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Thermosynechoccus elongatus DpsA binds Zn(II) at a unique three histidine-containing ferroxidase center and utilizes O2 as iron oxidant with very high efficiency, unlike the typical Dps proteins Flaminia Alaleona*, Stefano Franceschini*, Pierpaolo Ceci, Andrea Ilari and Emilia Chiancone C.N.R. Institute of Molecular Biology and Pathology, Department of Biochemical Sciences ‘A. Rossi-Fanelli’, University of Rome ‘La Sapienza’, Italy Keywords Dps proteins; ferroxidase center; ferroxidation reaction; protection from; reactive oxygen species; Thermosynechococcus elongatus Correspondence E. Chiancone, Department of Biochemical Sciences ‘A. Rossi-Fanelli’, University of Rome ‘La Sapienza’, 00185 Rome, Italy Fax: +39 06 4440062 Tel: +39 06 49910761 E-mail: emilia.chiancone@uniroma1.it Database The atomic coordinates for DpsA-Te have been deposited in the RCSB Brookhaven Protein Data Bank (http://www.rcsb.org) under accession code PDB ID 2VXX *These authors contributed equally to this work (Received 13 October 2009, revised 20 November 2009, accepted 4 December 2009) doi:10.1111/j.1742-4658.2009.07532.x The cyanobacterium Thermosynechococcus elongatus is one the few bacteria to possess two Dps proteins, DpsA-Te and Dps-Te. The present characterization of DpsA-Te reveals unusual structural and functional features that differentiate it from Dps-Te and the other known Dps proteins. Notably, two Zn(II) are bound at the ferroxidase center, owing to the unique substitution of a metal ligand at the A-site (His78 in place of the canonical aspartate) and to the presence of a histidine (His164) in place of a hydrophobic residue at a metal-coordinating distance in the B-site. Only the latter Zn(II) is displaced by incoming iron, such that Zn(II)–Fe(III) complexes are formed upon oxidation, as indicated by absorbance and atomic emission spectroscopy data. In contrast to the typical behavior of Dps proteins, where Fe(II) oxidation by H2O2 is about 100-fold faster than by O2, in DpsA-Te the ferroxidation efficiency of O2 is very high and resembles that of H2O2. Oxygraphic experiments show that two Fe(II) are required to reduce O2, and that H2O2 is not released into solution at the end of the reaction. On this basis, a reaction mechanism is proposed that also takes into account the formation of Zn(II)–Fe(III) complexes. The physiological significance of the DpsA-Te behavior is discussed in the framework of a possible localization of the protein at the thylakoid membranes, where photosynthesis takes place, with the consequent increased formation of reactive oxygen species. Structured digital abstract l MINT-7312099: DpsA (uniprotkb:Q8DL82) and DpsA (uniprotkb:Q8DL82) bind (MI:0407) by x-ray crystallography (MI:0114) Introduction The widely expressed bacterial Dps proteins (DNAbinding proteins from starved cells) are part of the complex defense system that bacteria use to combat stress conditions. The family prototype was identified in stationary-phase Escherichia coli cells, where it binds DNA and protects it from DNase cleavage, and also renders cells resistant to hydrogen peroxide stress [1]. Later observations established that E. coli Dps is also expressed during exponential growth in cells exposed to oxidative stress [2], and that it protects DNA from Abbreviations H-FtHu, recombinant human H-ferritin; ICP-AES, inductively coupled plasma atomic emission spectroscopy. FEBS Journal 277 (2010) 903–917 ª 2010 The Authors Journal compilation ª 2010 FEBS 903 The unusual Thermosynechoccus elongatus DpsA F. Alaleona et al. UV and gamma irradiation, and acid and base shock [3]. Furthermore, it was established that the DNAbinding capacity is shared only by those members of the family that possess a flexible N-terminus or C-terminus rich in positively charged residues or a positively charged molecular surface [4–8]. In contrast, all Dps proteins have iron oxidation ⁄ uptake capacity [9] and are characterized by a shell-like assembly [10–13], in both respects resembling ferritin. They were thus assigned to the ferritin superfamily. There are, however, several different structural and functional features between the two protein families. The ferritin oligomer has 432 symmetry, and in animals is built from 24 highly similar subunits, the L-chains and H-chain, with the latter harboring intrasubunit catalytic centers, whereas Dps proteins are formed from 12 identical subunits assembled with 23 tetrahedral symmetry, and contain unusual intersubunit ferroxidase centers, located at the dimer interfaces [9]. Importantly, whereas purified ferritins use O2 as iron oxidant, with the production of H2O2, Dps proteins typically prefer H2O2, which is about 100-fold more efficient than O2 [14]. The simultaneous consumption of Fe(II) and H2O2 reduces their potential toxicity, as it inhibits hydroxyl radical production via Fenton chemistry. It follows that Dps proteins are able to protect biological macromolecules from Fe(II)-mediated and H2O2-mediated stress more efficiently than ferritins. This functional disparity manifests itself in the different sensitivity of ferritin and Dps deletion mutants to O2-generated and peroxide-generated oxidative stress [15,16]. In turn, differences in the physiological roles of ferritins and Dps proteins are likely to underlie the significant variability in the type and number of ferritinlike proteins expressed in different bacteria. Thus, E. coli and Salmonella enterica possess two ferritins, one heme-containing ferritin (bacterioferritin) and a Dps protein [17,18], whereas Porphyromonas gingivalis [16] and Campylobacter jejuni [15] each contain one ferritin and a Dps protein. Only a few bacterial species express two Dps proteins, such as the radiation-resistant mesophilic eubacterium Deinococcus radiodurans [19,20] and several bacilli [12,21]. The presence of two dps genes appears to be more frequent in cyanobacteria, on the basis of the known genomes sequenced (http://genome. kazusa.or.jp/cyanobase/). Thermosynechococcus elongatus [22,23], Anabaena variabilis, Gloeobacter violaceus, Nostoc punctiforme, Prochlorococcus marinus, Synechococcus sp. and Trichodesmium erythraeum belong to this category. The coexistence of ferritins and Dps proteins is most intriguing, as the structural and functional properties of the Dps family members characterized to date appear to be very conserved. 904 Key to the physiological activity of all of these proteins is the ferroxidase center, which is highly conserved in both ferritins and Dps proteins. In ferritins, the center is bimetallic, as in all known proteins with ferroxidase activity; the two iron atoms are at a distance of about 3 Å, and are connected by an oxobridge. The so-called A-site typically uses a histidine and carboxylates as iron-coordinating ligands, and binds iron with higher affinity than the so-called B-site, where the metal is coordinated only by means of carboxylates [24]. Among Dps proteins, the ferroxidase center was identified in Listeria innocua Dps, where it contains one strongly bound iron coordinated by Glu62 and Asp58 from one subunit, by His31 from the symmetry-related subunit, and by a water molecule that is located about 3 Å from the iron and forms a hydrogen bond with His43 from the same monomer [11]. Ilari et al. [11] proposed that a second iron atom could replace the water molecule and give rise to a canonical bimetallic ferroxidase center. In the known X-ray structures of Dps proteins, the occupancy of the ferroxidase center with iron varies despite the conservation of the iron ligands, a fact that points to a significant influence of residues in the second ligation sphere. Thus, in E. coli Dps the center contains two water molecules, a fact ascribed to the presence of a lysine (Lys48) engaging Asp78, one of the iron ligands, in a salt bridge interaction [25]. For investigation of the physiological basis of the coexistence of two Dps proteins within a single bacterium, those expressed by T. elongatus appeared to be of special interest. T. elongatus is a thermophilic, unicellular, rod-shaped cyanobacterium that lives in hot springs at 55 C. The occurrence of oxygenic photosynthesis entails increased formation of reactive oxygen species as a result of the photosynthetic transport of electrons, such that, besides photosystems I and II, which are the main targets of photodamage, other cellular components are at risk. The T. elongatus genome contains the genes encoding for two Dps proteins, Dps-Te and DpsA-Te (IDs of the respective genes, tll2470 and tll0614), and one ferritin, but lacks catalase ⁄ peroxidase genes. Thus, Dps-Te and DpsA-Te, together with ferritin, must play an important role in alleviating the toxic effects of reactive oxygen species. The most interesting of the two T. elongatus Dps proteins is DpsA-Te. A sequence alignment (Fig. 1) shows that it is the only member of the family among those known that carries a substitution at the ferroxidase center, where a histidine (His78) replaces the canonical aspartate (Asp58 in L. innocua). Near the ferroxidase center, His164 replaces a hydrophobic residue (phenylalanine or methionine), and a phenylalanine (Phe52) FEBS Journal 277 (2010) 903–917 ª 2010 The Authors Journal compilation ª 2010 FEBS F. Alaleona et al. The unusual Thermosynechoccus elongatus DpsA Fig. 1. Alignment of representative sequences of Dps proteins. DpsA-Te from T. elongatus, Dps from H. salinarum (Dps-Hs), Dps from E. coli (Dps-Ec), Dps from B. brevis (Dps-Bb), Dps1 from B. anthracis (Dps1-Ba), Dps2 from B. anthracis (Dps2-Ba), MrgA from Bacillus subtilis (MrgA-Bs), Dps from L. innocua (Dps-Li), Dps-Te from T. elongatus (Dps-Te), and Nap protein from Helicobacter pylori (Nap-Hp). The residues at the ferroxidase center are indicated by arrows, the cysteines are in gray, and DpsA-Te His164 (see text) is in bold and underlined. replaces the highly conserved tryptophan (Trp32 in L. innocua). The structural and functional properties of DpsA-Te described here show features, such as the presence of two Zn(II) bound at the ferroxidase center and the high efficiency of O2 as iron oxidant, that render this protein unique among the Dps proteins characterized to date, and point to a distinct physiological role of DpsA-Te relative to the previously studied Dps-Te [23]. Results Sequence analysis of T. elongatus DpsA The DpsA-Te sequence was compared with those of the Dps family members of known three-dimensional structure (Fig. 1). A sequence similarity search performed with blast (http://blast.ncbi.nml.nih.gov/ Blasy.cgi) showed the highest identity (36%, 64 ⁄ 175 residues) with Halobacterium salinarum DpsA, 29% identity with Dps-Te (46 ⁄ 158 residues), 28% identity with Bacillus brevis Dps (40 ⁄ 139 residues), and 27% with Bacillus anthracis Dps2 (40 ⁄ 139 residues). The sequence identity with the prototypic E. coli Dps and L. innocua Dps was about 22%. DpsA-Te possesses a long N-terminal extension that has a partially hydrophobic character and lacks the DNA-binding signature characteristic of the E. coli Dps N-terminus, namely the positively charged lysines and arginines that interact with the negatively charged DNA backbone. On this basis, and given the lack of a long, positively charged C-terminal extension as in Mycobacterium smegmatis Dps [7], DpsA-Te is not predicted to bind DNA. FEBS Journal 277 (2010) 903–917 ª 2010 The Authors Journal compilation ª 2010 FEBS 905 The unusual Thermosynechoccus elongatus DpsA F. Alaleona et al. The most striking features emerging from the sequence comparison concern, as expected, the replacement of the otherwise conserved aspartate at the ferroxidase center with a histidine (His78), and the absence of tryptophans. Typically, Dps proteins contain two conserved tryptophans, one near the ferroxidase center (Trp52 in E. coli Dps, present in 90% of the known sequences) and the other (Trp160 in E. coli Dps, present in the majority of the known sequences) located at the three-fold interface. These two residues are replaced, respectively, by a phenylalanine and a tyrosine. A further unusual feature of DpsA-Te is the presence of five cysteines (Cys30, Cys69, Cys102, Cys103, and Cys114), as the other Dps sequences contain a maximum of one cysteine per monomer (e.g. E. coli Dps and H. salinarum DpsA). X-ray crystal structure of T. elongatus DpsA DpsA-TeHis yielded X-ray quality crystals, whereas all attempts to crystallize DpsA-Te failed. DpsA-TeHis forms cubic I23 crystals with the following cell dimensions: a = b = c = 174.504 Å, a = b = c = 90.00. The best crystal diffracted at 2.4 Å resolution (Table 1). The dataset collected from this crystal was used to determine the protein structure by molecular replacement, using as search model the H. salinarum DpsA tetramer (Protein Data Bank entry: 1MOJ), which displays 36% sequence identity with DpsA-Te. The final model contains four identical subunits that represent the asymmetric unit and are related by a two-fold and a three-fold symmetry axis. The coordinates and structure factors have been deposited in the Protein Data Bank (ID: 2VXX). As for the other members of the family, the DpsATeHis monomer is folded into a four-helix bundle and assembles into a shell-like dodecamer characterized by tetrahedral 23 symmetry, with external and internal diameters of about 90 Å and 45 Å, respectively. However, upon superimposition of the DpsA-TeHis monomer with those of Dps-Te and L. innocua Dps (rmsd values of 1.18 Å and 1.15 Å, respectively), the N-terminal part of the DpsA-TeHis D-helix appears to be slightly bent (about 5) towards the B-helix, a feature that has important ramifications at the interfaces (see below). The DpsA-TeHis N-terminal extension (1–15) is long and flexible as in E. coli and H. salinarum Dps. It is in a random coil conformation, and is visible apart from the first two residues. The next six amino acids of the extension assume a different conformation with respect to H. salinarum Dps, whereas the last seven have the same disposition. The five characteristic cysteines are located in the A-helix and 906 Table 1. Crystal parameters, data collection and refinement statistics of DpsA-TeHis. Values in parentheses are for the highest-resolution shell. Data reduction and crystal parameters Space group a = b = c (Å) No. of molecules in asymmetric unit Solvent content (%) Matthews coefficient (Å3.Da)1) Resolution range (Å) Unique reflections Completeness (%) Rmergea v2 Refinement Resolution range (Å) Reflections used for refinement Rcrys (%) Rfree (%) Correlation coefficient, Fo – Fc Correlation coefficient, Fo – Fc free Geometry rmsd bonds (Å) rmsd angles () Ramachandran plot Residues in core region of Ramachandran plot (%) Residues in most allowed region (%) Residues in generously allowed region (%) I23 174.504 4 52.7 2.62 100–2.4 (2.46–2.39) 34 749 99.9 (98.3) 0.18 (0.50) 0.9 (0.6) 10.8 (2.5) 100–2.4 (2.46–2.4) 32 937 (2426) 16.5 (21.3) 21.6 (28.8) 0.952 0.914 0.007 0.987 99.3 0.7 0 B-helix (Cys30 and Cys69, respectively) and in the BCloop (Cys102, Cys103, and Cys114). The X-ray crystal structure clearly shows that Cys30, Cys69 and Cys114 are completely buried in the monomer, and that the side chains of Cys102 and Cys103 are oriented towards the core of the protein and therefore cannot interact directly with solvent. The C-terminal extension (six residues long) assumes an extended conformation and is completely visible, whereas the 13 residues belonging to the His-tag are not. The symmetry of the dodecamer defines two nonequivalent interfaces and pores along the three-fold axes that have been named ‘Dps-type’ and ‘ferritinlike’, as the first are typical of Dps proteins, and the second resemble the trimeric interfaces of canonical ferritins with octahedral 432 symmetry [11]. In DpsA-Te, the subunits forming the pores at the ferritin-like interfaces have a slightly different orientation with respect to the three-fold symmetry axes than in the other Dps structures (Fig. 2A). This fact, taken together with the slight bending of the N-terminal part of the D-helix towards the C-helix, leads to a rearrangement of the ferritin-like interfaces that results in FEBS Journal 277 (2010) 903–917 ª 2010 The Authors Journal compilation ª 2010 FEBS F. Alaleona et al. The unusual Thermosynechoccus elongatus DpsA Fig. 2. Ferritin-like pore of DpsA-Te. (A) View of the pore perpendicular to the three-fold symmetry axis. The residues lining the pore are shown as sticks and colored according to atom type: N, blue; O, red; C, yellow, azure and green in the different three-fold symmetry-related subunits. (B) Schematic representation of the pore. View perpendicular to the three-fold symmetry axis. The residues lining the pore of a single subunit are indicated. (C) View of the pore in the dodecamer along the three-fold symmetry axis containing an iron ion (colored gray). Pictures were generated using PYMOL [41]. A B C the loss of the typical funnel shape of the pores and in an increase in their cross-section (Fig. 2B). Furthermore, the nature and spatial arrangement of the residues lining the pore change with respect to the other Dps family members. On the side facing the inner cavity, tyrosines (Tyr149) replace the three-fold symmetryrelated aspartes that typically form the ‘bottleneck’ of the pore. Furthermore, the orientation of the Tyr149 hydroxyl groups is such that the aromatic rings hinder access to the inner cavity. The opening of the pores on the external surface of the dodecamer is lined by Glu140, Arg145, Thr137, and Leu155. These amino acids replace the aspartates and glutamates that give rise to the negative electrostatic gradient characteristic of Dps proteins [10–13] and ferritins [24]. Interestingly, the entrance of the DpsA-Te ferritin-like pores is occupied by an ion (Fig. 2A,C) coordinated by the three symmetry-related Glu140 residues that is considered to be iron, given the presence in the X-ray fluorescence emission spectrum of a peak at 6500 eV typical of iron ions and the high affinity of glutamates for iron. Other distinctive features of the DpsA-Te ferritin-like interfaces concern the nature of the stabilizing interactions, which are mainly hydrophilic and comprise hydrogen bonds and a large number of salt bridges. The involvement of four arginines (Arg8, Arg83, Arg133, and Arg145) in establishing these interactions is noteworthy: Arg83, a conserved residue among the Dps family members, forms a salt bridge with Glu159 of a three-fold symmetry-related subunit (NH1–OE1 = 2.97 Å) and with Asp144 of the same subunit (NH2– OD1 = 3.0 Å). Arg133, another conserved residue, interacts with the Ile19 and Leu20 carbonyl groups (O Leu–NH1 = 3.1 Å), Arg8 interacts with the Asn171 carbonyl group (O Asn–NH1 = 2.76 Å), and Arg145 forms salt bridges with Asp152 (OD1–NH1 = 3.25 Å, OD2–NH1 = 3.0 Å) and Glu140 (OE–NH2 = 2.77 Å). The other residues that participate in hydrogen bond formation at the ferritin-like interfaces are: Tyr149 interacting with Gln153, His164 interacting with Glu82, and His167 interacting with Asn85. In FEBS Journal 277 (2010) 903–917 ª 2010 The Authors Journal compilation ª 2010 FEBS 907 The unusual Thermosynechoccus elongatus DpsA F. Alaleona et al. addition, the ferritin-like interface is stabilized by two hydrophobic patches: one formed by Ala162, Val18, Ile19, Leu122, and Ile129, and the other by the Ala146, Leu150, Leu155 and Leu156 side chains. The pores at the so-called Dps-type interfaces show marked variability in their dimensions and chemical nature among the Dps family members. In DpsA-Te, the external perimeter of the pore is lined by Asn171 and Val176 placed on the flexible C-terminal tail, the bottleneck by Glu58, Pro61, Asp75, and the internal perimeter by Gln64. The DpsA-Te ferroxidase center is unique, owing to the presence of a histidine (His78) in place of the canonical aspartate metal ligand (Asp58 in L. innocua). Furthermore, there is Phe52 in place of the nearby, highly conserved tryptophan (Trp32 in L. innocua), as shown in Fig. 1. The electron density map clearly shows that the ferroxidase center A-site and B-site are both occupied by a metal ion (Fig. 3A,B). The two ions are at a distance of about 3.0 Å, and are coordi- nated tetrahedrally by two histidines, a water molecule, and a bridging glutamate (Glu82). In particular, the A-site ion is coordinated by His78, His51 (His31 in L. innocua Dps), a water molecule, and Glu82 (Glu62 in L. innocua Dps), and the B-site ion is coordinated by Glu82, His63 (His43 in L. innocua Dps), a water molecule, and His164 belonging to the three-fold symmetry-related monomer (Fig. 3A,B). His164 is not conserved among the Dps family members, with the exception of H. salinarum DpsA, in which, however, the B-site does not contain a metal ion. The two strong peaks in the difference Fourier map, Fobs – Fcalc, that identify the two metals at the A-site and the B-site disappear when the map is contoured at 10r and 7r, respectively. The bound metal ions were assigned to Zn(II) on the basis of the presence of two strong peaks at 8800 eV and 10 300 eV in the X-ray fluorescence emission spectrum, and on inductively coupled plasma atomic emission spectroscopy (ICPAES) measurements on the soluble protein that A B C D Fig. 3. Ferroxidase center of DpsA-Te. (A) Overall view of the ferroxidase center. The residues of the first and the second Zn(II) coordination shell are shown as sticks and colored according to atom type: N, blue; O, red; C, yellow. The carbon atoms and the three different subunits are colored gray, blue, and yellow. Water molecules are shown as spheres and depicted in red; zinc ions are shown as spheres and depicted in gray. (B) Electron density map 2Fo – Fc of the ferroxidase center contoured at 1r. (C) Comparison between the DpsA-Te ferroxidase center (light blue), the G. intestinalis flavodiiron protein iron-binding site (dark blue), and the catalytic site of the Th. thermophilus RNA degradation protein (orange). (D) The two-fold symmetry interface. The tyrosines lining the interface are shown as sticks and colored according to atom type: N, blue; O, red. The carbon atoms of the tyrosines and the different subunits are colored gray, blue, and yellow. Pictures were generated using PYMOL [41]. 908 FEBS Journal 277 (2010) 903–917 ª 2010 The Authors Journal compilation ª 2010 FEBS F. Alaleona et al. indicate a zinc content of 24 per dodecamer. Assuming an occupancy of 1.0, the Zn(II) refinement gives reasonable mean thermal parameters of 30 and 48 Å2 in the A-site and the B-site, respectively, and thus points to tighter binding of the metal to the former site. Accordingly, the distances between Zn(II) and the protein ligands range between 2.0 and 2.2 Å for His51, His63, and His78, whereas those pertaining to Zn(II) at the B-site and His164 range between 2.2 and 2.5 Å in the four monomers present in the asymmetric unit. Interestingly, three tyrosines (Tyr60, Tyr70, and Tyr163) are placed in the second Zn(II) coordination shell with the hydroxyl groups oriented towards the internal cavity. Tyr60 and Tyr163 are, respectively, at 6.2 and 7.1 Å from the B-site Zn(II), and Tyr70 is at 6.4 Å from the A-site Zn(II). In some monomers, the phenol ring of Tyr60 displays an alternative conformation, with the side chain rotated about 30 in the direction of the Zn(II)-binding sites (Fig. 3A,B,D). The DpsA-Te ferroxidase center bears a striking similarity to the catalytic sites of the Thermus thermophilus RNA degradation protein and of the Giardia intestinalis flavodiiron protein (Fig. 3C). The first belongs to the metallo-b-lactamase superfamily and contains two Zn(II) in the catalytic site [26], whereas the second, which is believed to act as an oxygen scavenger, binds two irons in the catalytic site [27]. The unusual Thermosynechoccus elongatus DpsA of thermostability. This value is 20 C or 30 C higher than those measured for the mesophilic L. innocua and E. coli Dps proteins under the same experimental conditions [23]. Iron oxidation and incorporation kinetics The efficiency of O2 and H2O2 as Fe(II) oxidants was assessed by following the kinetics of the oxidation reaction spectrophotometrically at 350 nm and pH 7.0 in parallel experiments on DpsA-Te, DpsA-TeHis, and Dps-Te. Dps-Te, like nearly all Dps proteins so far characterized and as reported by Franceschini et al. [23], prefers H2O2 to O2 as an iron oxidant (Fig. 4A, inset). Thus, Structural characterization in solution As in all known Dps proteins, the DpsA-Te dodecamer is characterized by a sedimentation coefficient, s20,w, of 10.5 S. The CD spectrum in the near-UV region has major positive peaks around 280 nm that are attributable to tyrosines, and positive ellipticity in the 260– 270 nm region that can be assigned to phenylalanines (Fig. S1). Importantly, DpsA-Te and DpsA-TeHis show very similar spectra, an indication that the Histag at the C-terminus does not change the protein structure in solution. The ellipticity in the far-UV region was used to study DpsA-Te thermostability in comparison with that of Dps-Te. For both T. elongatus Dps proteins, the transition from the native to the denatured state could not be monitored over the pH range 7.0–3.0, owing to the extremely high protein stability even at 100 C. Thermal unfolding was followed at pH 2.0, a condition under which both DpsA-Te and Dps-Te preserve their native quaternary structure at room temperature (Fig. S2). At this pH, the denaturation process of both proteins was complete at  75–80 C (Fig. S2). As the transitions are irreversible, the midpoint of the denaturation process, Tm, was taken as a measure Fig. 4. Kinetics of iron oxidation ⁄ incorporation by DpsA-Te (A), using O2 or H2O2 as oxidant, and corresponding UV–visible spectra (B). (A) Oxidant, O2 (o), and H2O2 ( ). Traces were measured at 350 nm, which enables monitoring of the formation of the ferric core. Fe(II) was added to an Fe(II) ⁄ dodecamer ratio of 24 : 1. The inset depicts the behavior of Dps-Te. (B) Oxidant, O2 ( ), and H2O2 (—). The two spectra at the bottom were recorded at 1.5 s after addition of the oxidant. FEBS Journal 277 (2010) 903–917 ª 2010 The Authors Journal compilation ª 2010 FEBS • 909 The unusual Thermosynechoccus elongatus DpsA F. Alaleona et al. after the addition of 24 Fe(II) per dodecamer, the halftime of the reaction in the presence of H2O2 (0.5 : 1 molar ratio with respect to iron) was 2.5 s, as compared with 250 s in the presence of O2. Quite unexpectedly, in the parallel experiment on DpsA-Te containing 24 Zn(II) per dodecamer, ferroxidation by O2 was about 20-fold faster (t1 ⁄ 2 = 11 s). When the experiment was repeated on a DpsA-Te sample treated with 6 mm EDTA and containing only 12 Zn(II) per dodecamer on the basis of ICP-AES determinations, the same t1 ⁄ 2 value was obtained, and the rate of ferroxidation by H2O2 was only two-fold higher (t1 ⁄ 2 = 6 s; Fig. 4A). The DpsA-Te oxidation kinetics followed at different temperatures yielded the same results, in that H2O2 was approximately two-fold more efficient than O2 over the whole range studied. The activation energy, Ea, calculated from the Arrhenius plot, corresponded to 18.6 and 12.1 kcalÆmol)1 when H2O2 and O2 were used as oxidant, respectively (Fig. S3). The unusual reactivity of DpsA-Te called for a more extensive characterization of the ferroxidation reaction. As Fe–Zn complexes are known to display charge transfer absorption bands between 300 and 400 nm, the possible formation of oxidation intermediates was followed over the range 300–600 nm. During oxidation of 24 Fe(II) per dodecamer, similar bands at about 320 and 370 nm were observed 1.5 s after admission of O2 or H2O2, and persisted at the end of the reaction (Fig. 4B). In addition, to establish the reaction stoichiometry and the possible presence of H2O2 in solution at the end of the reaction, oxygraphic experiments were employed. Fe(II) solutions were added to 4 lm DpsA-Te or recombinant human H-ferritin (H-FtHu) [respective molar ratios: Fe(II) ⁄ docecamer, 12 : 1; or Fe(II) ⁄ 24mer, 14 : 1], and oxygen consumption was measured. When the Fe(II) ⁄ oligomer ratio was £ 24 : 1 for DpsA-Te or £ 48 : 1 for H-FtHu, the addition of Fe(II) to the protein resulted in fast oxygen consumption, according to an O2 ⁄ Fe(II) molar ratio of 1 : 2.0 to 1 : 2.1, in three different experiments (Fig. 5). This ratio shifted progressively towards 1 : 4 when the Fe(II) ⁄ protein ratio increased, and reached values of 1 : 3.8 to 1 : 4.0 (n = 3) at and beyond 96 Fe(II) per dodecamer (inset to Fig. 5). In the case of DpsA-Te, the addition of catalase at the end of the reaction did not cause O2 production, indicating that H2O2 was not released into solution. In contrast, O2 is produced in the presence of H-FtHu, where the ferroxidation reaction characterized by a 2 : 1 Fe(II) ⁄ O2 stoichiometry is known to result in the quantitative production of H2O2 [9]. 910 Fig. 5. Oxygen consumption during the DpsA-Te and H-FtHu Fe(II) oxidation reaction. A solution of Fe(II) was added (at about 1.5 min) to 4 lM apoDpsA-Te (—) or H-FtHu ( ) at an Fe(II) ⁄ protein molar ratio of 12 : 1 or 24 : 1, respectively. Buffer: 50 mM Mops ⁄ NaOH (pH 7.0), at 25 C. The addition of Fe(II) to both DpsA-Te and HFtHu results in fast oxygen consumption, according to an O2 ⁄ Fe(II) molar ratio of 1 : 2. The subsequent addition of catalase (light arrows) results in oxygen production only in the case of H-FtHu. The inset shows oxygen consumption when Fe(II) is added to apoDpsA-Te at an atom ⁄ protein molar ratio of 96 : 1. The formation of a ferric core by DpsA-Te and Dps-Te was followed in parallel at pH 7.0 in 50 mm Mops by using O2 as oxidant, as precipitation occurs in the presence of H2O2 when the added iron exceeds about 150 atoms per dodecamer. An Fe(II) ⁄ dodecamer molar ratio of 250 : 1 was achieved by adding five successive increments of 100 lm Fe(II) to 2 lm DpsA-Te or Dps-Te; the intervals between the iron additions were 60 min or 5 min, respectively. The increase in absorbance at 350 nm and analytical ultracentrifugation experiments indicated that all of the iron added was oxidized and incorporated. Thus, the sedimentation coefficient, s20,w, of apoDpsA-Te increased from 10.5 to 12.9 S after incorporation of 250 Fe(III) per dodecamer, as compared with an increase from 10.1 to 12.8 S in the case of apoDps-Te (Fig. S4). A minor component at  14.6 S and at  18.7 S, present respectively in apoDpsA-Te and mineralized DpsA-Te, can be assigned to dimers of dodecamers, as the protein is ‡ 99% pure upon SDS gel electrophoresis. DNA-binding assay and DNA protection against hydroxyl radical formation The possible interaction between DpsA-Te and DNA was assessed in agarose gel mobility shift assays, using supercoiled pET-11a DNA as a probe. Under the conditions employed, E. coli Dps forms Dps– FEBS Journal 277 (2010) 903–917 ª 2010 The Authors Journal compilation ª 2010 FEBS F. Alaleona et al. The unusual Thermosynechoccus elongatus DpsA DNA complexes that are too large to migrate into the gel matrix [4]. The reaction between DpsA-Te (3 lm) and DNA (20 nm) was allowed to proceed for 5 min in BAE or TAE (pH 6.5 or pH 7.5, respectively). At both pH values, no interaction was observed (data not shown). Dps-Te, analyzed in parallel as a control, likewise does not bind DNA, as reported in [20]. The ability to prevent hydroxyl radical-mediated DNA cleavage was determined by means of an in vitro damage assay [13]. Plasmid pET-11a DNA in 30 mm Tris ⁄ HCl (pH 7.3) (Fig. 6, lane 1) was fully degraded by the hydroxyl radicals formed by the combined effect of 50 lm Fe(II) and 1 mm H2O2 via a Fenton reaction (Fig. 6, lane 4). The efficient DNA protection resulting from the presence of Dps-Te (Fig. 6, lane 1) or DpsA-Te (Fig. 6, lane 2) is indicated by the essentially unaltered pattern of the plasmid bands. 1 2 3 4 Fig. 6. DNA protection by DpsA-Te and Dps-Te. Lane 1: plasmid DNA with 1 mM H2O2, 50 lM Fe(II), and 3 lM Dps-Te. Lane 2: plasmid DNA with 1 mM H2O2, 50 lM Fe(II), and 3 lM DpsA-Te. Lane 3: plasmid DNA. Lane 4: plasmid DNA with 1 mM H2O2 and 50 lM Fe(II). Discussion DpsA-Te is the sole known Dps protein carrying a substitution at the ferroxidase center, where a histidine (His78) replaces the highly conserved metal-coordinating aspartate at the A-site (Asp58, Listeria numbering). This aspartate fi histidine replacement is the basis for the unforeseen binding of Zn(II) at the ferroxidase center, and most likely for the high efficiency of O2 as Fe(II) oxidant. These properties differentiate DpsA-Te with respect to almost all characterized Dps proteins, and are suggestive of a distinctive role in the bacterium. Although the exceptionality of DpsA-Te can be traced back principally to the aspartate fi histidine replacement at the ferroxidase center, the possible effects of the few other substitutions of nearby, conserved residues cannot be discounted, although they are difficult to pinpoint in the absence of site-specific mutagenesis studies, e.g. Phe52 replacing Trp32 (Listeria numbering), Tyr163 replacing the other tryptophan at the three-fold symmetry axis (Trp144, Listeria numbering), and His164 replacing a hydrophobic residue (methionine in Listeria Dps) near the metal-binding B-site. The aspartate fi histidine replacement at the ferroxidase center impacts on the most intriguing characteristic of the DpsA-Te X-ray crystal structure, namely the presence of Zn(II) in both metal-binding sites. The two Zn(II) are coordinated tetrahedrally by two histidines, a water molecule, and a bridging glutamate. In particular, the A-site ion is coordinated by His78 and His51 (Asp58 and His31, respectively, in L. innocua Dps), Glu82 (Glu62 in L. innocua Dps), and a water molecule. The B-site ion is coordinated by Glu82, His63 (His43 in L. innocua Dps), and a water molecule, a fourth protein ligand being furnished by His164 belonging to the three-fold symmetry-related monomer. Among the known Dps family members, His164 is present only in H. salinarum DpsA, where, however, the B-site does not contain a metal ion. The coordination bond lengths between Zn(II) and the histidine ligands belonging to the two-fold symmetry-related subunits (His51, His63, and His78) are all in the range 2.0–2.2 Å, whereas the distance between His164 and the B-site Zn(II) is 2.2–2.5 Å. This observation indicates that Zn(II) is bound less strongly at the latter site, in accordance with the mean thermal parameters of the two metal ions [30 Å2 and 48 Å2, respectively, for Zn(II) bound at the A-site and the B-site]. In full agreement with the X-ray data, ICP-AES measurements showed that the zinc content of the sample used for determination of the X-ray structure corresponds to 24 Zn per dodecamer, and decreases to 12 Zn per dodecamer upon dialysis against 6 mm FEBS Journal 277 (2010) 903–917 ª 2010 The Authors Journal compilation ª 2010 FEBS 911 The unusual Thermosynechoccus elongatus DpsA F. Alaleona et al. EDTA. Importantly, upon exposure of the 12 Zn per dodecamer sample to 24 Fe(II) per dodecamer under air, rapid ferroxidation takes place that does not involve removal of the bound Zn(II). From a functional viewpoint, DpsA-Te stands out for the unusual efficiency of O2 as iron oxidant, such that the rates of ferroxidation by H2O2 and O2 are comparable (Fig. 4A). Thus, H2O2 is about two-fold more efficient than O2, in marked contrast to the 100-fold difference that characterizes Dps proteins, with the sole exception of B. anthracis Dps2 (also named Dlp2). B. anthracis Dps2 has canonical metal ligands at the ferroxidase center, but reacts with Fe(II) and H2O2 three-fold faster than with O2 [28]. However, the absolute rates are about 10-fold slower than in the case of DpsA-Te. To unravel the mechanism underlying DpsA-Te catalysis, two approaches were used: the ferroxidation rates of the proteins containing 24 or 12 Zn(II) were compared, and oxygraphic experiments were performed to establish the stoichiometry of the ferroxidation reaction. No differences ascribable to the Zn(II) content were detected. At an Fe(II) ⁄ dodecamer ratio of £ 24 : 1, the oxygraphic data showed that the protein uses two Fe(II) to reduce O2 and that H2O2 is not released into solution (Fig. 5). At higher Fe(II) ⁄ dodecamer ratios, H2O2 is likewise undetectable at the end of the reaction, but the number of Fe(II) required to reduce O2 increases progressively to reach a value of 4. This indicates that crystal growth, whose contribution increases progressively with increases in the Fe(II) ⁄ dodecamer ratio, leads to the production of water, as in all Dps proteins and ferritins [9,14]. The findings just described can be rationalized on the basis of the following overall scheme: 2Fe(II) þ O2 þ 2Hþ ! 2Fe(III) þ H2 O2 ð1Þ H2 O2 þ 2Fe(II) þ 2Hþ ! 2Fe(III) þ 2H2 O ð2Þ Several comments are in order. The similarity of the rate of ferroxidation by O2 and H2O2 suggests that reaction (2) is rate-limiting. Furthermore, the fact that H2O2 is produced, as shown by the observed Fe ⁄ O2 stoichiometry, but is undetectable is related to its reduction to water, although its entrapment by the protein moiety cannot be excluded. The most intriguing aspect, however, concerns the mechanism that allows reduction of one O2 by two Fe(II) at a ferroxidase center that contains a permanently bound Zn(II) at the A-site. After entry of Fe(II) via the ferritin-like pores (Fig. 2A,C), the Fe(II)-binding 912 step involves the B-site, with the concomitant displacement of Zn(II) and the formation of Zn–Fe complexes, as indicated by the ICP-AES and optical absorbance data. Thus, upon addition of oxygen or H2O2, absorption bands at 320 and 370 nm appear, and persist during the course of the reaction (Fig. 4B). These bands can be assigned to Fe–Zn charge transfer [29], with a possible contribution of charge transfer between oxygen and either metal at 320 nm [30]. Two different scenarios can be envisaged for the subsequent iron oxidation step, which must entail the successive oxidation of two Fe(II) bound either to the same ferroxidase center or to two distinct centers located at the same dimeric interface. The first hypothesis requires formation of an oxygen radical intermediate, and the second that the two ferroxidase centers be connected by an efficient electron transfer pathway along the dimeric interface, a task that can probably be performed by the Tyr44 and Tyr70 lining it (Fig. 3D). The significant ferroxidase activity of DpsA-Te despite the concomitant presence of iron and zinc at the catalytic center is yet another manifestation of its uniqueness. Thus, in other members of the Dps family, notably L. innocua Dps [31] and Streptococcus suis Dpr [32], binding of Zn(II) at the ferroxidase center leads to inhibition of the iron oxidation ⁄ uptake reaction. Significantly, despite the distinctive ferroxidation mechanism and the lack of DNA-binding capacity, DpsA-Te protects this macromolecule against Fe(II)mediated and H2O2-mediated damage just as efficiently as the previously characterized Dps-Te (Fig. 6). At this point of the discussion, the question arises of the physiological relevance of the present data obtained with recombinant DpsA-Te. Given the resemblance between the zinc uptake systems in bacteria [33], DpsA-Te is expected to be saturated with Zn(II) also in its physiological environment, and O2 is expected to act as the preferred Fe(II) oxidant. The long hydrophobic N-terminal tail may be indicative of DpsA-Te localization at the thylacoid membranes, where photosynthesis takes place and O2 is produced. If so, the specific role of DpsA-Te would be to protect photosystems I and II from this oxidant. In contrast, Dps-Te would have the canonical Dps function of inhibiting the Fe(II)-mediated and H2O2-mediated production of hydroxyl radicals via Fenton chemistry. These ideas will be verified in ad hoc immune-localization experiments, using antibodies directed against DpsA-Te. The possible binding of substrates other than O2 could occur, and DpsA-Te could catalyze other types of reaction, as water is a metal ligand, as in all catalytic zinc sites [34,35]. This possibility is suggested by FEBS Journal 277 (2010) 903–917 ª 2010 The Authors Journal compilation ª 2010 FEBS

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