Crystal structure of the cambialistic superoxide dismutase
from Aeropyrum pernix K1 – insights into the enzyme
mechanism and stability
Tsutomu Nakamura
1
, Kasumi Torikai
1,2
, Koichi Uegaki
1
, Junji Morita
2
, Kodai Machida
3,4
,
Atsushi Suzuki
5
and Yasushi Kawata
3,4
1 National Institute of Advanced Industrial Science and Technology, Ikeda, Osaka, Japan
2 Department of Food Science and Nutrition, Faculty of Human Life and Science, Doshisha Women’s College of Liberal Arts, Kyoto, Japan
3 Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, Japan
4 Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science,
Tottori University, Japan
5 Power Train Material Engineering Division, Toyota Motor Corporation, Aichi, Japan
Keywords
Aeropyrum pernix; cambialistic; metal
coordination; stability; superoxide dismutase
Correspondence
T. Nakamura, National Institute of Advanced
Industrial Science and Technology, 1-8-31
Midorigaoka, Ikeda, Osaka 563-8577, Japan

3AK1 (apo-form), 3AK2 (Mn-bound form) and 3AK3 (Fe-bound form)
Structured digital abstract
l
MINT-8075688: Superoxide dismutase (uniprotkb:Q9Y8H8) and Superoxide dismutase
(uniprotkb:
Q9Y8H8) bind (MI:0407)bycosedimentation in solution (MI:0028)
l
MINT-8075667: Superoxide dismutase (uniprotkb:Q9Y8H8) and Superoxide dismutase
(uniprotkb:
Q9Y8H8) bind (MI:0407)byx-ray crystallography (MI:0114)
l
MINT-8075678: Superoxide dismutase (uniprotkb:Q9Y8H8) and Superoxide dismutase
(uniprotkb:
Q9Y8H8) bind (MI:0407)bymolecular sieving (MI:0071)
Abbreviations
ApeSOD, superoxide dismutase from Aeropyrum pernix; SOD, superoxide dismutase; TthSOD, superoxide dismutase from
Thermus thermophilus.
598 FEBS Journal 278 (2011) 598–609 ª 2010 The Authors Journal compilation ª 2010 FEBS
Introduction
Superoxide dismutases (SODs; EC 1.15.1.1) play a pro-
tective role against oxidative stress by catalyzing dis-
proportionation of the superoxide anion radical (O
2
Æ
)
)
to hydrogen peroxide (H
2
O
2

cofactor, respectively. SODs are grouped into four
classes according to their metal cofactors: copper and
zinc-containing SOD (Cu,Zn-SOD), iron-containing
SOD (Fe-SOD), manganese-containing SOD (Mn-
SOD) and nickel-containing SOD (Ni-SOD). These
four types of SOD are divided into three groups based
on amino acid sequence homology; Fe- and Mn-SODs
are homologous [2].
Although Mn-SOD and Fe-SOD are closely related
in amino acid sequence and tertiary structure, they are
generally active only in the presence of their specific
metals. For example, although the Fe-SOD and
Mn-SOD of Escherichia coli have 45% sequence identity
and can bind each other’s metals, they are inactive
when the wrong metal is incorporated at the active site
[3]. However, several SODs are active in the presence
of either Fe or Mn. These types of SODs are referred
as to cambialistic SODs. In addition to the tertiary
structures of metal-specific SODs [4], crystal structures
of several cambialistic SODs have been reported,
including those from Porphyromonas gingivalis [5] and
Propionibacterium shermanii [6]. The metal-specificity
of cambialistic SODs can be suppressed by mutagene-
sis at a site 11 A
˚
away from the reaction center, as
reported for the P. gingivalis SOD [7]. This supports
the hypothesis that cambialism is a consequence of
multiple factors rather than the result of a unique type
of active site structure [8].

purified in the presence of EDTA. An assay of the
ApeSOD preparation revealed that the enzyme (referred
to as the apo-enzyme) had low activity (Table 1). This
was attributed to the Mn or Fe ions incorporated into
the enzyme as it accumulated in the E. coli cells. Indeed,
the activity of the apo-enzyme was significantly lower
than the metal-containing enzyme.
Metal cofactors were incorporated into the enzyme;
when the growth medium contained MnSO
4
or FeSO
4
,
the activity of the obtained enzymes was 20-fold or
six-fold higher, respectively, than that of the apo-
enzyme (Table 1), indicating that the metals had suc-
cessfully been incorporated during bacterial expression.
When the metal cofactors were added to the purified
apo-enzyme and incubated at 70 °C, the enzyme
became significantly more active (Table 1). Incubation
with the metal at 37 °C did not raise the activity of
Table 1. SOD activity.
Enzyme
Activity
(unitÆmg
)1
)
a
Mn
(molÆmol

ApeSOD (data not shown); similar temperature depen-
dence has been reported for the cambialistic SOD from
Pyrobaculum aerophilium [17]. This indicates that pro-
teins in solution need to have structural flexibility to
successfully incorporate metal cofactors. Because the
metal contents of the reconstituted enzymes were
higher (Table 1), the crystallographic studies were per-
formed on apo- and metal-reconstituted ApeSODs.
Crystallization and determination of structure
The crystals of ApeSOD were grown for 2–3 days in
the presence of polyethylene glycol. The crystals
belonged to the space group P2
1
, with four polypep-
tides in the asymmetric unit. After collection of diffrac-
tion data, the crystal structures were refined to 1.56,
1.35 and 1.45 A
˚
resolutions for apo, Mn-bound and
Fe-bound ApeSODs, respectively. Data collection and
refinement statistics are summarized in Table 2.
All three ApeSODs had essentially the same struc-
ture (Fig. 1A). The polypeptides consisted of seven
a-helices, a three-stranded antiparallel b-sheet and loops
connecting these secondary structure elements. The con-
tents of the a-helix and b-strand were 50% and 11%,
respectively. The monomer structure comprised two
domains: the rod-shaped N-terminal domain consisting
Table 2. Data collection and refinement statistics.
Protein Data Bank code 3AK1 3AK2 3AK3

(A
˚
3
ÆDa
)1
) 1.95 1.94 1.92
R
merge
(%)
a,b
7.3 (39.6) 4.8 (36.1) 7.7 (38.3)
Completeness (%)
a
98.0 (92.4) 98.0 (97.0) 95.9 (88.9)
Total reflections 454833 1179512 732659
Unique reflections 106085 164370 124631
Redundancy
a
4.4 (2.7) 7.3 (6.9) 6.1 (5.2)
I ⁄ r(I)
a
18.1 (2.9) 15.9 (5.8) 16.6 (3.6)
B-factors of data from Wilson plot (A
˚
2
) 19.8 13.8 18.3
Refinement
Resolution range (A
˚
)

b
R
merge
¼
P
hkl
P
j
jI
hkl;j
À<I
hkl
>j=
P
hkl
P
i
I
Ihkl;j
, where I
hkl,j
is the intensity of
observation I
hkl,j
and <I
hkl
> is the average of symmetry-related observations of a unique reflection.
c
R
cryst

ble surface area of each monomer. These two dimers
associated to form a tetramer in the asymmetric unit
(Fig. 1B). The tetramerization buried 13% of the
accessible surface area of each dimer. Neighboring tet-
ramers came into loose contact with each other in the
crystal packing. Similar molecular arrangements have
been found in the crystal structures of SODs from sev-
eral sources, such as Sulfolobus solfataricus [18], Myco-
bacterium tuberculosis [19], Aquifex pyrophilus [20] and
P. shermanii [6]. These SODs are assumed to be tetra-
meric in solution. Figure 1C illustrates the superimpo-
sition of ApeSOD with the cambialistic SOD from
P. shermanii. The overall structures of these enzymes
were similar; the rmsd of the 747 Ca atoms was
0.695 A
˚
.
ApeSOD eluted from the gel-filtration column with
the elution volume for a molecular mass of 57 kDa
(Fig. 2A). Because the calculated molecular mass of
the ApeSOD monomer is 24 577 Da, the gel filtration
results suggested a dimeric association; similar results
have previously been reported for the same protein
[16,21]. A second gel filtration through a Superdex 200
column also suggested that ApeSOD has a dimeric
structure in solution (data not shown). These findings
are in contrast to the results obtained in the crystallo-
graphic study (described above), which demonstrated
that ApeSOD has a tetrameric structure. To deter-
mine whether ApeSOD polypeptide associations were

rate measurement, we conclude that ApeSOD exists as
a tetramer in solution.
Active site geometry
Each monomer had an independent metal-binding site
at the interface of the two domains, which consists
of four side chains: two (His31 and His79) from the
N-terminal domain and two (Asp165 and His169) from
the C-terminal domain (Fig. 3). In Mn-reconstituted
ApeSOD, the metal ion was five-coordinate in trigonal
bipyramidal geometry (Fig. 3A). Three of the ligands,
OD2 of Asp165, NE2 of His79 and NE2 of His169,
formed an equatorial plane. The other protein ligand,
NE2 of His31, bound to the metal, in the company of
a water oxygen, from the apical positions. The manga-
nese was only 0.06 A
˚
out of the equatorial plane
(Table 3). The angles around the metal cofactor sug-
gested that the ligation form of Mn in ApeSOD is tri-
gonal bipyramidal.
In Fe-reconstituted ApeSOD, the metal was coordi-
nated with six ligands: the five same ligands in the
Mn-reconstituted enzyme and an additional water oxy-
gen, which, together with the OD2 of Asp165, the
NE2 of His79 and the NE2 of His169, formed an
equatorial plane (Fig. 3B). The metal ion and the addi-
tional water oxygen were only 0.03 and 0.04 A
˚
, respec-
tively, out of the equatorial plane defined by the other

MW (kDa)
131211109
Elution volume (mL)
252015
10
5
0
Elution volume (mL)
1.5
–0.05
0.00
0.05
Residuals
0.5
1.0
A
280
A
280
6.956.85 6.90 7.107.057.00
0.0
Radius (cm)
A
B
Fig. 2. Assembly of ApeSOD chains in solution. (A) Representative
gel filtration chromatogram of ApeSOD and calibration curve (inset).
The standard proteins are albumin (1), ovalbumin (2), chymotrypsin-
ogen A (3) and ribonuclease A (4). The estimated molecular mass
of ApeSOD is 57.0 kDa. (B) Sedimentation equilibrium distribution
of ApeSOD. The line reflects the best fit of the data and indicates

WAT
H169 (NE2)
D165 (OD2)
WAT
H31 (NE2)
H79 (NE2)
WAT
H31 (NE2)
H79 (NE2) WAT
H169 (NE2)
D165 (OD2)
T. Nakamura et al. Crystal structure of SOD from A. pernix K1
FEBS Journal 278 (2011) 598–609 ª 2010 The Authors Journal compilation ª 2010 FEBS 603
active site residues between Mn- and Fe-bound ApeS-
ODs, although slight changes in His31, His79 and
Asp165 were observed between the metal-bound and
apo ApeSODs. The shift of the conserved Tyr residue
depending on the metal cofactor does not appear to be
a common feature among cambialistic SODs because,
in the case of the cambialistic SOD from P. shermanii,
no significant difference was found between the active
sites of the Mn- and Fe-bound forms (Fig. 3E).
Stability in organic medium
After we elucidated the tertiary structure of ApeSOD,
we were able to compare the number of ion pairs
among thermophilic SODs. Table 4 summarizes the
data obtained from two species each of archaea and
bacteria. A single ApeSOD polypeptide was found to
have seven intrasubunit ion pairs, whereas 24 intersub-
unit ion pairs were found in the ApeSOD tetrameric

Distance (A
˚
)
Metal – equatorial plane 0.06 ± 0.01 0.03 ± 0.02
Wat
a
– equatorial plane 0.04 ± 0.03
Angle (°)
D165-M-H79 104.8 ± 1.3 97.2 ± 2.9
H79-M-H169 136.1 ± 0.8 151.5 ± 2.1
H79-M-wat
a
77.1 ± 1.9
Wat
a
-M-H169 74.5 ± 0.6
H169-M-D165 118.8 ± 0.8 111.1 ± 1.5
H31-M-D165 82.7 ± 0.6 82.1 ± 1.2
H31-M-wat
b
170.1 ± 1.5 169.5 ± 1.5
a
Water on the equatorial plane.
b
Water at the apical position.
A
B
C
Fig. 4. Thermal stability of ApeSOD and TthSOD. Residual activi-
ties of ApeSOD (closed circles) and TthSOD (open circles) after

from S. solfataricus [23] and P. shermanii [24]. Ursby
et al. [18] reported that the estimated molecular mass
of the S. solfataricus SOD increased when the column
was recalibrated with thermophilic proteins from the
same source. In cases such as these, analytical ultra-
centrifugation, rather than gel filtration, is a powerful
tool for accurately determining the oligomeric struc-
ture of SOD enzymes.
We observed five-coordinate and six-coordinate struc-
tures of metal ions in Mn-bound and Fe-bound Ape-
SOD crystal structures, respectively. In the six-
coordinated Fe-bound ApeSOD, an additional water
molecule was found in the equatorial plane (Fig. 3B).
Although trigonal bipyramidal 5-coordinate structures
are often observed around bound metals of SODs,
the octahedral six-coordinate crystal structures are
detected only in exceptional cases; for example, in the
cryo-trapped form of E. coli Mn-SOD [25], in Fe-substi-
tuted Mn-SOD of E. coli [3], in peroxide-soaked Mn-
SOD of E. coli [26] and in TthSOD complexed with
azide, a SOD inhibitor [27]. It remains unclear why the
six-coordinated structure was observed in Fe-bound
ApeSOD, but not in the Mn-bound form, because the
crystal contained neither superoxide substrate, nor anio-
nic inhibitors. The answer to this question will shed light
on the reaction mechanism of this cambialistic SOD.
Several enzymological properties known for Ape-
SOD [16] can be related to the structural characteris-
tics in the active site. Cambialistic SODs can be
divided into two groups: one with almost the same

ies to play a critical role in catalysis [29,30]. The OH
of Tyr39 was found to have shifted toward the apical
water molecule upon Fe binding (Fig. 3D). This shift
was analogous to that of Tyr34 in E. coli Mn-SOD
upon binding of hydrogen peroxide to the central
metal [26]. Peroxide-bound E. coli Mn-SOD represents
a product-inhibited form of the reduction step from
superoxide to hydrogen peroxide [26]. These findings
lead to the hypothesis that Fe-bound ApeSOD mimics
the product-inhibited form and the shift of Tyr39 sup-
presses the release of the peroxide product. This may
be one of the reasons why ApeSOD is less active in its
Fe-bound form. It is noteworthy that this shift of the
Tyr residue is not observed in the cambialistic SOD
from P. shermanii (Fig. 3E), which exhibits almost the
same activity in the presence of Mn and Fe as cofac-
tors [24].
Thermophilic bacteria, as well as thermophilic
archaea, produce thermophilic enzymes. Comparison
of two thermophilic SODs, ApeSOD (an archaeon)
and TthSOD (a bacterium) revealed that ApeSOD
contained more ion pairs, especially intersubunit ion
pairs, than TthSOD (Table 4). Because electrostatic
T. Nakamura et al. Crystal structure of SOD from A. pernix K1
FEBS Journal 278 (2011) 598–609 ª 2010 The Authors Journal compilation ª 2010 FEBS 605
interactions are dominant under organic conditions,
we expected that the difference in number of ion pairs
would be reflected in a corresponding difference in the
stability of the proteins in organic solvents. Indeed,
although the stabilities of the two SODs were indistin-

a cation exchange column (HiTrap SP; GE Healthcare,
Piscataway, NJ, USA). The protein was eluted by a linear
gradient of 0–1 m NaCl in the same buffer. The fractions
containing ApeSOD were collected, concentrated and gel-
filtered with a Superdex 75 column equilibrated with
20 mm Tris-HCl (pH 8.1), with 150 mm NaCl as the final
step. The purified protein was dissolved in 20 mm Tris-HCl
(pH 8.1). The protein concentration was determined from
its absorbance at 280 nm [32].
Gel filtration
Analytical gel filtration chromatography was performed
using a Superdex75GL (10 ⁄ 30) column (GE Healthcare)
with a buffer containing 20 mm Tris-HCl (pH 8.1) and
150 mm NaCl. The flow rate was 0.8 mLÆmin
)1
. The
column was calibrated using a Gel Filtration Calibration
Kit LMW (GE Healthcare).
Ultracentrifugation
ApeSOD solution in 20 mm Tris-HCl (pH 8.1) containing
150 mm NaCl was subjected to a sedimentation equilibrium
analysis using a Beckman Optima XL-A analytical ultracen-
trifuge with an An-60 Ti rotor (Beckman Coulter, Fullerton,
CA, USA). Samples were centrifuged at 9500 g. for 41 h at
20 °C. The molecular mass of the protein was calculated
from the sedimentation equilibrium plot.
Incorporation of metals
Metal cofactors were incorporated into the enzyme by one
of two procedures. To incorporate metals during growth
of bacterial host cells, 1 mm MnSO

ApeSOD was crystallized by hanging drop vapor diffu-
sion, with a reservoir solution containing 100 mm Hepes-
NaOH (pH 7.5) 10% (w ⁄ v) poly(ethylene glycol) 6000 and
8% (v ⁄ v) ethylene glycol at 20 °C. The crystal was cryo-
protected with modified reservoir solution containing
22.5% ethylene glycol, cooled in a nitrogen gas stream
(100 K) and subjected to X-ray diffraction measurements
with synchrotron radiation at SPring-8 (Harima, Japan).
The collected data were integrated and scaled with
hkl2000 [35]. The initial phase was determined by molec-
ular replacement with molrep in the ccp4 suite [36,37].
Fe-SOD from S. solfataricus [18] (Protein Data Bank
code: 1WB8) was used as the search model. The resulting
structure was subjected to simulated annealing using cns
Crystal structure of SOD from A. pernix K1 T. Nakamura et al.
606 FEBS Journal 278 (2011) 598–609 ª 2010 The Authors Journal compilation ª 2010 FEBS
[38], followed by further refinement with refmac in the
ccp4 suite [37,39].
The secondary structure of proteins was assigned by dssp
[40]. Stereochemical analysis was performed using pro-
check [41]. Ion pairs were analyzed using contact in the
ccp4 suite [37] and confirmed visually using coot [42]. Ion-
pair interactions were identified using distances < 4 A
˚
.
When we counted the interactions, we excluded all residues
involved in the binding of the metal cofactor.
ApeSODs in different forms were superimposed with the
least square fit of Ca atoms of the residues in the range
10–200. ApeSOD and P. shermanii SOD were superimposed

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