Tài liệu Báo cáo Y học: Thermostability of manganese- and iron-superoxide dismutases from Escherichia coli is determined by the characteristic position of a glutamine residue pdf

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

activity compared with wild types. FeSOD double mutants
(FeSOD[Q69G/A141Q]), which mimic the glutamine posi-
tion in MnSOD, elicited 25% the activity of wild-type
FeSOD while the activity of the corresponding MnSOD
double mutant (MnSOD[G77Q/Q146A]) increased to 150%
(relative to wild-type MnSOD). Both double mutants
showed reduced selectivity toward their metal. Differences
exhibited in the thermostability of SOD activity was most
obvious in the mutants that contained two glutamine resi-
dues (FeSOD[A141Q] and MnSOD[G77Q]), where the
MnSOD mutant was thermostable and the FeSOD mutant
was thermolabile. Significantly, the MnSOD double mutant
exhibited a thermal-inactivation profile similar to that of
wild-type FeSOD while that of the FeSOD double mutant
was similar to wild-type MnSOD. We conclude therefore
that the position of this glutamine residue contributes to
metal selectivity and is responsible for some of the different
physicochemical properties of these SODs, and in particular
their characteristic thermostability.
Keywords: superoxide dismutase; site-directed mutagenesis;
metal specificity; thermostability.
Iron superoxide dismutases (FeSOD, E.C.1.15.1.1) and
manganese superoxide dismutases (MnSOD) constitute a
class of structurally equivalent metalloenzymes prevalent in
prokaryotes and in eukaryotic mitochondria, respectively.
They exhibit a very high degree of homology in both
sequence and structure (Fig. 1). The SODs are active only in
dimeric association and all share structural homology in this
respect [1]. The metal cofactors are required to catalyse the
disproportionation of the superoxide radical into hydrogen

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,

the resulting enzyme is active only with the authentic metal at
its active centre [4–7]. A small number of cambialistic SODs
have been shown to be active with either iron or manganese,
though only those of Propionibacterium shermanii [8],
Bacteroides gingivalis [9] and Bacteroides fragilis [10]
demonstrate similar specific activities with either metal.
In all structures solved for the mononuclear SODs, the
metal ion is held in place by an absolutely conserved
quartet of residues comprising three histidines and one
aspartic acid which act as ligands to the metal (H26, H81,
D167 and H171 for Escherichia coli MnSOD, Fig. 1B.
This numbering will be used throughout except where
indicated) [11–21]. This conservation is also reflected in all
sequences elucidated for this large group of ubiquitous
enzymes. A fifth metal ligand, either water or hydroxide,
present in all structures produces a trigonal-pyramidal
geometry at the active site. A distorted-octahedral geo-
metry is assumed during catalytic turnover or inhibition,
Correspondence to G. J. Hunter, Department of Physiology and
Biochemistry, University of Malta, Msida, MSD 06, Malta.
Fax: + 356 21310577, Tel.: + 356 21316655,
E-mail:
Abbreviations: Fe[Q69G], Fe[A141Q] and Fe[Q69G/A141Q],
Escherichia coli FeSOD mutated at glutamine 69 to glycine, alanine
141 to glutamine, or both, respectively; FeSOD, MnSOD, the iron- or
manganese-containing SOD, respectively; FXa, active form of
restriction protease factor X; GSH, reduced glutathione; GSt, gluta-
thione S-transferase; IPTG, isopropyl thio-b-
D
-galactoside;

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

sole source of protons for the dismutation reaction,
although this residue has been shown to be a source of
the pH dependence of FeSOD activity [26]. Moreover, these
mutants show an increased sensitivity to the inhibitor azide
[24] and also, in the case of human MnSOD, to product
inhibition [28], processes thought to be analogous. Y34 is
hydrogen bonded to Q146 and thus forms part of an
extensive hydrogen-bonding network involving various
residues as well as the coordinated solvent.
Q146 is of interest as it represents one of two residues
originally identified to distinguish between FeSOD and
MnSOD [7]. The position of the glutamine residue, which is
structurally equivalent in the enzymes, is contributed by the
N domain of FeSODs (Q69) and the C domain of MnSODs
(Q146). Exceptions to this scheme include the substitution
of Gln by His in two MnSODs and some FeSODs which
have substituted His at the equivalent position of A141 for
the glutamine at position 69 of most other FeSODs.
Substitution of Q143 (equivalent to Q146) in human
MnSOD with Asn drastically reduced enzymatic activity
and effectively opened the active site of the enzyme by
reducing the occupied internal volume, allowing the intro-
duction of an extra water molecule [29]. Replacement of the
same residue by Ala similarly maintains the same charac-
teristic enzyme fold but with a concomitant introduction of
further water molecules, in this case two solvent molecules
occupy positions equivalent to the Oe1andNe2ofthe
missing Gln30. Interestingly, replacement of this residue by
Glu had little effect but replacement by Lys yielded an
enzyme too unstable to purify [30]. In E. coli, MnSOD

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

and Q146A), which change in vivo metal selectivity, specific
activity and thermal stability of these isoenzymes.
MATERIALS AND METHODS
Chemicals and enzymes
Superoxide dismutases (iron-containing or manganese-
containing enzymes from E. coli) were purchased from
Sigma (Poole, Dorset, UK). Xanthine oxidase (from cow
milk), all restriction endonucleases (used according to the
manufacturers instructions in the buffers provided) and
protease factor Xa cleavage and removal kit, were pur-
chased from Boehringer Mannheim (Mannheim, Germany).
Nitro Blue tetrazolium and isopropyl thio-b-
D
-galactoside
(IPTG) were obtained from United States Biochemicals
(Cleveland, Ohio USA). T4 DNA ligase (FPLCpure)and
reduced glutathione (GSH)-sepharose were purchased from
Pharmacia Biotech (Vienna, Austria).
Visualization and analysis of molecular structures
Molecular structures whose coordinates were obtained
from the RCSB database were visualized using the
programs MOLMOL [33] or GeneMine [34]. Addition-
ally, POVRAY () was used to
produce Fig. 1. Structural alignment of SODs was
maximized using the combinatorial extension program
CE [35] while mutational analyses were carried out using
the CARA and ENCAD algorithms included in the
GENEMINE
program.
Bacterial strains and vectors

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

[37] using ABI prism dye-terminator DNA sequencing
reagents and an Applied Biosystems model 800 Catalyst
sequencing station followed by detection and analysis on
an Applied Biosystems model 373A automated DNA
sequencer.
The cloned wild-type and mutant sod genes were fully
sequenced in both directions using the sequencing primers
PGEXPLUS, d(5¢-GTTTGGTGGTGGCGACCATCCT)
and PGEXMINUS, d(5¢-GAGGCAGATCGTCAGCAG
TCA) and various mutagenic primers (see below).
Construction of pGH-MnSOD and pGH-FeSOD
Wild-type sodAandsodB genes were isolated by PCR.
FeSOD was cloned using the primers ECF-5¢ d(5¢-TCATT
CGAATTACCTGCACTAC) and ECF-3¢ d(5¢-TTATGC
AGCGAGATTTTTCGCT) and the sodB plasmid, pHS1-8
(supplied by D. Touati, Institut Jacques Monod, Paris,
France) as template DNA. MnSOD was cloned using the
primers ECM-5¢ d(5¢-AGCTATACCCTGCCATCCCTG)
and ECM-3¢ d(5¢-TTATTTTTTCGCCGCAAAACGTG)
and E. coli genomic DNA as template. PCR was carried out
using Amplitaq enzyme according to the manufacturer’s
instructions (Perkin-Elmer corporation) although the exten-
sion reaction was omitted. Instead, the DNA products were
incubated in the presence of 1 Unit of Klenow DNA
polymerase enzyme for 30 min at 30 °C. This step greatly
improved the efficiency of blunt-end cloning of the PCR
products into the vector [36]. Constructs were designated
pGH-FeSOD and pGH-MnSOD.
In vitro
site-directed mutagenesis

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

100 lgÆmL
)1
ampicillin, sodium salt, 50 l
M
iron(III) sulfate
and 50 l
M
manganese sulfate. When the D
600
of the culture
reached a value of 0.4, IPTG was added to a final
concentration of 10 m
M
and the culture incubated for a
further 6 h.
Protein purification
Cells from IPTG-induced cultures were harvested by low-
speed centrifugation and resuspended in approximately
35 mL of NaCl/P
i
buffer (20 m
M
sodium phosphate buffer
pH 7.2, 150 m
M
sodium chloride) containing SDS (0.03%)
and Triton X-100 (1%). All sample volumes were then
adjusted to give an equal D
600
. Resuspended cells were lysed

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

M
) and incubated overnight with
the active form of restriction protease factor X (FXa; 6 lg,
biotinylated) at 4–22 °C in a final volume of 100 lL. The
digest was subjected to a further round of GSH-sepharose
affinity chromatography after addition of protein A-agarose
(50 lL) to remove FXa, the purified SOD being present in
the through-wash. Buffer exchange and concentration was
performed as described above.
Assay for superoxide dismutase activity
The specific activity of SOD was measured spectrophoto-
metrically by its inhibitory action on the superoxide-
dependent reduction of cytochrome c by xanthine-xanthine
oxidase as described by McCord and Fridovich [39] and
Ysebaert-Vanneste and Vanneste [40]. The reduction of
cytochrome c was followed at a wavelength of 550 nm using
a Beckman Diode Array DU7500 spectrophotometer in KP
buffer at 25 °C and a final volume of 1 mL. A blank
measurement was recorded in the absence of sample over
1min(V
b
). SOD proteins were diluted 200- to 6000-fold
depending on the activity of the enzyme and cytochrome c
reduction was followed over 1 min (V
s
) for a range of
sample dilutions (200 lL sample per reaction). The slope of
a plot of the reciprocal of the sample volume against V
b
/V

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

(FeSODs) or 5 m
M
(MnSODs) hydrogen peroxide. Aliqu-
ots (200 lL) were added to catalase (100 U, 1 lL) and then
used to measure SOD activity as described above, SOD
concentrations having been calculated to yield 1 U SOD
activity in a standard 1 mL assay. Activities were expressed
as a percentage of SOD activity at 25 °C without hydrogen
peroxide. Blanks were performed with hydrogen peroxide
and catalase to ensure there were no adverse effects on the
SOD assay.
Polyacrylamide gel electrophoresis (PAGE)
Native 8% polyacrylamide gels (acrylamide : N,N¢-methyl-
enebisacrylamide, 29 : 1, w/w) containing NaCl/Tris,
pH 8.8 utilized a Tris/glycine electrophoresis buffer system
consisting of 25 m
M
Tris, 250 m
M
glycine, pH 8.3. Samples
contained 50 m
M
Tris/HCl, pH 6.8, 0.1% bromophenol
blue and 10% glycerol prior to gel application. Denaturing
polyacrylamide gel electrophoresis (SDS/PAGE) was carried
out in 15% polyacrylamide gels essentially by the procedure
of Laemmli [41] utilizing a 5% stacking gel. Samples were
pretreated by boiling for 4 min in 100 m
M
Tris/HCl, pH 6.8,

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

600
of 0.03. Cultures were grown in
250 mL flasks with shaking at 37 °C and 1 mL aliquots
were removed regularly to measure optical density.
Metal analysis
Concentrations of iron and manganese in protein samples
were determined by atomic absorbance with a Hitachi
Z-9000 atomic spectrophotometer (Showa Woman’s
University, Japan) by Professor F. Yamakura (Juntendo
University School of Medicine, Japan) and Professor
T. Matsumoto (Showa Woman’s University).
RESULTS
Mutation of FeSOD and MnSOD
The sodB and sodA genes of E. coli were amplified by PCR
from either the sodB-containing plasmid pHS1-8 or E. coli
genomic DNA, respectively, and subcloned (by blunt-end
ligation) into the novel expression vector pGHX(–) [36].
Single-stranded DNA was produced from pGH-SOD
clones and used to carry out site-directed mutagenesis
(Materials and methods). The introduction of the correct
unique mutations was confirmed by dideoxy DNA sequen-
cing of the entire SOD-coding region of each expression
construct (results not shown).
Expression of pGH-SOD derivatives
In pGHX(–), SOD gene expression is under the control of
the powerful tac promoter [44]. We developed this vector
specifically to be able to purify authentic SOD proteins via a
GST fusion protein intermediate similar to that reported
previously [24]. Authenticity of the N-terminal amino acid
residues is ensured by the inclusion of a factor Xa cleavage

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

of SOD proteins containing authentic N-terminal amino
acid residues. SOD is released from GST-SOD fusion
proteins by cleavage with FXa (Fig. 2C). Although this
method is less efficient than cleavage by thrombin [24] the
released SOD proteins contain no additional N-terminal
Fig. 2. Purification and superoxide dismutase activity of FeSOD and
MnSOD mutants. (A) SDS/PAGE (15%) of GST-SOD fusion pro-
teins from single-column affinity purification. Lanes 1–8 are Fe[wt],
Fe[Q69G], Fe[A141Q], Fe[Q69G/A141Q], Mn[wt], Mn[G77Q],
Mn[Q146A] and Mn[G77Q/Q146A], respectively. (B) Native-PAGE
(8%) stained for superoxide dismutase activity. Lanes 1–8 as for A
(8 lg per lane), lanes 9–12 are Fe[A141Q], Fe[Q69G/A141Q],
Mn[G77Q] and Mn[G77Q/Q146A] (20 lg per lane). (C) Cleavage and
purification of authentic SODs. SDS/PAGE (15%) of GST-SOD
fusion proteins (examples shown are Fe[A141Q], Fe[Q69G/A141Q],
Mn[G77Q] and Mn[G77Q/Q146A] in lanes 1–4, respectively) were
cleaved by treatment with the restriction protease FXa (lanes 5–8,
samples as for lanes 1–4 after FXa treatment), Samples subjected to
further affinity chromatography to remove uncut fusion protein and
released GST, to leave pure, authentic SOD (lanes 9–12 as for lanes 1–
4, 9 and 10, 1 lg each, 11 and 12, 5 lg each). Lane 13 contains FeSOD
and MnSOD (from Sigma, 4 lg each). (D) Native-PAGE (8%) of
purified SOD samples stained for SOD activity. Lane 1 contains
FeSOD and MnSOD markers (from Sigma, arrowed, 4 lg each), lanes
2–9 as for lanes A1 to A8, lanes 10–13 as for lanes C1 to C4.
Ó FEBS 2002 Active-site glutamines in SODs (Eur. J. Biochem. 269) 5141
amino acid residues sometimes present due to cloning [36].
The efficiency of FXa cleavage varied between 50% and
70% as measured by laser densitometry (results not shown).
Biotinylated FXa was removed by the addition of protein

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

the existing glutamine and SOD activities are reasonably
similar between the two mutants. In contrast, however,
addition of a second glutamine residue to the manganese
enzyme (Mn[G77Q]) leaves the mutant with about half the
specific activity of the wild-type enzyme. A final contrast can
be seen between the double mutant enzymes where the
glutamine residue has been removed from the wild-type
location and replaced by a glutamine at the location
corresponding structurally to the position found in its
isoenzyme (Fe[Q69G/A141Q] and Mn[G77Q/Q146A]). In
this case the FeSOD demonstrates a reduction in activity to
about a quarter of its wild-type level but the MnSOD
increases by half as much again (Table 1).
We measured the metal content of the mutant proteins by
atomic absorbance spectroscopy. The results of this analysis
are presented in Table 2, with both iron and manganese
levels reported for each mutant as the number of metal ions
present per subunit of protein. In wild-type enzymes, the
metal sites of the purified proteins are either completely
(FeSOD, 1.17) or nearly fully occupied by the metal ion
from which the SOD derives, and very little of the contrary
metal is present (Table 2). This result demonstrates the
selectivity of each of the wild-type enzymes for its respective
Table 1. Specific activities of mutant superoxide dismutases. Results are expressed as the mean from at least three individual measurements
(duplicate measurements were within 5% of each other). Mn[Q146A] gave no discernible activity even at high protein concentrations.
SOD expressed GST-SOD activity
a
SOD activity
a
(unitsÆmg

)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
)

Ratio
a
Iron Manganese
Iron superoxide dismutase mutants
Fe[wt] 1.17 0.07 16
Fe[Q69G] 1.06 0.02 49
Fe[A141Q] 0.80 0.05 15
Fe[Q69G/A141Q] 0.83 0.16 5
Manganese superoxide dismutase mutants
Mn[wt] 0.03 0.71 23
Mn[G77Q] 0.04 0.39 10
Mn[Q146A] 0.00 0.50 100
Mn[G77Q/Q146A] 0.23 0.87 3
a
Ratio is given as Fe : Mn content (iron superoxide dismutase
mutants) and Mn : Fe content (manganese superoxide dismutase
mutants).
5142 T. Hunter et al. (Eur. J. Biochem. 269) Ó FEBS 2002
metal in vivo and corresponds to a ratio around 20 times in
favour of the ÔcorrectÕ metal. This ratio of correct to
incorrect metal in the active site of the enzyme is greatly
increasedbymorethantwofoldtogreaterthan50times
when the active-site glutamine residue is removed
(Fe[Q69G] and Mn[Q146A], Table 2). Although all of the
sites are metallated in the Fe[Q69G] mutant, only half of the
sites appear to be occupied in the Mn[Q146A] mutant.
Selectivity for metal is somewhat reduced in Mn[G77Q] and
similar to wild type in Fe[A141Q] but is greatly reduced in
the double mutants Fe[Q69G/A141Q] and Mn[G77Q/
Q146A] where the ratio of correct to incorrect metal is

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

conditions. As can be seen by comparison of Fig. 3A with
Fig. 3B, increases in growth rates of the induced cultures
varies similarly between the different mutants of FeSOD
and MnSOD, although absolute growth rates are some-
what different (results not shown). Cells harbouring the
double mutants (Fe[Q69G/A141Q] and Mn[G77Q/
Q146A]) grow with the fastest rates (circles, Fig. 3), with
wild-type enzymes and SODs lacking a glutamine residue
(Fe[Q69G] and Mn[Q146A]) demonstrating equivalent
growth rates representative of the slowest of the SOD-
containing cells (squares and diamonds, respectively,
Fig. 3). SOD mutants which contain an extra glutamine
(Fe[A141Q] and Mn[G77Q]) exhibit growth rates between
wild-type and double mutants (triangles, Fig. 3). This is a
somewhat surprising result as we had previously shown
that under equivalent conditions, cellular growth rates are
proportional to the specific activity of the SOD being
expressed by the cell type [24]. In this experiment, this is
clearly not the case. Several repeated experiments under
slightly different conditions lead to the same conclusive
result.
Effect of hydrogen peroxide on enzyme activity
Hydrogen peroxide, the product of the SOD reaction, is
an inhibitor of SODs and an inactivator of FeSOD. It is
often used to distinguish between the two types of
isoenzyme, particularly in conjunction with native PAGE
gels stained for SOD activity, where 5 m
M
concentrations
inhibit FeSODs but leave MnSODs virtually unaffected

[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(·).

(B) MnSOD samples. Cells expressing GST-SOD fusion proteins for Mn[wt] (j), Mn[G77Q] (m), Mn[Q146A] (r) and Mn[G77Q/Q146A] (d)or
thepGHX(–)vectoralone(·).
Ó FEBS 2002 Active-site glutamines in SODs (Eur. J. Biochem. 269) 5143
hydrogen peroxide (Fig. 4). Most sensitive was the mutant
containing two glutamines, Fe[A141Q] (50% inhibited
after 2 min), wild-type FeSOD was similarly inhibited to
50% after 5 min, the double mutant Fe[Q69G/A141Q]
after 9 min and the least sensitive was Fe[Q69G] (50%
inhibited after 11 min) (Fig. 4).
Hydrogen peroxide inactivates FeSODs via a Fenton
reaction with the iron metal at the active site. The presence
of iron is therefore a prerequisite for its effect and suggests
that the absence of reactivity with the MnSOD mutants is
due to the lack of activity in these mutants with iron at the
active site, despite there being some 20% iron in the
Mn[G77Q/Q146A] mutant (Tables 1 and 2). The differen-
tial effects of hydrogen peroxide on the FeSOD mutants
may be explained by an alteration of reactivity induced by
the electronic configuration of active site residues, in this
case glutamine 69 (or 141). Sensitivity to hydrogen peroxide
was observed to increase in the order: no glutamine
<Q141 < Q69 < two glutamines.
Effect of azide on enzyme activity
Azide has similarly been used to discriminate between
different SOD types [47], FeSOD exhibiting a higher
sensitivity than MnSODs. In our assay procedure (Mate-
rials and Methods) there appeared to be little to distinguish
any of the FeSOD mutants from the wild-type enzyme,
exhibiting a K
i

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.

The most dramatic differences in thermostability were
observed for those enzymes that have gained a second
glutamine residue. Mn[G77Q] was found to be the most
thermostable of the enzymes studied, and maintained
almost full activity for over one hour (Fig. 6). In contrast,
Fe[A141Q] was found to be the most thermolabile of the
enzymes. Its SOD activity was reduced by half within
4 min of incubation at 50 °C and reducing to zero within
20 min (Fig. 6). The Fe[Q69G] mutation demonstrated a
thermal stability profile very similar to that of Fe[wt], and
0
20
40
60
80
100
Residual SOD Activity (%)
020304050
Time (min.)
10
Fig. 4. Effect of hydrogen peroxide on the activity of SODs. Samples of
purified SOD were incubated with hydrogen peroxide (0.25 m
M
for
FeSODs and 5 m
M
for MnSODs) at 22 °C. At the indicated times,
aliquots were removed for analysis. These 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 measure-

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

counterpart. Fe[Q69G/A141Q] exhibited a half-time of
9 min similar to Mn[wt], and Mn[G77Q/Q146A] exhibited a
half-time of 50 min similar to that of Fe[wt] (Fig. 6). These
results suggest a structural role for these glutamine residues
in the mononuclear SODs.
DISCUSSION
The basis for metal cofactor selectivity in vivo and metal ion
reaction specificity in the homologous FeSODs and
MnSODs has been difficult to ascertain by comparative
analyses of sequence and/or structural information. Several
MnSODs and FeSODs have been crystallized [11–21],
MnSOD has been crystallized containing an iron ion [48] and
cambialistic SOD holoenzyme structures have been com-
pared reconstituted with different metals [21]. Furthermore,
a plethora of SOD sequences is available from the DNA
and protein sequence databases. There appears, therefore
to be no obvious sequence or structural signal that
determines selectivity or specificity in this important class
of metalloenzymes.
Despite recent reports of changes in metal specificity in
engineered SOD enzymes [31,32], the results have been
unspectacular, resulting in small increases in enzyme activity
in SOD holoenzymes reconstituted with the ÔwrongÕ metal.
Unfortunately, the H145E mutation of Mycobacterium
tuberculosis FeSOD which produces an active MnSOD [49]
bears no relevance to the central question of specificity as no
other SODs with this mutation exist naturally. The nature
of this phenomenon still remains elusive.
Metal specificity may be considered as consisting of two,
presumably distinct, stages. First, selectivity in vivo of the

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

Mn[G77Q/Q146A] mutant actually exceeding that of wild-
type MnSOD to 150% (Table 1). This is in contrast to the
activity of the same mutant reported previously as 75% [32].
However, methodological differences exist in these two
studies which make accurate and direct comparisons
extremely unreliable. Also, these authors observed an
iron-supported activity in this mutant of some 7%. We
have observed no changes in the hydrogen peroxide
sensitivity of this mutant compared to wild type (Fig. 3),
though the high manganese-supported activity may mask
any changes in our experiments. Furthermore, in vivo
experiments which produced iron-containing enzymes
Fe[wt], Fe[Q69G/A141Q], Mn[wt] and Mn[G77Q/Q146A]
using anaerobic culture conditions in the presence of iron
and absence of manganese salts [3], generated only active
Fe[wt] and Fe[Q69G/A141Q] enzymes (results not shown).
Both Mn[wt] and Mn[G77Q/Q146A] were inactive when
cultured under these conditions which favour the insertion
of iron into the active site of SODs [3] (results not shown).
Hydrogen peroxide inhibits MnSOD as it is the product
of the enzymatic dismutation of the superoxide radical. In
addition to this, however, FeSODs are inactivated by
hydrogen peroxide [46]. A Fenton reaction with the
coordinated iron produces a peroxide radical which then
attacks critical residues near the iron active centre. This
residue has been shown to be a tryptophan in other SODs
[46,51–53]. Reduction of SOD activity at relatively low
0
20
40

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

a more sensitive enzyme, whereas the absence of any
glutamineresidueintheactivesiterenderstheenzymemore
sensitive). The reason for this is unclear.
Azide, like hydrogen peroxide, is also often used to
discriminate between FeSOD and MnSODs as the latter are
usually much less sensitive to its inhibitory action [47]. The
effect of azide, a substrate analogue, showed no prominently
different behaviour between mutant SODs, though
Mn[G77Q/Q146A] was significantly less sensitive to azide
than wild type or Mn[G77Q]. In addition, all FeSOD
mutants were significantly, though only slightly, more
sensitive to azide than wild type. The order in which azide
effects the FeSOD mutant proteins may reflect the acces-
sibility of the active site to this compound (Fe[wt] <
Fe[Q69G/A141Q] < Fe[A141Q] < Fe[Q69G], Fig. 4) as
this trend matches the degree of steric hindrance to be
expected from these mutations, if azide were to approach
the active site via the substrate funnel. The orientation of
azide bound in the active site appears to be different for
FeSOD and MnSOD enzymes [11]. Bound azide is
contained within a pocket formed by no less than six
residues all within 4 A
˚
(azide N1 comes within 2.1 A
˚
of the
metal ion). Azide binding residues include the three His
metal ligands (H26, H81 and H171) and gateway residues
H30 and H34. A further gateway residue, H31, is involved
in FeSOD but not in MnSOD. Only in MnSOD is the active

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

to N74 also disrupts the active site residues, causing a shift in
the position of Y34 and a twist in the ring of the metal-
binding H81. Thermostability is not a new phenomenon
amongst SODs; those isolated from hyperthermophilic
organisms, for example, exhibit similarly high thermosta-
bility [16,17,54,55]. Recent mutational studies of the FeSOD
from Aquifex pyrophilus demonstrate the importance of
hydrogen bonding patterns on thermal stability [54]. It is not
possible to predict which of the glutamine residues will be
distorted out of the active site, or for which of the mutants,
but it may be more likely that the FeSOD[A141Q] with
lowest activity has the largest disruption around the active
site.
Although hydrogen bond pairs and bond strengths may
have been altered in the mutations discussed, other effects
due to repackaging of the active site around the hydropho-
bic stem of this residue and the corresponding alanine
replacement also should not be ignored. Nor too should the
possible inclusion of water molecules into the active site as
has been reported for human MnSOD[Q143A] [30]. Exten-
sion of these effects through the dimer interface via local
residues may also be important, though all these possibilities
remain highly speculative at this time. Elucidation of the
structures of the mutants presented here should help to
explain the chemical nature of the physical effects observed.
It seems certain, however, that metal selectivity and
specificity in the mononuclear SODs is not governed by
one or even two residues, but is most likely accomplished by
the concerted effects of a combination of key residues.
Further analysis of these and other mutant SODs is

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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