases, such as stearoyl-acyl carrier protein 9-fatty acyl desaturase, ribonucleotide reductase, and methane monooxygenase, except that in the ferritins the iron is a substrate (3). In maxiferritins, the first step in iron mineralization is the oxidation of coupled diferrous ions with O₂ via a diferric peroxo complex (19–23), with partly characterized decay products such as diferric hydroperoxide complex (24) to form diferric oxy mineral precursors. Then, by mechanisms unknown, the mineral precursors move through the protein cage to the cavity and are concentrated by clustered carboxylates on the inner surface of the protein cavity and, later, at the mineral surface; inorganic hydrolysis and polymerization in the ferritin cavity form the hydrated ferric oxide minerals. Bacterial Dps proteins are miniferritins with 12 subunits (2, 3, 25) that contrast with the 24 in eukaryotic and bacterial maxi-ferritins. The location of the catalytic site in the protein nanocage is also different as are the active site ligands and the oxidant substrate (2, 3, 22, 26–28). dps genes occur in organisms inhabiting a wide range of environmental niches, including those comparable with primitive, terrestrial atmospheres in at least 130 bacterial and Archaea species (8, 29). The stable self-assembled ferritins, 4 -helix-bundle subunits that interact to create the internal cavity (diameters of 5 nm in Dps proteins and 9 nm in maxi-ferritins) for the mineral (17, 22, 25, 27, 30–35) also have gated pores at the junctions of triple subunit sets, with highly conserved gates thought to control the entry and exit of iron through the protein shell (3, 36). Structural features specific to the Dps mini-ferritins include location of the Fe²⁺ oxidation sites between two subunits at the surface of the cavity rather than in the middle of the subunit as in maxi-ferritins (2, 17, 25, 27, 30–32, 37, 38) and the consumption of H₂O₂ during iron uptake, oxidation, and mineralization rather than the release of H₂O₂ as in eukaryotic ferritins (22, 26). In the E. coli and Listeria innocua Dps protein dodecamers, H₂O₂ is the preferred oxidant based on ferroxidase rates (26–28).

The two dps genes, common in Bacillus spp., have different regulatory properties (6, 15, 39, 40) and, as explored in this report, functional differences. MrgA in B. subtilis, for example, now known to be a dps gene, is regulated by PerR and responds to peroxide-specific transcriptional regulation. DpsA, the second dps gene in B. subtilis, is regulated by the SigmaB factor in stationery phase and Fur in log phase, responding to glucose starvation, metal, heat, and osmotic stresses. To compare biochemical properties of Dps protein pairs, we chose to study the Dps protein dodecamers from Bacillus anthracis, since they are the only Bacillus Dps proteins for which the crystal structures are known (31) (crystal structures for two Dps proteins from Lactococcus lactis, which have no recognizable Fe²⁺ oxidation sites, have been solved recently (41)). Based on sequence similarities to Dps proteins in other Bacilli (see Fig. 1), B. anthracis Dps1 (also called Dlp-1) (31) is analogous to DpsA, and Dps2 (also called Dlp-2) (31) is analogous to MrgA.

We now report that Ba² Dps1 and Ba Dps2 protein dodecamers have different reactions with Fe²⁺/H₂O₂ and similar reaction rates with Fe²⁺/O₂. In Ba Dps2 the rate increased 3-fold with H₂O₂, but with Ba Dps1 no protein-dependent rate was detected. In anaerobic conditions an A_{650 nm} intermediate (diferric peroxo) characteristic of the animal maxi-ferritin Fe²⁺/O₂ reaction was observed in the Ba Dps2 Fe²⁺/H₂O₂ reaction. Both Ba Dps1 and Ba Dps2 protected DNA from oxidative damage (Fe²⁺ + H₂O₂ in air), but Ba Dps1 was less effective than Ba Dps2. The different activities of Ba Dps1 and Ba Dps2 with Fe²⁺ and H₂O₂ in air and the enhanced activity of Ba Dps2 in the absence of oxygen suggest that each protein contributes to bacterial survival under different sets of conditions in vivo and together provide protection against a wide range of environmental stresses.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cloning—To clone the dps genes from B. anthracis dlp-1 (Ba dps1) and dlp-2 (Ba dps2), coding regions for Ba Dps1 and Ba Dps2 were amplified from the chromosomal DNA of B. anthracis (Sterne 34-F2) by PCR with the following primers: Ba Dps1F, 5'-CAAACACATGAACAAACAAGTAATC; Ba Dps1R, 5'-CAACTGGATCCTTATTGATTCAAGGATCCAAC; Ba Dps2F, 5'-CAAACACATGGAGTACGAAAACAAATG; Ba Dps2R, 5'-TTTAAGAACGCACTTAGCATGGATCCAAC. NdeI and BamH1 sites (underlined) were created during amplification for subcloning into an E. coli expression vector. Amplified PCR products were cloned into Topo vectors using the TA cloning kit (Invitrogen) to create plasmids Topo II-Ba Dps1 and Topo II-Ba Dps2. The DNA fragments of Ba dps1 and Ba dps2 were then excised from Topo II-Ba Dps1 and Topo II-Ba Dps2 by restriction digestion with NdeI and BamH1 and ligated into the multiple cloning sites of the protein expression vector pET-3a (Invitrogen), yielding plasmids pET-3a-Ba Dps1 and pET-3a-Ba Dps2. Both plasmids were sequenced with T7 primer to confirm that the open reading frame contained the correct sequences.

Expression, Purification, and Properties of Recombinant Ba Dps1 and Ba Dps2—Protein expression used cultures of E. coli competent cells BL21 (DE3)-Plyss strain (Novae) and conditions previously described (37, 42, 43). The expression vectors were pET-3a-Ba Dps1 or pET-3a-Ba Dps2, and washed cells were stored at –20 °C overnight. After sanitation, cell extracts were clarified by centrifugation for 2 h at 4 °C at 43,000 x g, and ammonium sulfate was added to the supernatant solution to a concentration of 60%, conditions where Ba Dps1 and Ba Dps2 remained soluble. Buffer exchange and dialysis against two changes of 25 mM Bis-trispropane (pH 7.5) was followed by ion-exchange chromatography (Mono Q from GE Healthcare) (buffer, 25 mM Bis-trispropane (pH 7.5); linear gradient, 0 to 1 M NaCl). Fractions containing Ba Dps1 or Ba Dps2, identified by SDS-PAGE (15% acrylamide), were pooled, concentrated, and applied to a Sephacryl S300 (GE Healthcare) gel-filtration column. Purified Ba Dps1 and Ba Dps2 proteins dialyzed against 100 mM Mops and 100 mM NaCl (pH 7.0) and stored at 4 °C appeared to be stable for up to 2 weeks for iron-free proteins and 2 months for mineralized proteins.

Protein concentrations were determined with the Bradford Assay (Bio-Rad), and the endogenous or mineralized iron content of the purified, recombinant proteins was analyzed with a colorimetric assay for the detection of Fe²⁺-phenanthroline at 510 nm (44). Analysis of protein purity used electrophoresis in SDS denaturing gels (12%) and in native (4–15%) polyacrylamide gels calibrated with the BroadRange protein marker from Bio-Rad and stained with Coomassie Blue. Proteins with apparent sizes of 200 kDa were observed by native gel electrophoresis, gel filtration, and mass spectrometry (see below) and are consistent with the dodecameric assemblies of Dps subunits. Protein purity was >95%, and the endogenous iron content was <0.1 iron atoms/protein nanocage, as previously observed (31).

To confirm the identity of the expressed protein, peptides produced by denaturation and trypsin digestion of recombinant B. anthracis Ba Dps1 and Ba Dps2 (Trypsin Gold, mass spectrophotometer grade, Promega Inc., at a 1:100 molar ratio (pH 7.8) with incubation for 18 h at 37 °C, before adding 0.5 µg of phenylmethylsulfonyl fluoride) were analyzed by MALDI-TOF (Bruker Biflex III, in the negative reflector mode, N₂ laser) and calibrated with external standards (Bruker Daltonics). Data were analyzed with SpectroTYPER (sequenom) software; the error of the observed masses was estimated to be 0.1%. Predicted sites of tryptic digestions and masses were obtained with Pepcutter (ExPAcy) software. Four Ba Dps1 tryptic peptides had the following masses: residues 42–59, 2153.0 Da (theoretical) and 2153.3 Da (experimental); residues 12–22, 1293.7 Da (theoretical) and 1293.5 Da (experimental); residues 32–41, 1203.6 Da (theoretical) and 1203.4 Da (experimental); residues 4–11, 942.6 Da (theoretical) and 942.1 Da (experimental); undigested subunit, 16910.43 Da (theoretical) and 16877.5 Da (experimental). Three tryptic peptides from Ba Dps2 had the following masses: residues 44–61, 2196.0 Da (theoretical) and 2195.5 Da (experimental); residues 62–74, 1384.8 Da (theoretical) and 1384.6 Da (experimental); residues 5–13, 1015.6 Da (theoretical) and 1015.3 Da (experimental); undigested subunit, 16648.8 Da (theoretical) and 16771.4 Da (experimental).

Formation of the Iron Mineral—Determination of the number of iron atoms that can be accommodated inside the Dps dodecameric protein cages used solutions of recombinant Ba Dps1 and Ba Dps2 proteins in a buffer of 200 mM Mops (pH 7.5) with 200 mM NaCl. Protein solutions were mixed with an equal volume of a freshly prepared solution of FeSO₄ in 1 mM HCl to minimize spontaneous oxidation and hydrolysis; 50-fold increments of Fe²⁺ atoms/protein dodecameric nanocage were added up to 1000 iron atoms. The Fe²⁺ and protein mixture was incubated for 1 h at room temperature followed by incubation overnight at 4 °C to complete mineralization (conversion of ferric mineral precursors to mineral), as previously described for maxi-ferritins (36). After the incubation, Fe²⁺/protein mixtures were passed through G-25 columns to trap insoluble iron hydroxide precipitates on the top of the gel that were only observed at >500 Fe²⁺/protein dodecamers. The iron content of mineralized Dps proteins was determined by incubating with dithiothreitol (5 mM) and 2,2'-bipyridyl (5 mM) for 24 h at 4 °C; the Fe²⁺-bipyridyl complex was analyzed spectrophotometrically (molar extinction coefficient: 8300 M^–1 cm^–1 at 522 nm).

Fe²⁺ Oxidation Kinetics—To measure the kinetics of iron oxidation by O₂, solutions of ferrous substrate (0.12–4.8 mM FeSO₄ in 1 mM HCl) were rapidly mixed (1 ms) with an equal volume of Ba Dps1 or Ba Dps2 protein dodecamers (2 mg/ml, 10 µM) in 200 mM Mops/200 mM NaCl at pH 7.0 aerobically or anaerobically using a stopped-flow, UV-visible spectrophotometer ( *, Applied Photophysics, Surrey, UK); the molar ratio of Fe²⁺:protein ranged from 2 to 480 iron atoms per Dps protein dodecamer (1–40 iron per subunit) as previously described (26, 37). Data were collected at 350 nm (e.g. Refs. 3, 4, 25, 32, and 45), nonspecific absorbance of ferric oxy species, and at 650 nm, the absorbance maximum of the diferric peroxo reaction intermediate observed in maxi-ferritin ferroxidase reactions (19, 36, 43). Kinetic studies with H₂O₂ as the oxidant for Fe²⁺ under aerobic and anaerobic conditions used a small volume of H₂O₂ added to protein solutions (2 mg/ml (10 µM) protein dodecamers) to a final H₂O₂ of 10 mM followed by rapid mixing with various [Fe²⁺] solutions at room temperature (20 °C). In a few experiments anaerobic mixtures of protein dodecamers (10 µM) and Fe²⁺ were mixed with anaerobic solutions of H₂O₂ to initiate the reaction, but the change in the order of addition had no effect. The concentration of 10 mM H₂O₂, chosen to ensure complete Fe²⁺ oxidation, is consistent with previous studies (46). O₂ was removed by purging all solutions with argon under intermittent vacuum for 10 min as described in Mabrouk et al. (47). In the absence of protein and O₂, a control sample of Fe²⁺ showed no oxidation (change in absorbance) during the course of the experiments.

Initial rates of Fe²⁺ oxidation (ferroxidase activity) were calculated by fitting the linear portion of the ascending part of each kinetic trace (with R 0.98) over a range of iron:protein dodecamer ratios obtained by varying the iron concentration. The initial rates at 480 Fe²⁺/Dps were taken as V_max for Fe²⁺ oxidation analyzed at 350 or 650 nm. All data presented were computed from measurements on at least three independent protein preparations, each analyzed three times, as previously described (37).

Effects of Ba Dps1 and Ba Dps2 on DNA Degradation by Fe²⁺ + H₂O₂—Supercoiled plasmid DNA from pBR322 or pUC18 was purified with the Qiaprep Midi kit (Qiagen, Chatsworth, CA) followed by ethanol precipitation. Samples for supercoiled and relaxed pBR322 were obtained from Topogen. Effects of Ba Dps1 and Ba Dps2 on DNA mobility during electrophoresis and resistance to hydroxyl radical degradation used methods similar to those in Martinez and Kolter (48). Briefly, to analyze Dps protein protection of DNA from hydroxyl radical degradation, 4 nM DNA and Dps protein were incubated at 37 °C for 10 min followed by the sequential addition of Fe²⁺ (50 µM final concentration), H₂O₂ (10 mM final concentration) with incubation for an additional 15 min at 37 °C. DNA integrity was examined with 1% agarose gels in TAE buffer (40 mM Tris acetate, 1 mM EDTA (pH 8.0)), calibrated with pBR322 (supercoiled, linear (NdeI digest), and relaxed (topoisomerase digest) obtained from Topogen (Port Orange, FL)). Agarose gels were stained with ethidium bromide and were analyzed with an Alpha-imager (Alpha Innotech Corp.).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Sequence Comparisons of Bacillus Dps Pairs—Pairs of translated dps genes in each Bacillus spp. have 60–62% sequence identity (Fig. 1), which is similar to the sequence similarities between the animal H and L ferritin subunits that have different catalytic activities but superimposable secondary and quaternary structures (49). An array of known, translated Bacillus Dps sequences are aligned in Fig. 1 by decreasing similarity to Ba Dps1 using ClustalW. The sequence similarity for B. anthracis, Bacillus cereus, and Bacillus thuringiensis Dps is 99% and parallels the close genomic relationship of the species (50); each species has a distinct environmental niche.

The primary sequences of Dps proteins are less conserved than the protein nanocage structure (Fig. 1) (11, 31, 32). For example, the quaternary structures of E. coli, B. anthracis (Ba Dps1 and Ba Dps2), and Bacillus brevis Dps protein dodecamers in crystals are very similar, but the sequence conservation between E. coli Dps and B. brevis Dps is only 30%. For the two B. anthracis Dps proteins and E. coli, the sequence identity is only 22%. B. subtilis Dps2 (MrgA) has the lowest sequence similarity to E. coli Dps protein in the array, with only 19% sequence identity.

View larger version (55K): [in this window] [in a new window]   FIGURE 1. Comparison of pairs of B. anthracis Dps proteins Ba (Dps1 (Dlp-1) and Ba Dps2 (Dlp-2)) to other Bacillus Dps sequences. Conserved residues proposed as Fe2+ substrate sites for oxidation, by analogy to other Dps proteins, are shaded. Ba Dps1 (Dlp-1), the Dps protein of B. anthracis strain Ames (gi: 20278904); Ba Dps2 (Dlp-2), the second Dps protein of B. anthracis strain Ames (gi: 20278906). The proteins are 99% identical to the Dps1 of B. cereus(Bc) (gi: 29895698), Dps2 of B. cereus (gi: 29898643), Dps1 from B. thuringiensis (Bt) strain 97–27 (gi: 49481572) and Dps2 from B. thuringiensis strain 97–27 (gi: 49481226). Bs Dps1 is MrgA from B. subtilis(Bs) strain 168 (gi:16080351), Bs Dps2 is DpsA from B. subtilis strain 168 (gi:16080117); Bb Dps is the single identifiable Dps in B. brevis (Bb) (gi:31615599), and Ec Dps, the first Dps protein identified (1) (gi:33357272) is from E. coli (Ec). Protein crystal structures are known for three Bacillus spp. Dps proteins: B. anthracis Dps1 (Dlp-1), B. anthracis Dps2 (Dlp-2), and B. brevis (11, 31, 32).

Ferroxidase activity of Ba Dps1 and Ba Dps2 protein dodecamers with O₂ as the oxidant was significant and contrasted with the E. coli Dps protein previously studied (26, 28). The Fe²⁺ oxidation rates in the two Ba Dps protein dodecamers were 20% that of Fe²⁺ oxidation in 24 subunit maxi-ferritins (Table 1) assuming 1 active site/subunit in both maxi- and mini-ferritins. The rates of Fe²⁺ oxidation by O₂ for Ba mini-ferritins were, by contrast, 50- and 65-fold faster than that of catalytically inactive L maxi-ferritin and E. coli Dps protein dodecamers (26, 37). The rate of oxidation in both Ba Dps proteins changed at approximately 24 Fe²⁺/protein dodecamer, indicating that all 12 putative di-iron ferroxidase sites had been filled (Fig. 2, C and D). Such a rate increase in Fe²⁺ oxidation above 24 Fe²⁺/protein dodecamer suggests either an increased Dps ferroxidase turnover rate caused by the phase transition to mineral, to Fe²⁺ oxidation at other types of sites such as the mineral surface, or both.

Differences between B. anthracis Dps1 and Dps2 in the Reactivity with Fe²⁺ and H₂O₂—There was a qualitative difference between Ba Dps1 and Ba Dps2 in the reactions with Fe²⁺ + H₂O₂ (Fig. 3, B and C). Ba Dps1 had no detectable, protein-dependent Fe²⁺/H₂O₂ reaction under anaerobic or aerobic conditions, in contrast to Ba Dps2 (Fig. 3). However, Ba Dps1 inhibited the solution reaction between Fe²⁺ and H₂O₂ (Fig. 3, A and B) (note that when Fe²⁺ was added to H₂O₂ without protein, the results as measured here were the same aerobically or anaerobically, but only the aerobic reaction is shown in Fig. 3). Fe²⁺ oxidation was so fast that the reaction was apparently complete during the mixing time (dead time 1 ms), and the A_{350 nm}, was constant during the reaction period but increased with increasing concentrations of Fe²⁺ (Fig. 3A). When Ba Dps1 protein dodecamers were present there was also no change in the A_{350 nm} absorbance after mixing, but unlike the reaction of Fe²⁺ and H₂O₂ without protein, the A_{350 nm} was low and independent of the Fe²⁺ concentration (Fig. 3B). The apparent inhibition by Ba Dps1 protein dodecamers of the inorganic Fe²⁺/H₂O₂ reaction may reflect Fe²⁺ binding to the protein that decreases Fe²⁺/H₂O₂ reaction. The reaction of Ba Dps2 with Fe²⁺ and H₂O₂ was 3-fold faster than with O₂, in contrast to Ba Dps1, with a hyperbolic progress curve similar to the reaction with O₂ (Fig. 3C) (Table 1). This difference is smaller than the >100-fold rate difference between Fe²⁺/H₂O₂ and Fe²⁺/O₂ with E. coli Dps protein (26). Differences in Fe²⁺/H₂O₂ reactivity among Ba Dps2, E. coli Dps (26), and L. innocua Dps protein dodecamers (28) may be a result of different active site conformations and ordered H₂O molecules involved in the reaction. The absence of any reactivity with H₂O₂ observed in Ba Dps1 is unique among known Dps protein dodecamers.

View larger version (35K): [in this window] [in a new window]   FIGURE 2. Similarity of Fe2+ oxidation by B. anthracis Dps1 and Dps2 proteins in air. Rapid mixing of solutions of ferrous sulfate and protein, Dps1 (panel A) and Dps2 (panel B), and absorbance measurements used an Applied PhotoPhysics *, UV/visible spectrophotometer; the data were analyzed as described under "Experimental Procedures." Final protein concentrations were 5 µM (1 mg/ml) in 100 mM Mops, 100 mM NaCl (pH 7.0). Reactions were followed at 350 nm for the appearance of [Fe3+O] products with Fe2+ ranging from 12 to 240 Fe2+ per protein nanocage (12 sites/protein cage) as shown in panel A (Ba Dps1) and panel B (Ba Dps2). The apparent initial rates of Fe2+ oxidation for Ba Dps1 (panel C) and Ba Dps2 (panel D) are plotted versus Fe2+ concentration; the arrow indicates a rate change at 24 Fe2+/protein dodecamer (2Fe2+/active site).

View larger version (33K): [in this window] [in a new window]   FIGURE 3. Different reactions of B. anthracis Dps1 and Dps2 proteins with Fe2+ and H2O2. Progress curves are displayed for Ba Dps1 and Ba Dps2 in the presence and absence of O2. Final protein concentrations were 5 µM (1 mg/ml) in 100 mM Mops, 100 mM NaCl (pH 7.0). Progress curves of the appearance of [Fe3+O] products, absorbing at 350 nm, were obtained with Fe2+ concentrations ranging from 0.06 to 1.2 mM (12–240 Fe2+ per protein nanocage). H2O2 concentration was 5 mM in all reactions. A, solution reaction between Fe2+ and H2O2, aerobic; B, Ba Dps1, Fe2+ + O2 +H2O2; C, Ba Dps2, Fe2+ + O2 + H2O2; D, Ba Dps 2, Fe2+ + H2O2. No protein-dependent reaction was detected with Dps1 with or without O2 (not shown), although inhibition to the solution reaction was apparent (panel B). Ba Dps2 showed faster rates with H2O2 than with O2 alone (panel C; compared with Fig. 2B) and still faster rates with H2O2 in the absence of O2 (panel D), indicating that O2 inhibited the Fe2+/H2O2 reaction with Dps2.

View larger version (16K): [in this window] [in a new window]   FIGURE 4. Appearance of an A650 nm intermediate in Fe2+ oxidation by B. anthracis Dps2 with H2O2 in the absence of O2. Progress curves show the reaction of Fe2+/H2O2 reaction with Ba Dps2 protein dodecamers (4 Fe2+ per site; 48 Fe2+/protein dodecamer) in the absence of O2. O2 was removed by exhaustive argon purges with intermittent evacuation as described under "Experimental Procedures" and Mabrouk et al. (47); the stability of purged and control solutions of Fe2+ during the time measurements demonstrated the effective removal of O2. Each trace represents 5000 data points, and the traces presented are representative of 11 sets of measurements using 3 different protein preparations. The transient species absorbing at 650 nm, reaching a maximum accumulation at 108 ms, had similar kinetics to the diferric peroxo intermediate in maxi-ferritins (19). The biphasic reaction path in the formation of Fe3+O species had a broad absorption, measured at A350 nm. The progress curves at 650 and 350 nm were similar for the first 100 ms, when the A650 species began to decay; there is no UV-visible spectral signature that distinguishes among diferric peroxo, diferric oxo, and ferric oxo mineral, explaining why the A350 nm does not decrease.

Formation of the A_{650 nm} Reaction Intermediate (diferric peroxo) by B. anthracis with Fe²⁺ and H₂O₂ in the Absence of Air—Fe²⁺/O₂ reactions in animal maxi-ferritins proceed via a well characterized diferric peroxo intermediate, the first reaction product detected after the addition of Fe²⁺ (19–23, 52), with an absorbance maximum at 650 nm (19, 53). In the case of Dps protein dodecamers, no evidence of the diferric peroxo intermediate has been previously reported to our knowledge. Nor was the characteristic blue, A_{650 nm}-absorbing species observed in this study in the presence of O_2. However, with H₂O₂ in the absence of O₂, the formation and decay of a species absorbing at 650 nm was clear (Fig. 4, Table 1). The molar extinction coefficient is 1987 ± 283 M^–1·cm^–1 for the A_{650 nm} species in Ba Dps2, assuming it is a diferric peroxo species, as it is in maxi-ferritins, where the extinction coefficient is approximately half that in mini-ferritins. The progress curves at 350 and 650 nm for the Fe²⁺ + H₂O₂ reaction of Ba Dps2 protein dodecamer in the absence of air were a parallel for the first 100 ms (Fig. 4) when the A_{650 nm} absorbing species began to decay. As the reaction proceeded the rate of Fe³⁺ formation measured at 350 nm decreased, as shown in the progress curve, but the absorbance continued to increase as the diferric peroxo (A_{650 nm}) decayed, indicating the conversion of the diferric peroxo complex to a Fe³⁺O product, which has similar but not identical spectral properties to the diferric peroxo precursor at 350 nm.

The formation and decay of the diferric peroxo reaction intermediate (A_{650 nm}) in the Fe²⁺/H₂O₂ reaction permitted computation of kinetic parameters impossible from the absorbance of Fe³⁺O at 350 nm because of the spectral similarities among intermediates, products, mineral precursors, and mineral in UV-visible spectroscopy that obscures the decay of intermediates (Fig. 4). The kinetic parameters are similar to those for vertebrate maxi-ferritin kinetics (23, 37, 53, 54), where DFP, diferric oxo/hydroxo products/mineral precursors, and ferric oxy mineral have been differentiated by UV-visible (A_{650 nm}), Mössbauer, resonance Raman, and extended x-ray absorption fine structure spectroscopies (19–21). The k_cat for the A_{650 nm} species in Ba Dps2 was 638 ± 118 mol of Fe³⁺-O-O-Fe³⁺ formed/mol of Dps2/s; the approximate t for formation was 54 ms and for decay was 1.5 s. A K_app of 0.067 mM for the DFP in Ba Dps2 in the absence of air is larger than the K_d for Fe²⁺ binding in the L. innocua Dps (10^–7 M, determined by isothermal calorimetry) (28) in the absence of air.

The Hill coefficient for formation of the diferric peroxo complex in Ba Dps2 reaction was 0.75 ± 0.02 (Table 1), contrasting with the positive cooperativity observed in maxi-ferritins, (37, 45, 55, 56). When the initial rates were plotted versus Fe²⁺/molecule, the sharp transition at 2.0 Fe²⁺/site characteristic of maxi-ferritin di-iron sites was absent.

Ba Dps Protection of DNA from Fe²⁺/H₂O₂ (Hydroxyl Radical Degradation)—The protection of DNA from degradation by hydroxyl radical is analyzed as changes in DNA mobility during electrophoresis in agarose gels in air using supercoiled plasmid DNA of defined sizes (e.g. Refs. 10, 26–28, 30, and 48). In the absence of protein the well known sensitivity of pBR322 supercoiled DNA to radical damage is illustrated in Fig. 5, lanes 3–6; DNA was linearized by Fe²⁺, relaxed by H₂O₂ and destroyed by Fe²⁺ and H₂O₂, as determined by comparison to DNA standards for the different supramolecular states.

Ba Dps2, which can use H₂O₂ as a substrate (Figs. 2 and 3; Table 1), provides essentially complete protection from Fe²⁺/H₂O₂ degradation (Fig. 5, lane 8). With Ba Dps1, DNA decreased (Fig. 5, lane 7), indicating partial protection. Similar results were obtained with pUC18 plasmid DNA. The difference in DNA protection by the two Ba Dps proteins is likely due to the fact that Ba Dps1 will consume Fe²⁺ and Ba Dps2 will consume both H₂O₂ and Fe²⁺ in the ferroxidase reaction (Figs. 2 and 3).

View larger version (30K): [in this window] [in a new window]   FIGURE 5. Ba Dps1 (Dlp-1) and Ba Dps2 (Dlp-2) protection of DNA from degradation by hydroxyl radical. Supercoiled pBR322 DNA (4 nM) was incubated with Dps protein (protein:DNA molar ratio, 100:1). Fe2+ 50µM and H2O2 (10 mM) were added as indicated. Mixtures were incubated at 37 °C in air and fractionated on 1% agarose gels; staining used ethidium bromide (see "Experimental Procedures"). Lane 1, relaxed DNA (R); lane 2, linear (L) DNA; lane 3, supercoiled (SC) DNA; lane 4, SC + Fe2+; lane 5, SC + H2O2; lane 6, SC + Fe2++ H2O2; lane 7, SC + Fe2+ + H2O2 + Ba Dps1; lane 8, SC + Fe2+ + H2O2 + Ba Dps2.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

H₂O₂ is generally the preferred substrate over O₂, at least in Dps proteins from organisms that have only one dps gene (26, 27). It is the reaction with H₂O₂ that is considered the main factor in Dps protein dodecamer DNA protection and resistance to oxidant stress (2). However, we found that Ba Dps2 used O₂ or H₂O₂ in air, with rates that differed only 3-fold (Table 1), not the 100-fold change observed in E. coli Dps protein dodecamers (26). Moreover, Ba Dps1 had no detectable ferroxidase activity with H₂O₂ either aerobically or anaerobically (Fig. 3), although the activity with O₂ was comparable with Ba Dps2 (Fig. 2, Table 1). Nevertheless, both Ba Dps1 and Ba Dps2 protect DNA (Fig. 5). The difference in the activity with H₂O₂ in the ferroxidase reactions of the two B. anthracis Dps proteins indicates that DNA protection by Ba Dps1 is restricted to removing Fe²⁺ and O₂, whereas Ba Dps2 can remove Fe²⁺, O₂, and H₂O₂, which may explain the apparently greater DNA protection by Ba Dps2 than by Ba Dps1 (Fig. 5).

Ferroxidase reactions in Ba Dps1 and Ba Dps2 differ from the reactions in other Dps proteins or maxi-ferritins in several ways. For example, the ferroxidation rates of Ba Dps1 and Ba Dps2 with O₂ were faster than E. coli and L. innocua Dps proteins (26, 28). In addition, rates of the anaerobic Fe²⁺/H₂O₂ reaction in Ba Dps2 are faster than in air but equivalent to rates for maxi-ferritin in air and with the diferric peroxide reaction intermediate (A_{650 nm}) of maxi-ferritins. The different Dps2 reactions of Fe²⁺ may reflect the ability of Bacillus to grow under a variety of oxygen concentrations.

Detection of a diferric peroxo complex in the anaerobic reaction of Ba Dps2 with Fe²⁺ and H₂O₂ is without precedent among ferritins, as is the extinction coefficient; positive cooperativity is also absent. A stoichiometry of 1.94 ± 0.11 Fe²⁺/site can be computed from the data but does not reflect the increase in rate when the Fe²⁺ added is >2Fe²⁺/site. The active site structure in Dps proteins, mainly inferred from metal sites observed in protein crystals, has a small number of metal ligands. Less information on iron ligands is currently available from mutagenesis studies than will be needed to understand the spectral observations reported here. Thus, a structural explanation of the source of the distinctive features of the A_{650 nm} species in Ba Dps 2 is premature but might include multiple aromatic residues nearby, accessibility to solvent/substrate, or proximity to the cavity (11, 27, 31). In a recent study that appeared online after submission of this manuscript and where the results in solution were related to oxidant protection by maxi-ferritin in cells (Ref. 62 and references therein), the overall reaction proposed for the maxi-ferritin catalytic site with H₂O₂ as a substrate was 2 Fe²⁺ + H₂O₂ + 2H₂O 2Fe(O)OH_core + 4H⁺. How this reaction relates to the A_{650 nm} intermediate observed here with Ba Dps remains to be determined in the future.

The putative active site residues in Ba Dps1 and Ba Dps2 (metal sites in protein crystals (31)), which are potential drug targets to control pathogenic bacteria, are similar to those in other Dps proteins (see Fig. 1; for review, see Ref. 2) but have yet to be studied directly. However, the similarity of the metal sites in protein crystals suggests that the catalytic differences between Bacillus Dps, mini-ferritin proteins, and those in other bacteria are due to "second shell" residues yet to be identified. A role for second shell residues in maxi-ferritin ferroxidase activity has also been indicated recently (37). Different conformations and metal binding ligands at or around ferroxidase sites (including ordered H₂O molecules), which result from variation in Dps primary sequences, may contribute to both the mechanistic and kinetic variability observed here and among other ferritins (e.g. Refs. 24, 37, 46, and 63).

Distributing Dps functions between the two Bacillus Dps proteins Ba Dps1 and Ba Dps2 in B. anthracis may relate to different regulatory signals. For example, in B. anthracis, Dps1 is most similar in sequence to B. subtilis DpsA, which is regulated by Sigma factor B and general stress, whereas B. anthracis Dps2, the protein which uses H₂O₂ as a substrate, is most similar in sequence to MrgA in B. subtilis, which is regulated by PerR and H₂O_2. Separation of function in paired Dps mini-ferritins of Bacilli could be analogous to the two animal maxi-ferritin H and L proteins, which have different catalytic activities and signal responses (3, 45, 64–66) and which co-assemble in physiologically dependent ratios (3, 45, 51). Understanding how paired Dps proteins of Bacillus function in vivo and how the proteins selectively use O₂ and H₂O₂ will clarify the contributions of Dps protein nanocages to DNA protection and iron/oxidant metabolism in host/pathogen interactions as well as identify potential drug targets in bacterial infection.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grant DK-20251 (to X. L. and E. C. T.), Cooley's Anemia Foundation (to X. L.), and Defense Advanced Research Projects Agency (to K. K. and T. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Children's Hospital Oakland Research Institute, 5700 Martin Luther King, Jr. Way, Oakland, CA 94609. Tel.: 510-450-7670; Fax: 510-597-7131; E-mail: etheil{at}chori.org.

² The abbreviations used are: Ba, B. anthracis; Bis-tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; Mops, 4-morpholinepropanesulfonic acid.

	ACKNOWLEDGMENTS

 
We thank Christina Yi for her contribution in vector construction, recombinant protein expression, and protein purification and Dr. Mark Shigenaga for technical help in MALDI-TOF analysis.

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