Notably, two ZnII 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
Trang 1unique three histidine-containing ferroxidase center and
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 H2O2is 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 )
Abbreviations
H-FtHu, recombinant human H-ferritin; ICP-AES, inductively coupled plasma atomic emission spectroscopy.
Trang 2UV 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 O2 as iron
oxidant, with the production of H2O2, Dps proteins
typically prefer H2O2, which is about 100-fold more
effi-cient than O2 [14] The simultaneous consumption of
Fe(II) and H2O2 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
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
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-coccussp 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 55C 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 phenyl(phenyl-alanine (Phe52)
Trang 3replaces 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 O2as 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.
Trang 4The 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 coliDps 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
(Table1) 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
No of molecules in asymmetric unit 4
Matthews coefficient (A˚3.Da)1) 2.62 Resolution range (A ˚ ) 100–2.4 (2.46–2.39)
Refinement Resolution range (A ˚ ) 100–2.4 (2.46–2.4) Reflections used for refinement 32 937 (2426)
Correlation coefficient, Fo– Fc 0.952 Correlation coefficient, Fo – Fc free 0.914 Geometry
Ramachandran plot Residues in core region of Ramachandran plot (%)
99.3 Residues in most allowed region (%) 0.7 Residues in generously allowed
region (%)
0
Trang 5the 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].
Trang 6addition, 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, 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 (ICP-AES) measurements on the soluble protein that
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 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].
Trang 7indicate 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 charactdodecam-erized 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
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
100C 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, Tm, was taken as a measure
of thermostability This value is 20C or 30 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 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 character-ized and as reported by Franceschini et al [23], prefers
H2O2 to O2 as an iron oxidant (Fig 4A, inset) Thus,
Fig 4 Kinetics of iron oxidation ⁄ incorporation by DpsA-Te (A), using O2or H2O2as 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.
Trang 8after the addition of 24 Fe(II) per dodecamer, the
half-time of the reaction in the presence of H2O2 (0.5 : 1
molar ratio with respect to iron) was 2.5 s, as
com-pared with 250 s in the presence of O2 Quite
unex-pectedly, 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
fer-roxidation 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 H2O2was 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
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 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,
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
O2⁄ 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 O2
production, indicating that H2O2 was not released
into solution In contrast, O2 is produced in the
pres-ence 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]
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 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, 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 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 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 apo-DpsA-Te at an atom ⁄ protein molar ratio of 96 : 1.
Trang 9DNA 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 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
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 numbermetal-coordinat-ing) This aspartatefi histidine replacement is the basis for the unforeseen binding of Zn(II) at the ferroxidase center, and most likely for the high efficiency of O2as 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 aspartatefi 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 aspartatefi 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
Fig 6 DNA protection by DpsA-Te and Dps-Te Lane 1: plasmid
DNA with 1 m M H2O2, 50 l M Fe(II), and 3 l M Dps-Te Lane 2:
plas-mid DNA with 1 m M H2O2, 50 l M Fe(II), and 3 l M DpsA-Te Lane
3: plasmid DNA Lane 4: plasmid DNA with 1 m M H2O2 and 50 l M
Fe(II).
Trang 10EDTA 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 H2O2three-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
per-formed to establish the stoichiometry of the
ferroxida-tion reacferroxida-tion 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 O2and that H2O2 is not
released into solution (Fig 5) At higher Fe(II)⁄
dode-camer 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 O2þ 2Hþ! 2Fe(III) þ H2O2 ð1Þ
H2O2þ 2Fe(II) þ 2Hþ! 2Fe(III) þ 2H2O 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
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 H2O2, 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 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 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 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 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 O2
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