Thermostability of manganese- and iron-superoxide dismutases
from
Escherichia coli
is determined by the characteristic position
of a glutamine residue
The
´
re
`
se Hunter
1
, Joe V. Bannister
1,2
and Gary J. Hunter
1
1
Department of Physiology and Biochemistry, University of Malta, Msida, Malta;
2
Cranfield Biotechnology Centre, Institute of
BioScience and Technology, Cranfield University, Silsoe, Bedfordshire, UK
The structurally homologous mononuclear iron and man-
ganese superoxide dismutases (FeSOD and MnSOD,
respectively) contain a highly conserved glutamine residue in
the active site which projects toward the active-site metal
centre and participates in an extensive hydrogen bonding
network. The position of this residue is different for each
SOD isoenzyme (Q69 in FeSOD and Q146 in MnSOD of
Escherichia coli). Although site-directed mutant enzymes
lacking this glutamine residue (FeSOD[Q69G] and
MnSOD[Q146A]) demonstrated a higher degree of selec-
tivity for their respective metal, they showed little or no

peroxide and molecular oxygen [2] in a cyclic, two-stage
oxidation-reduction mechanism:
M
3þ
þ O
À
2
! M
2þ
þ O
2
ð1Þ
M
2þ
þ O
À
2
þ 2H
þ
! M
3þ
þ H
2
O
2
ð2Þ
where M represents either iron or manganese.
Selectivity of the proteins for their metal cofactor has been
demonstrated in vivo [3] and although apoenzymes of each
type of SOD may be reconstituted by the addition of metals,

Mn[G77Q], Mn[Q146A] and Mn[G77Q/Q146A], E. coli MnSOD
mutated at glycine 77 to glutamine, glutamine 146 to alanine, or both,
respectively; SOD, superoxide dismutase; wt, wild type.
Enzymes: iron superoxide dismutase from E. coli; SODF_ECOLI,
manganese superoxide dismutase from E. coli;SODM_ECOLI
(E.C. 1.15.1.1).
(Received 11 March 2002, revised 10 July 2002,
accepted 22 August 2002)
Eur. J. Biochem. 269, 5137–5148 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03200.x
where a sixth ligand, presumed to be hydroxide, is bound
to the metal [11,22,23].
Beyond the metal ligand residues and within 10 A
˚
of the
metal there are few significant differences between iron- and
manganese-containing SODs. During catalysis substrate
and products must pass through a ÔfunnelÕ made up of
residues from each subunit [11,24] and include so-called
Ôgateway residuesÕ His30, Tyr34, Trp169 and Glu170, the
latter from the second subunit of the functional dimer [25].
Studies of the highly conserved residues within this outer
sphere have revealed structural or chemical roles for these
residues and highlighted the importance of a hydrogen-
bonded network between various residues and the water (or
hydroxide) coordinated to the metal ion (participating
residues are shown in Fig. 1B). Site-directed mutations of
Y34 in E. coli FeSOD [24,26], MnSOD [27] and human
MnSOD [28] show that the phenolic hydroxyl is not
necessary for maximal activity and mutants display no
overall change in structure. Importantly, Y34 can not be the

mutation of Q146 to Glu generated an apoprotein only,
Fig. 1. Comparison of E. coli mononuclear superoxide dismutase molecular features. (Top) Stereo view of the superposition of the backbone peptide
chainofonesubunitofFeSOD(black)andMnSOD(grey)ofE. coli (coordinates taken from database entries 1ISB (10), chain A, and 1VEW [18],
chain C, respectively). The iron ion is shown as a black sphere and amino acid sidechains are shown in ball and stick for FeSOD residues Q69 and
A141, relevant to this study. The corresponding sites in MnSOD are occupied by G77 and Q146, respectively (not shown). Superposition was
calculated using the combinatorial extension method to maximize backbone contacts [42]. Labels indicate the positions of the N- and C-termini, the
iron ion and residues Q69 and A141 in FeSOD. (Bottom) Stereo view of selected residues around the metal centres. Superposition, orientation and
colour are the same as above. Metal and hydroxyl ions are shown as light coloured spheres (only those of FeSOD are labelled) and sidechains of
residues relevant to mutation studies here are shown as ball and stick (FeSOD Q69, A141 and MnSOD G77, Q146). Hydrogen bonds and metal
contacts between residues of FeSOD are shown as dashed lines.
5138 T. Hunter et al. (Eur. J. Biochem. 269) Ó FEBS 2002
whereas mutation to Leu or His reduced activity to less than
10% with little or no structural changes in the mutants [27].
Although low with either metal, the Q146H mutation was
reported to give similar activities with either iron or
manganese in the reconstituted enzyme [27].
Double mutations have been introduced into P. gingi-
valis and E. coli SODs with the intention of altering their
metal specificities. In the former cambialistic enzyme,
mutations Q70G/A142Q reduced the iron-supported
activity of the enzyme and altered the ratio of Mn : Fe
from 1.4 to 3.5 [31]. In the latter MnSOD, the equivalent
mutation G77Q/Q146A was demonstrated to reduce
specific activity to 71% and introduce an iron-supported
SOD activity which did not exist in the wild-type enzyme,
though this was only 6% of manganese-supported activity
in the wild type [32].
Here we present data for FeSOD mutations Q69G and
A141Q, both single and double mutations, and a compari-
son with data on equivalent mutations in MnSOD (G77Q

The mutagenesis and expression phagemid, pGHX(–) was
produced in our laboratory and is described elsewhere [36].
E. coli K12 strain TG1 [sup E, hsd D5, thi, D(lac
–
proAB),
F¢(tra D36 pro A
+
B
+
lac I
q
lac ZDM15)], was supplied
with the Sculptor Oligonucleotide-Directed In Vitro Muta-
genesis System kit obtained from Amersham International,
UK, which was used to generate site-directed mutations.
E. coli OX326A (DsodA DsodB) was kindly supplied by
H. Steinman, Albert Einstein College of Medicine, New
York, USA.
Oligonucleotide synthesis
Oligonucleotides were synthesized on an Applied Biosys-
tems model 392 DNA synthesizer and purified by prepar-
ative gel electrophoresis in 20% polyacrylamide gel
containing 7
M
urea. Before use in mutagenesis protocols
the oligonucleotides were first used as primers in dideoxy
sequencing [37] to confirm the position of their unique
binding site within the sodA or sodB gene (see below).
Dideoxy DNA sequencing
DNA sequencing was carried out by the dideoxy method

Oligonucleotide site-directed mutagenesis was carried out
by the phosphorothioate DNA method of Eckstein [38]
utilized in the Sculptor in vitro mutagenesis kit from
Amersham International, UK. Single-stranded DNA tem-
plate was produced from the pGH-SOD constructs using
VCS-M13 helper phage (Stratagene). One microgram was
utilized in mutagenesis reactions together with oligonucleo-
tides ECF-Q69G d(5¢-AACAACGCAGCT
GGGCTCTG
GAACCAT), ECF-A141Q d(5¢-TCAACCTCTAAC
CAG
GCTACTCCGCTG) ECM-G77Q d(5¢-AACAACGCTGG
C
CAGCACGCTAACCAC) and ECM-Q146A d(5¢-TCT
ACTGCTAAC
GCGGATTCTCCGCTG) following the
manufacturer’s instructions (mutagenic nucleotide are
underlined). Mutated plasmids were designated pGH-
FeSOD[Q69G], pGH-FeSOD[A141Q], pGH-MnSOD
[G77Q] and pGH-MnSOD[Q146A]. ssDNA produced
from cells harbouring pGH-FeSOD[Q69G] and
Ó FEBS 2002 Active-site glutamines in SODs (Eur. J. Biochem. 269) 5139
pGH-MnSOD[G77Q] was used as the template for the
production of the double mutants using oligonucleotides
ECF-A141Q and ECM-Q146A, respectively.
Induced expression of SOD
E. coli OX326A (DsodA DsodB) harbouring the pGH-SOD
plasmids was grown at 30 °C with shaking in 2 L culture
flasks containing 500 mL 2TY medium (1.6% tryptone, 1%
yeast extract and 0.5% sodium chloride) supplemented with

by passage through a French pressure cell (Amicon) at
16 000 psi. The cell lysates were clarified by centrifugation at
10 000 r.p.m. (SS-34 rotor, Sorval RC-5C centrifuge) for
20 min and supernatants were mixed by gentle shaking in
batch at 4 °C overnight with 3–5 mL of GSH-sepharose
resin prewashed with buffer. The resin was then packed into
columns and the unbound protein washed through the
column with 25 mL NaCl/P
i
followed by 2 mL GSH
(10 m
M
in 50 m
M
Tris/HCl pH 8.0). GSH was used to elute
the bound fusion protein which usually eluted in the first 6–
10 mL. Buffer-exchange using KP buffer (50 m
M
potassium
phosphate, 0.1 m
M
EDTA, pH 7.8) and concentration was
carried out using Microcon 30 (Amicon) centrifugal
concentrators and recovered proteins were stored at )80 °C.
To obtain pure SOD enzymes with authentic N-termini,
the glutathione S-transferase (GST)-fusion proteins (50 lg)
were diluted into Tris/HCl buffer (50 m
M
, pH 8.3) contain-
ing calcium chloride (2 m

s
was used to calculate SOD activity [40]. All assay constit-
uents were dissolved in KP buffer before use and the
amount of xanthine oxidase required was adjusted to give a
blank value (V
b
) of approximately 0.025 DAÆmin
)1
.All
spectrophotometric measurements were used after subtrac-
tion of a blank containing SOD sample but no xanthine
oxidase to ensure lack of interference with the assay
constituents by mutant proteins.
For activity measurements at different temperatures or in
the presence of sodium azide, an initial dilution of SOD was
adjusted to give V
s
equal to half V
b
(equal to 1 unit of SOD
activity under standard conditions). After incubation of
aliquots at the required temperature or after addition of
sodium azide at the required concentration, V
s
was meas-
ured again. Activities were expressed as a percentage of
SOD activity at 25 °C without azide.
For measurements of hydrogen peroxide inactivation, the
SOD sample (1.6 mL) was incubated at 23 °C with 0.25 m
M

100 m
M
dithiothreitol, 2% SDS, 0.1% bromophenol blue
and 10% glycerol prior to application to the gel.
Superoxide dismutase activity stain
Native PAGE (8%) gels were stained for SOD activity
by the Nitro Blue tetrazolium reaction as described by
Beauchamp and Fridovich [42].
Protein concentration
Estimation of the concentration of purified protein or in the
lysates was by the method of Bradford using BSA as
standard [43].
Protection against paraquat-induced stress
Overnight cultures (5 mL) of E. coli OX326A transformed
with the appropriate plasmid were diluted 1 : 100 in 2TY
medium to a final volume of 5 mL, grown with shaking at
5140 T. Hunter et al. (Eur. J. Biochem. 269) Ó FEBS 2002
37 °C for 1–2 h and used to inoculate 50 mL of 2TY media
containing 100 lgÆmL
)1
ampicillin, 50 l
M
ferric sulfate,
50 l
M
manganese sulfate, 250 l
M
paraquat and 0.1 m
M
IPTG to an initial D

site appropriately situated with respect to the SalI cloning
site in this vector. Our oligonucleotide primers utilized for
PCR were designed to encode the SODs from the second
codon (i.e. after the ATG start codon). When cloned into
pGHX(–) as described these SOD genes are rendered
in-frame with the GST gene, separated by the FXa
recognition sequence.
Expression of GST-SOD proteins was high, correspond-
ing to approximately 40% of the total cell protein (results
not shown). Single column purification on glutathione-
sepharose yielded proteins of the expected size (47 300 Da
for GST-FeSOD and 49 000 Da for MnSOD, Fig. 2A).
Purity of the fusion proteins was estimated to be 98% as
measured by laser densitometry of Coomassie-stained SDS/
PAGE gels (results not shown). Perhaps surprisingly, the
GST-SOD fusion proteins exhibited SOD activity on native
PAGE (Fig. 2B), although only GST-FeSOD[wt] (wild
type), GST-Mn[wt] and GST-Mn[G77Q/Q146A] showed
any appreciable activity on native gels and higher protein
concentrations were required to visualize SOD activity of
GST-Fe[A141Q] and GST-Mn[G77Q] (Fig. 2B). This result
suggests the formation of active dimers between the SOD
domains of the fusion proteins products. Slower-migrating
bands also visualized by SOD activity staining may have
been derived from further interactions of the fusion protein
GST domains (Fig. 2B). Although not visualized in these
zymograms, all of the fusion proteins had detectable SOD
activity in a spectrophotometric assay (Table 1).
Purification of authentic SOD
Utilization of the pGHX(–) vector enabled the purification

A-agarose to the digest prior to further affinity chromato-
graphy on GSH-sepharose which then removes both
undigested GST-SOD and released GST proteins (Fig. 2C).
All purified SOD mutants comigrated on SDS/PAGE with
authentic FeSOD or MnSOD obtained from Sigma
Chemical Company, Poole, UK (Fig. 2C and results not
shown).
Samples of pure SOD exhibited a similar pattern of SOD
activity on native-PAGE as the GST-SOD fusion proteins
(Fig. 2D). Higher protein concentrations were necessary to
visualize Fe[A141Q], Fe[Q69G/A141Q] and Mn[G77Q],
however, both Fe[A141Q] and Mn[G77Q] were observed to
stain a light red colour rather than achromatically as is the
case for SOD in this system (Fig. 2D). This aberrant
staining has been observed before with high protein
concentrations in the system used [24].
Enzyme activity and metal content
The specific activity of superoxide dismutase mutants was
measured in a spectrophotometric assay. Both fusion and
purified proteins reflect the same differences in activity
between the mutant enzymes. Assay results for GST-SOD
fusion derivatives are lower than for pure SOD proteins but
are proportional to the difference in size between the
proteins (Table 1). Wild-type SODs show a similar level of
specific activity (per mg protein basis) while mutants lacking
a glutamine at the active site (Fe[Q69G] and Mn[Q146A])
exhibit a large reduction, the manganese enzyme being
reduced to an undetectable level (Table 1). The addition of a
second glutamine to the active site location of the iron
enzyme (Fe[A141Q]) has a very similar effect to removal of

)1
)
SOD activity
b
(unitsÆmetal ion
)1
)
Iron superoxide dismutase mutants
Fe[wt] 1100 (100) 3048 (100) 2605 (100)
Fe[Q69G] 86 (7.8) 241 (8) 227 (8)
Fe[A141Q] 122 (11) 366 (12) 457 (17)
Fe[Q69G/A141Q] 363 (33) 801 (26) 965 (37)
Manganese superoxide dismutase mutants
Mn[wt] 1560 (100) 3500 (100) 4929 (100)
Mn[G77Q] 650 (42) 1620 (46) 4153 (84)
Mn[Q146A] 7 (0.5) 0 (0) 0 (0)
Mn[G77Q/Q146A] 2190 (140) 5285 (151) 8257 (167)
a
Activity of SOD as measured by the xanthine-xanthine oxidase assay with percentage activity relative to wild type in parentheses.
b
Activity of SOD expressed on a per-iron metal basis (iron superoxide dismutase mutants) and per-manganese metal basis (manganese
superoxide dismutase mutants).
Table 2. Metal contents of mutant superoxide dismutases. Metal con-
tent was measured by atomic absorbance and is given as the number of
metal ions per subunit protein.
SOD
Metal content expressed
(mol metalÆmol SOD
)1
)

lower than 5 (Table 2).
As SODs are active only when a metal ion is present in at
least one active site of the dimeric enzyme, we recalculated
the specific activity of each mutant enzyme on a Ôper metal
ionÕ basis using values obtained for the correct metal.
Relative results do not vary significantly from the specific
activities on a Ôper mg proteinÕ basis, except for Mn[G77Q]
which was not very well metallated and its specific activity
becomes very similar to that of the wild-type enzyme
(Table 1).
Protection against paraquat-induced stress
As the GST-SOD fusion proteins are active SODs, we
tested the ability of the mutant enzymes to protect SOD
minus E. coli cells from the effects of oxidative stress. The
herbicide paraquat was used to induce oxidative stress in
E. coli and acts via the electron transport chain to
produce superoxide anions intracellularly [45]. Both stress
and protein expression were induced simultaneously after
inoculation of media with cultures grown to exponential
phase in the absence of paraquat and IPTG (Materials
and methods [24]). The final concentration of paraquat
and IPTG used for the experiment presented were chosen
empirically to give differential growth rates between the
mutant enzymes. Expression of each mutant SOD was
similar (approximately 40% of total protein) and did not
change throughout the time course of the experiment,
being similar to expression levels observed in overnight
cultures (results not shown). As illustrated in Fig. 3A,B,
growth rates of OX326A (DsodA DsodB) cells harbouring
the expression vector are very slow under the selected

[46]. We found that there was very little difference between
any of three active MnSOD types when incubated with
hydrogen peroxide at a concentration of 5 m
M
prior to
spectrophotometric assay of SOD activity (Materials and
methods and Fig. 4). FeSOD mutants, however, could be
distinguished by their different sensitivities to 0.25 m
M
0.0
0.2
0.4
0.6
0.8
OD
600
0.0 2.5 5.0
A
7.5 10.0
Time (Hr)
0.0 2.5 5.0 7.5 10.0
B
Fig. 3. Effect of GST-SODs expressed in E. coli OX326A DsodA, DsodB cells under paraquat-induced oxidative stress. E. coli OX326A (DsodA,
DsodB) cells harbouring the appropriate plasmid were grown to exponential phase and used to inoculate media containing IPTG (0.1 m
M
)and
paraquat (250 l
M
). Cell growth was then followed by measuring the optical density at 600 nm (A) FeSOD samples. Cells expressing GST-SOD
fusion proteins for GST-Fe[wt] (h), GST-Fe[A141Q] (n), GST-Fe[Q69G] (e) and GST-Fe[Q69G/A141Q] (s)orthepGHX(–)vectoralone(·).

of between 1.0 and 2.0 m
M
(Fig. 5). All the
FeSOD mutants, however, appeared to be more inhibited at
higher azide concentrations than did wild type (Fig. 5).
Fe[Q69G] was inhibited to a greater degree at lower azide
concentrations than other FeSODs, but was not affected to
thesameextentat10m
M
azide, a concentration which
virtually eliminated any activity from Fe[A141Q] or
Fe[Q69G/A141Q] (Fig. 5). Although not affected to the
same extent as FeSOD derivatives, the Mn[G77Q/Q146A]
mutant showed a similar sensitivity to azide as Mn[wt] (K
i
approx. 12.0 m
M
), and Mn[Q146A] was apparently the least
sensitive of all SODs tested (K
i
approx. 40 m
M
, results not
shown and Fig. 5).
Effect of temperature on enzyme activity
The thermostability of the enzymatic activity of the SOD
mutants was investigated at 50 °C (Fig. 5). Mn[wt] is
inherently less thermostable than Fe[wt], as shown in
Fig. 6, where the relative activity of Fe[wt] takes 50 min
to reduce to 50% while Mn[wt] takes only 9 min.

ments were made for each data point. Samples shown are Fe[wt] (h),
Fe[A141Q] (n), Fe[Q69G] (e) Fe[Q69G/A141Q] (s), Mn[wt] (j),
Mn[G77Q] (m) and Mn[G77Q/Q146A] (d). Mn[Q146A] is not rep-
resented due to its lack of measurable activity even at high protein
concentration.
0
20
40
60
80
100
Residual Activity (%)
0246810
Sodium Azide (mM)
Fig.5.EffectofazideontheactivityofSOD.Samples of purified
SODs were adjusted to give 1 unit of SOD activity under standard
assay conditions. Changes in the observed activity in the presence of
azide were normalized to this as a percentage. Aliquots of each SOD
were added to sodium azide at the appropriate concentration to yield
the required concentration of azide in the complete assay solution, and
assayed immediately. At least three independent measurements were
made for each data point. Samples shown are Fe[wt] (h), Fe[A141Q]
(n), Fe[Q69G] (e), Fe[Q69G/A141Q] (s), Mn[wt] (j), Mn[G77Q]
(m) and Mn [G77Q/Q146A] (d). Mn[Q146A] is not represented due to
its lack of measurable activity even at high protein concentrations.
5144 T. Hunter et al. (Eur. J. Biochem. 269) Ó FEBS 2002
the Mn[Q146A] mutant could not be assayed due to its
lack of activity.
Most significantly, however, the double mutant enzymes
demonstrated thermal profiles similar to their isoenzyme

proteins for their metal cofactor is a prerequisite, as the
proteins must fold de novo about their corresponding metal
ion. Even at this stage it is unclear as to whether the enzymes
utilize a divalent or trivalent metal ion during folding, or
whether, like their copper- and zinc-containing SOD
counterparts, they utilize a chaperone to transfer only the
correct metal [50]. Mononuclear SODs appear to fold with
only the correct metal when presented with both, whether
in vivo or in vitro [3,4]. Second, specificity of the reaction is
determined by the metal ion: the enzyme is active only with
the ÔcorrectÕ metal. Although, in the complete absence of the
ÔcorrectÕ metal, each type of SOD may be forced to fold with
the ÔincorrectÕ metal at its active centre, the resulting enzyme
is no longer an active SOD [3,4].
Here we report differences in metal selectivity in muta-
tions that affect residues which contribute to the hydrogen
bonding network around the metal and metal-ligand
residues (Table 2). Selectivity appears to follow a pattern
dependent upon the presence and orientation of the active-
site Q69 (FeSOD) or Q146 (MnSOD). The ratio of metals
incorporated into SOD mutants examined showed a
reduction in selectivity in the order: no glutamine > one
glutamine[wt] > two glutamines > one glutamine (the
double mutants). Thus the presence and position of this
residue affects in vivo, the selectivity for metal ion (Table 2).
Changes in metal selectivity were not apparently related
to the activity of the mutant enzymes (Table 1). Mutants
lacking a glutamine residue showed the highest selectivity
but either low or no discernible SOD activity. Double
mutants showed reasonable levels of activity, with the

60
80
100
Residual SOD activity (%)
020406 080
Time (min.)
Fig. 6. Effect of temperature on SOD activity. Samples of SOD at the
appropriate concentrations were incubated at 50 °Cfortheindicated
times. Aliquots were removed for analysis of SOD activity and were
calculated to give 1 unit of SOD activity in the standard SOD assay
conditions used and all values were normalized to this. At least three
independent measurements were made for each data point. Samples
shown are Fe[wt] (h), Fe[A141Q] (n), Fe[Q69G] (e) Fe[Q69G/
A141Q] (s), Mn[wt] (j), Mn[G77Q] (m) and Mn[G77Q/Q146A] (d).
Mn[Q146A] is not represented due to its lack of measurable activity
even at high protein concentration.
Ó FEBS 2002 Active-site glutamines in SODs (Eur. J. Biochem. 269) 5145
levels of hydrogen peroxide has therefore been used as an
indicative marker for the presence of iron at the active site
and, indeed, is frequently used to determine the type of SOD
isoenzyme present in a sample [49]. Both wild-type and
mutant MnSODs were affected to some extent by hydrogen
peroxide but showed no significant differences (Fig. 3).
FeSODs were affected to differing degrees, but in general all
demonstrated a similar inactivation to wild type. The least
affected was Fe[Q69G] followed by Fe[Q69G/A141Q],
Fe[wt] with Fe[A141Q] being the most affected (Fig. 3).
Although steric effects may need to be considered, the
glutamine appears to play a role in hydrogen peroxide
sensitivity (the presence of two glutamine residues generates

site glutamine (Q146) also in proximity to the bound azide.
Azide binding could therefore be affected by electrostatic
interactions as well as steric interference by the mutational
changes studied.
The most striking change in physical characteristics of the
mutant SODs was revealed in temperature-sensitivity stud-
ies (Fig. 5). These showed that double mutations in each
enzyme (Fe[Q69G/A141Q] and Mn[G77Q/Q146A])
endowed the mutant with a similar temperature-deactiva-
tion profile to that of its opposing wild-type enzyme
(Mn[wt] and Fe[wt], respectively). We show therefore that
the position of this glutamine residue in the active site is
responsible for this behaviour, presumably primarily
through its participation in a hydrogen-bonding network
involving other residues and solvent molecules. An unex-
pected result was the extremely high thermostability of
Mn[G77Q] and the very low thermostability of Fe[A141Q]
(Fig. 5). Each of these mutants has two glutamine residues
in their active sites; one from the Ônaturally occurringÕ
residue, the other engineered to mimic its counterpart.
Molecular modelling [34] indicates that the active sites of
both MnSOD and FeSOD would most probably not be
able to accommodate a second glutamine residue (results
not shown). The most stable conformations of these
mutants leave one glutamine extended away from the active
site and stabilized by hydrogen bonding to an Asn residue.
Q77 is capable of hydrogen bonding with N74 or Q146 with
N73. As the numbering indicates, N73 and N74 lie adjacent
to each other, and both are conserved in FeSOD and
MnSOD sequences. Simulation suggests that Q77 bonding

currently underway.
ACKNOWLEDGEMENTS
We are indebted to G. Peplow, F. Yamakura and T. Matsumoto for the
analyses of iron and manganese in protein samples. We also wish to
thank H. Steinman for the gift of E. coli OX326A. We finally thank Mr
M. Farrugia for photographic assistance.
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