Thermosynechoccus elongatus DpsA binds Zn(II) at a unique three histidine-containing ferroxidase center and utilizes O 2 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 Introduction The widely expressed bacterial Dps proteins (DNA- binding 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 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 character- ization 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 substi- tution 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 hydro- phobic 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) com- plexes are formed upon oxidation, as indicated by absorbance and atomic emission spectroscopy data. In contrast to the typical behavior of Dps pro- teins, where Fe(II) oxidation by H 2 O 2 is about 100-fold faster than by O 2 , in DpsA-Te the ferroxidation efficiency of O 2 is very high and resembles that of H 2 O 2 . Oxygraphic experiments show that two Fe(II) are required to reduce O 2 , and that H 2 O 2 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) 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 UV and gamma irradiation, and acid and base shock [3]. Furthermore, it was established that the DNA- binding 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 ani- mals is built from 24 highly similar subunits, the L-chains and H-chain, with the latter harboring intra- subunit 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 O 2 as iron oxidant, with the production of H 2 O 2 , Dps proteins typically prefer H 2 O 2 , which is about 100-fold more effi- cient than O 2 [14]. The simultaneous consumption of Fe(II) and H 2 O 2 reduces their potential toxicity, as it inhibits hydroxyl radical production via Fenton chemis- try. It follows that Dps proteins are able to protect bio- logical macromolecules from Fe(II)-mediated and H 2 O 2 -mediated stress more efficiently than ferritins. This functional disparity manifests itself in the different sensitivity of ferritin and Dps deletion mutants to O 2 -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 ferritin- like 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 fer- ritin 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 elonga- tus [22,23], Anabaena variabilis, Gloeobacter violaceus, Nostoc punctiforme, Prochlorococcus marinus, Synecho- coccus sp. and Trichodesmium erythraeum belong to this category. The coexistence of ferritins and Dps proteins is most intriguing, as the structural and functional prop- erties of the Dps family members characterized to date appear to be very conserved. Key to the physiological activity of all of these pro- teins is the ferroxidase center, which is highly con- served 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 dis- tance of about 3 A ˚ , and are connected by an oxo- bridge. 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 carb- oxylates [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 A ˚ 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 conser- vation of the iron ligands, a fact that points to a sig- nificant 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 bacte- rium, those expressed by T. elongatus appeared to be of special interest. T. elongatus is a thermophilic, uni- cellular, rod-shaped cyanobacterium that lives in hot springs at 55 °C. The occurrence of oxygenic photo- synthesis 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 cel- lular 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 cata- lase ⁄ 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 pro- teins 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 cen- ter, where a histidine (His78) replaces the canonical aspartate (Asp58 in L. innocua). Near the ferroxidase center, His164 replaces a hydrophobic residue (phenyl- alanine or methionine), and a phenylalanine (Phe52) The unusual Thermosynechoccus elongatus DpsA F. Alaleona et al. 904 FEBS Journal 277 (2010) 903–917 ª 2010 The Authors Journal compilation ª 2010 FEBS 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 O 2 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. 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. F. Alaleona et al. The unusual Thermosynechoccus elongatus DpsA FEBS Journal 277 (2010) 903–917 ª 2010 The Authors Journal compilation ª 2010 FEBS 905 The most striking features emerging from the sequence comparison concern, as expected, the replace- ment of the otherwise conserved aspartate at the ferr- oxidase center with a histidine (His78), and the absence of tryptophans. Typically, Dps proteins con- tain two conserved tryptophans, one near the ferroxi- dase 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 con- tain 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 dimen- sions: a = b = c = 174.504 A ˚ , a = b = c = 90.00°. The best crystal diffracted at 2.4 A ˚ 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 coordi- nates and structure factors have been deposited in the Protein Data Bank (ID: 2VXX). As for the other members of the family, the DpsA- TeHis 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 A ˚ and 45 A ˚ , respectively. However, upon superimposition of the DpsA-TeHis monomer with those of Dps-Te and L. innocua Dps (rmsd values of 1.18 A ˚ and 1.15 A ˚ , respectively), the N-terminal part of the DpsA-TeHis D-helix appears to be slightly bent (about 5°) towards the B-helix, a fea- ture 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. salina- rum 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 con- formation with respect to H. salinarum Dps, whereas the last seven have the same disposition. The five char- acteristic cysteines are located in the A-helix and B-helix (Cys30 and Cys69, respectively) and in the BC- loop (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 res- idues 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 non- equivalent interfaces and pores along the three-fold axes that have been named ‘Dps-type’ and ‘ferritin- like’, 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 orienta- tion 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 rear- rangement of the ferritin-like interfaces that results in Table 1. Crystal parameters, data collection and refinement statis- tics of DpsA-TeHis. Values in parentheses are for the highest-reso- lution shell. Data reduction and crystal parameters Space group I23 a = b = c (A ˚ ) 174.504 No. of molecules in asymmetric unit 4 Solvent content (%) 52.7 Matthews coefficient (A ˚ 3 .Da )1 ) 2.62 Resolution range (A ˚ ) 100–2.4 (2.46–2.39) Unique reflections 34 749 Completeness (%) 99.9 (98.3) R merge a 0.18 (0.50) v 2 0.9 (0.6) <I ⁄ r(I)> 10.8 (2.5) Refinement Resolution range (A ˚ ) 100–2.4 (2.46–2.4) Reflections used for refinement 32 937 (2426) R crys (%) 16.5 (21.3) R free (%) 21.6 (28.8) Correlation coefficient, F o – F c 0.952 Correlation coefficient, F o – F c free 0.914 Geometry rmsd bonds (A ˚ ) 0.007 rmsd angles (°) 0.987 Ramachandran plot Residues in core region of Ramachandran plot (%) 99.3 Residues in most allowed region (%) 0.7 Residues in generously allowed region (%) 0 The unusual Thermosynechoccus elongatus DpsA F. Alaleona et al. 906 FEBS Journal 277 (2010) 903–917 ª 2010 The Authors Journal compilation ª 2010 FEBS the loss of the typical funnel shape of the pores and in an increase in their cross-section (Fig. 2B). Further- more, the nature and spatial arrangement of the resi- dues lining the pore change with respect to the other Dps family members. On the side facing the inner cav- ity, tyrosines (Tyr149) replace the three-fold symmetry- related 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 occu- pied 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 interac- tions, 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 A ˚ ) and with Asp144 of the same subunit (NH2– OD1 = 3.0 A ˚ ). Arg133, another conserved residue, interacts with the Ile19 and Leu20 carbonyl groups (O Leu–NH1 = 3.1 A ˚ ), Arg8 interacts with the Asn171 carbonyl group (O Asn–NH1 = 2.76 A ˚ ), and Arg145 forms salt bridges with Asp152 (OD1–NH1 = 3.25 A ˚ , OD2–NH1 = 3.0 A ˚ ) and Glu140 (OE–NH2 = 2.77 A ˚ ). 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 A B C Fig. 2. Ferritin-like pore of DpsA-Te. (A) View of the pore perpen- dicular 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 sym- metry-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]. F. Alaleona et al. The unusual Thermosynechoccus elongatus DpsA FEBS Journal 277 (2010) 903–917 ª 2010 The Authors Journal compilation ª 2010 FEBS 907 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 A ˚ , 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 sym- metry-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, F obs – F calc , 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 (ICP- AES) measurements on the soluble protein that AB CD 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 2F o – F c of the ferroxidase center contoured at 1r. (C) Comparison between the DpsA-Te ferroxidase cen- ter (light blue), the G. intestinalis flavodiiron protein iron-binding site (dark blue), and the catalytic site of the Th. thermophilus RNA degrada- tion 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]. The unusual Thermosynechoccus elongatus DpsA F. Alaleona et al. 908 FEBS Journal 277 (2010) 903–917 ª 2010 The Authors Journal compilation ª 2010 FEBS indicate a zinc content of 24 per dodecamer. Assuming an occupancy of 1.0, the Zn(II) refinement gives rea- sonable mean thermal parameters of 30 and 48 A ˚ 2 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 pro- tein ligands range between 2.0 and 2.2 A ˚ for His51, His63, and His78, whereas those pertaining to Zn(II) at the B-site and His164 range between 2.2 and 2.5 A ˚ 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 A ˚ from the B-site Zn(II), and Tyr70 is at 6.4 A ˚ from the A-site Zn(II). In some monomers, the phenol ring of Tyr60 displays an alternative conforma- tion, with the side chain rotated about 30° in the direc- tion 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 thermo- philus 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 scav- enger, binds two irons in the catalytic site [27]. Structural characterization in solution As in all known Dps proteins, the DpsA-Te dodecam- er is characterized by a sedimentation coefficient, s 20,w , of 10.5 S. The CD spectrum in the near-UV region has major positive peaks around 280 nm that are attribut- able 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 His- tag 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 pre- serve their native quaternary structure at room temper- ature (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, T m , was taken as a measure of thermostability. This value is 20 °Cor30°C higher than those measured for the mesophilic L. innocua and E. coli Dps proteins under the same experimental con- ditions [23]. Iron oxidation and incorporation kinetics The efficiency of O 2 and H 2 O 2 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 character- ized and as reported by Franceschini et al. [23], prefers H 2 O 2 to O 2 as an iron oxidant (Fig. 4A, inset). Thus, Fig. 4. Kinetics of iron oxidation ⁄ incorporation by DpsA-Te (A), using O 2 or H 2 O 2 as oxidant, and corresponding UV–visible spectra (B). (A) Oxidant, O 2 (o), and H 2 O 2 ( • ). 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, O 2 ( ), and H 2 O 2 (—). The two spectra at the bottom were recorded at 1.5 s after addition of the oxidant. F. Alaleona et al. The unusual Thermosynechoccus elongatus DpsA FEBS Journal 277 (2010) 903–917 ª 2010 The Authors Journal compilation ª 2010 FEBS 909 after the addition of 24 Fe(II) per dodecamer, the half- time of the reaction in the presence of H 2 O 2 (0.5 : 1 molar ratio with respect to iron) was 2.5 s, as com- pared with 250 s in the presence of O 2 . Quite unex- pectedly, in the parallel experiment on DpsA-Te containing 24 Zn(II) per dodecamer, ferroxidation by O 2 was about 20-fold faster (t 1 ⁄ 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 t 1 ⁄ 2 value was obtained, and the rate of fer- roxidation by H 2 O 2 was only two-fold higher (t 1 ⁄ 2 = 6 s; Fig. 4A). The DpsA-Te oxidation kinetics followed at different temperatures yielded the same results, in that H 2 O 2 was approximately two-fold more efficient than O 2 over the whole range studied. The activation energy, E a , calculated from the Arrhenius plot, corresponded to 18.6 and 12.1 kcalÆmol )1 when H 2 O 2 and O 2 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 interme- diates was followed over the range 300–600 nm. Dur- ing oxidation of 24 Fe(II) per dodecamer, similar bands at about 320 and 370 nm were observed 1.5 s after admission of O 2 or H 2 O 2 , and persisted at the end of the reaction (Fig. 4B). In addition, to establish the reaction stoichiometry and the possible presence of H 2 O 2 in solution at the end of the reaction, oxy- graphic 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 oxy- gen 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 O 2 ⁄ Fe(II) molar ratio of 1 : 2.0 to 1 : 2.1, in three different experiments (Fig. 5). This ratio shifted pro- gressively 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 O 2 production, indicating that H 2 O 2 was not released into solution. In contrast, O 2 is produced in the pres- ence of H-FtHu, where the ferroxidation reaction characterized by a 2 : 1 Fe(II) ⁄ O 2 stoichiometry is known to result in the quantitative production of H 2 O 2 [9]. 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 O 2 as oxidant, as precipitation occurs in the presence of H 2 O 2 when the added iron exceeds about 150 atoms per dodecamer. An Fe(II) ⁄ dodecamer molar ratio of 250 : 1 was achieved by adding five suc- cessive 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 ultracentrifuga- tion experiments indicated that all of the iron added was oxidized and incorporated. Thus, the sedimenta- tion coefficient, s 20,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 pro- tein 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– 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 l M apoDpsA-Te (—) or H-FtHu ( ) at an Fe(II) ⁄ protein molar ratio of 12 : 1 or 24 : 1, respectively. Buffer: 50 m M Mops ⁄ NaOH (pH 7.0), at 25 °C. The addition of Fe(II) to both DpsA-Te and H- FtHu results in fast oxygen consumption, according to an O 2 ⁄ 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 apo- DpsA-Te at an atom ⁄ protein molar ratio of 96 : 1. The unusual Thermosynechoccus elongatus DpsA F. Alaleona et al. 910 FEBS Journal 277 (2010) 903–917 ª 2010 The Authors Journal compilation ª 2010 FEBS 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, respec- tively). At both pH values, no interaction was observed (data not shown). Dps-Te, analyzed in par- allel 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 H 2 O 2 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. Discussion DpsA-Te is the sole known Dps protein carrying a sub- stitution at the ferroxidase center, where a histidine (His78) replaces the highly conserved metal-coordinat- ing 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 O 2 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 replace- ment at the ferroxidase center, the possible effects of the few other substitutions of nearby, conserved resi- dues cannot be discounted, although they are difficult to pinpoint in the absence of site-specific mutagenesis studies, e.g. Phe52 replacing Trp32 (Listeria number- ing), Tyr163 replacing the other tryptophan at the three-fold symmetry axis (Trp144, Listeria numbering), and His164 replacing a hydrophobic residue (methio- nine in Listeria Dps) near the metal-binding B-site. The aspartate fi histidine replacement at the ferrox- idase center impacts on the most intriguing characteris- tic 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 partic- ular, 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 mole- cule. 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 mono- mer. 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 coordina- tion 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 A ˚ , whereas the distance between His164 and the B-site Zn(II) is 2.2–2.5 A ˚ . This observation indi- cates that Zn(II) is bound less strongly at the latter site, in accordance with the mean thermal parameters of the two metal ions [30 A ˚ 2 and 48 A ˚ 2 , respectively, for Zn(II) bound at the A-site and the B-site]. In full agreement with the X-ray data, ICP-AES measure- ments showed that the zinc content of the sample used for determination of the X-ray structure corre- sponds to 24 Zn per dodecamer, and decreases to 12 Zn per dodecamer upon dialysis against 6 mm 1234 Fig. 6. DNA protection by DpsA-Te and Dps-Te. Lane 1: plasmid DNA with 1 m M H 2 O 2 ,50lM Fe(II), and 3 lM Dps-Te. Lane 2: plas- mid DNA with 1 m M H 2 O 2 ,50lM Fe(II), and 3 lM DpsA-Te. Lane 3: plasmid DNA. Lane 4: plasmid DNA with 1 m M H 2 O 2 and 50 lM Fe(II). F. Alaleona et al. The unusual Thermosynechoccus elongatus DpsA FEBS Journal 277 (2010) 903–917 ª 2010 The Authors Journal compilation ª 2010 FEBS 911 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 O 2 as iron oxidant, such that the rates of ferroxidation by H 2 O 2 and O 2 are comparable (Fig. 4A). Thus, H 2 O 2 is about two-fold more efficient than O 2 , 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 H 2 O 2 three-fold faster than with O 2 [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 per- formed to establish the stoichiometry of the ferroxida- tion 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 pro- tein uses two Fe(II) to reduce O 2 and that H 2 O 2 is not released into solution (Fig. 5). At higher Fe(II) ⁄ dode- camer ratios, H 2 O 2 is likewise undetectable at the end of the reaction, but the number of Fe(II) required to reduce O 2 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 O 2 þ 2H þ ! 2Fe(III) þ H 2 O 2 ð1Þ H 2 O 2 þ 2Fe(II) þ 2H þ ! 2Fe(III) þ 2H 2 O ð2Þ Several comments are in order. The similarity of the rate of ferroxidation by O 2 and H 2 O 2 suggests that reaction (2) is rate-limiting. Furthermore, the fact that H 2 O 2 is produced, as shown by the observed Fe ⁄ O 2 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 O 2 by two Fe(II) at a ferroxidase center that contains a perma- nently bound Zn(II) at the A-site. After entry of Fe(II) via the ferritin-like pores (Fig. 2A,C), the Fe(II)-binding step involves the B-site, with the concomitant displace- ment 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 H 2 O 2 , absorp- tion bands at 320 and 370 nm appear, and persist dur- ing 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 oxy- gen and either metal at 320 nm [30]. Two different sce- narios can be envisaged for the subsequent iron oxidation step, which must entail the successive oxida- tion 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 forma- tion 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 concomi- tant 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 H 2 O 2 -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 resem- blance between the zinc uptake systems in bacteria [33], DpsA-Te is expected to be saturated with Zn(II) also in its physiological environment, and O 2 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 O 2 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 H 2 O 2 -mediated pro- duction of hydroxyl radicals via Fenton chemistry. These ideas will be verified in ad hoc immune-localiza- tion experiments, using antibodies directed against DpsA-Te. The possible binding of substrates other than O 2 could occur, and DpsA-Te could catalyze other types of reaction, as water is a metal ligand, as in all cata- lytic zinc sites [34,35]. This possibility is suggested by The unusual Thermosynechoccus elongatus DpsA F. Alaleona et al. 912 FEBS Journal 277 (2010) 903–917 ª 2010 The Authors Journal compilation ª 2010 FEBS [...]... Supporting information The following supplementary material is available: Fig S1 Near-UV CD spectra of DpsA- Te and DpsAHis The unusual Thermosynechoccus elongatus DpsA Fig S2 Thermal denaturation of Dps- Te and DpsATe Fig S3 Iron oxidation kinetics of DpsA- Te as a function of temperature Fig S4 Sedimentation velocity of Dps- Te and DpsATe before and after oxidation ⁄ incorporation of 250 Fe per dodecamer This... sequence The factor Xa cleavage site was created using the QuikChange Site-Directed Mutagenesis Kit (Stratagene La Jolla, CA, USA) Removal of the His-tag was achieved by incubating DpsA- TeHis overnight at room temperature with bovine factor Xa protease (GE Healthcare) at 10 units per mg DpsA- TeHis The reaction was performed in 50 mm Tris ⁄ HCl (pH 8.0), 1 mm CaCl2, and 0.1 m NaCl, at 25 °C DpsA- Te was obtained... protparam (http://www.expasy.org) Removal of the His-tag A factor Xa cleavage site was created between the last amino acid (valine) and the His-tag Cleavage by factor Xa occurs after an arginine, and the preferred cleavage site is Asp (or Glu or Ile)-Gly-Arg Factor Xa was chosen as protease instead of the more common thrombin, because there is a thrombin cleavage site at position 8 (MTTSALPR) of the DpsA- Te... compilation ª 2010 FEBS 913 The unusual Thermosynechoccus elongatus DpsA F Alaleona et al process and calculated by plotting the first derivative of the molar ellipticity values as a function of temperature Protein crystallization, data collection, and data processing Crystallization experiments, performed at 298 K by the hanging drop vapor diffusion method, yielded X-ray-quality crystals only with DpsA- TeHis... ELETTRA (Trieste, Italy), using a MAR CCD detector at a temperature of 100 K The dataset was processed with denzo and scaled with scalepack [36] The autoindexing procedure indicates that the crystals are cubic On the basis of the scaling procedure, the crystals belong to the I23 space ˚ group, with cell parameters a = b = c = 174.504 A The data are 99.9% complete, with an Rmerge value of 16% at ˚ 2.4 A. .. Q-Sepharose HP cellulose column (GE Healthcare, Uppsala, Sweden) equilibrated with the same buffer DpsATeHis was eluted with 100 mm NaCl The relevant fraction was dialyzed overnight against 30 mm Tris ⁄ HCl (pH 7.8), 10 mm imidazole, and 300 mm NaCl, and loaded onto a HisTrap HP column (GE Healthcare) equilibrated with the same buffer DpsA- TeHis was eluted with 350 mm imidazole; it was dialyzed against...F Alaleona et al the similarity between the DpsA- Te ferroxidase center and those of the Th thermophilus RNA degradation protein and the G intestinalis flavodiiron protein, and could account for the unusual features of the ferritinlike pores, which remain unexpected, namely their size, shape, and the distinct nature of the lining residues (Fig 2B) In conclusion, the present work on DpsA- Te has disclosed... disclosed unique structural and functional properties that point to a different physiological role than that of Dps- Te and warrant further investigation Priority will be given to the localization of the protein in the bacterium, as it will allow us to validate the suggestion that the unusual efficiency of O2 as iron oxidant is related to the occurrence of photosynthesis Experimental procedures Strains and. .. H 2O2 was monitored by addition of 2 mgÆmL)1 bovine liver catalase (Sigma-Aldrich) Measurements were performed at 25 °C in air-equilibrated 50 mm Mops (pH 7.0) The software datlab 4.2, furnished by the manufacturers, was used for data acquisition and analysis Analytical ultracentrifugation Sedimentation velocity studies were performed on a Beckman-Coulter XLI analytical ultracentrifuge, using absorbance... supernatant was treated for 30 min at 37 °C with 0.1 mgÆmL)1 DNase The unusual Thermosynechoccus elongatus DpsA (Sigma-Aldrich, St Louis, MO, USA) was supplied with 10 mm MgCl2, heated to 75 °C for 10 min, cooled on ice, and then centrifuged at 10 000 g for 15 min to remove denatured proteins The recovered supernatant was dialyzed overnight against 30 mm Tris ⁄ HCl (pH 7.8), and loaded onto a HiTrap Q-Sepharose . Thermosynechoccus elongatus DpsA binds Zn(II) at a unique three histidine-containing ferroxidase center and utilizes O 2 as iron oxidant with very high. supplementary material is available: Fig. S1. Near-UV CD spectra of DpsA- Te and DpsA- His. Fig. S2. Thermal denaturation of Dps- Te and DpsA- Te. Fig. S3. Iron