From the Department of Biology, University of Rome "Tor
Vergata," Via della Ricerca Scientifica, 00133 Roma and
Department of Biochemical Sciences "A. Rossi Fanelli"
and Consiglio Nazionale delle Ricerche Center of Molecular Biology,
University of Rome "La Sapienza," P. le Aldo Moro 5, 00185 Roma, Italy
To investigate the structural/functional role of
the dimeric structure in Cu,Zn superoxide dismutases, we have studied
the stability to a variety of agents of the Escherichia
coli enzyme, the only monomeric variant of this class so far
isolated. Differential scanning calorimetry of the native enzyme showed
the presence of two well defined peaks identified as the metal free and
holoprotein. Unlike dimeric Cu,Zn superoxide dismutases, the unfolding
of the monomeric enzyme was found to be highly reversible, a behavior that may be explained by the absence of free cysteines and the highly
polar nature of its molecular surface. The melting temperature of the
E. coli enzyme was found to be pH-dependent
with the holoenzyme transition centered at 66 °C at pH 7.8 and at
79.3 °C at pH 6.0. The active-site metals, which were easily
displaced from the active site by EDTA, were found to enhance the
thermal stability of the monomeric apoprotein but to a lower extent
than in the dimeric enzymes from eukaryotic sources. Apo-superoxide
dismutase from E. coli was shown to be nearly as stable as
the bovine apoenzyme, whose holo form is much more stable and less
sensitive to pH variations. The remarkable pH susceptibility of the
E. coli enzyme structure was paralleled by the slow
decrease in activity of the enzyme incubated at alkaline pH and by
modification of the EPR spectrum at lower pH values than in the case of
dimeric enzymes. Unlike eukaryotic Cu,Zn superoxide dismutases, the
active-site structure of the E. coli enzyme was shown to be
reversibly perturbed by urea. These observations suggest that the
conformational stability of Cu,Zn superoxide dismutases is largely due
to the intrinsic stability of the
-barrel fold rather than to the
dimeric structure and that pH sensitivity and weak metal binding of the
E. coli enzyme are due to higher flexibility and
accessibility to the solvent of its active-site region.
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INTRODUCTION |
Cu,Zn superoxide dismutases
(Cu,Zn-SODs)1 are
metalloenzymes involved in the mechanisms of cellular defense against
oxidative damage. They have been found in the cytoplasm of all the
eukaryotic cells and in the periplasm of several bacterial species
(1-2). Eukaryotic Cu,Zn-SODs are homodimers that contain one atom of zinc and one atom of copper per subunit and catalyze the dismutation of
the superoxide anion at a diffusion-limited rate enhanced by electrostatic guidance of the substrate to the active site (3). Moreover, Cu,Zn-SODs possess a very compact structure that is highly
resistant to denaturing agents such as urea and SDS and to attack by
proteolytic enzymes. Several factors are thought to contribute to the
enzyme stability, including the prosthetic metal ions (4), the
intrasubunit disulfide bond (5), and the close packing of the
hydrophobic interface between the subunits and the two halves of the
-barrel core (6). Structural and functional properties of bacterial
Cu,Zn-SODs have not yet been studied in detail, but amino acid
comparisons (7-9) and the analysis of the three-dimensional structure
of the dimeric enzyme from Photobacterium leiognathi (10)
have shown that prokaryotic and eukaryotic Cu,Zn-SODs share a conserved
ligand stereochemistry and a very similar monomer fold, based on a
flattened Greek-key eight-stranded
-barrel. However, despite these
similarities, in P.leiognathi Cu,Zn-SOD the dimer interface
is formed from
-strands that are different with respect to the
eukaryotic enzymes (10). This finding suggests that, starting from a
putative monomeric SOD precursor, prokaryotic and eukaryotic Cu,Zn-SODs
have convergently evolved toward a dimeric structure, which may be
important for the enzyme biological function.
Since the discovery of Cu,Zn-SOD, there has been interest in
understanding if the dimeric structure contributes to the high catalytic efficiency and to the remarkable stability of this class of
enzymes. All attempts to obtain monomeric Cu,Zn-SODs by treatments with
detergents (11) or site-directed substitutions of hydrophobic residues
at the dimer interface (12) have provided enzymes that display very low
catalytic activity and gross alterations of the spectroscopic
properties. These dramatic changes probably reflect changes in the
tertiary structure consequent to rearrangements of the solvent-exposed
hydrophobic dimer interface.
A valuable tool to investigate the role of the dimeric structure in
Cu,Zn-SODs is represented by the recently discovered enzyme from
Escherichia coli (13), which we have shown to be monomeric (9, 14, 15) and which possesses a catalytic activity very close to that
of the human and bovine enzymes (9, 14, 16). This finding does not
exclude the possibility of a more subtle regulation of activity in the
dimeric enzymes but demonstrates that the dimeric structure is not
necessary to ensure efficient catalytic activity. Moreover, the
different organization of the dimer interface of P. leiognathi and eukaryotic Cu,Zn-SODs strongly argues against the
existence of a common mechanism of communication between the
subunits.
An alternative explanation that could account for a selective advantage
of subunit association is that the dimeric structure provides a
substantial contribution to the stability of the enzyme. The stability
of the tertiary structure of the enzyme has been the focal point of
several investigations. It was shown that eukaryotic Cu,Zn-SODs are
characterized by a high conformational melting temperature and undergo
irreversible denaturation at temperature values higher than 70 °C
(17-21). To understand if the dimeric structure substantially
contributes to the stability of Cu,Zn-SODs, we have undertaken an
investigation of the thermal unfolding of the monomeric E. coli enzyme by differential scanning calorimetry (DSC). We have
found that denaturation of this enzyme is reversible and that the
temperature of unfolding is largely affected by small variations of pH.
This pH-dependent thermal stability is consistent with a
similar dependence of activity upon incubation of the enzyme at
alkaline pH and by sensitivity of metal binding and EPR spectrum to pH
variations. These findings, together with the observation that urea is
able to induce modifications of the copper site, at variance with the
insensitivity of the eukaryotic enzymes, suggest that the monomeric
structure allows the solvent to alter the microenvironment of the
active site more readily than in the dimeric enzymes.
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EXPERIMENTAL PROCEDURES |
Protein Expression and Purification--
E. coli
Cu,Zn-SOD was purified from E. coli TOP10 cells harboring
pPSEcSOD1 (9) as described previously, except that ion-exchange chromatographic steps were performed at pH 7.4 instead than 7.8. Purified Cu,Zn-SOD was dialyzed against 5 mM potassium
phosphate, pH 7.0. Protein concentration was evaluated by the method of
Lowry et al. (22) using bovine serum albumin as standard.
Bovine Cu,Zn-SOD (obtained from Sigma) was dialyzed against 20 mM Tris-HCl, pH 7.0, and further purified by ion-exchange
chromatography with a Mono-Q HR 5/5 fast protein liquid chromatography
column (Pharmacia) equilibrated with the same buffer using a 0-0.1
M NaCl linear gradient. Bovine Cu,Zn-SOD concentration was
determined spectrophotometrically using the extinction coefficient
= 1.03 × 104 M
1
cm
1 (23). Copper content of protein samples was
determined by double integration of the EPR spectra using a
Cu2+-EDTA solution as a standard (24).
Preparation of Apo-superoxide Dismutase--
Apo E. coli Cu,Zn-SOD (devoid of both the copper and zinc ions) was
prepared as follows using solutions prepared with Chelex 100 (Bio-Rad)-treated water and nitric acid-treated glassware. Samples
containing 20 mg/ml recombinant Cu,Zn-SOD were initially dialyzed for
24 h at 4 °C 1:2,000 against 50 mM sodium acetate buffer, pH 3.8, 2 mM EDTA and then for 24 h against 50 mM sodium acetate, pH 3.8, 0.1 M NaCl to remove
excess EDTA. Metal-devoid protein samples were subsequently dialyzed
twice against 50 mM sodium acetate buffer, pH 5.0, and
finally against 100 mM potassium phosphate buffer, pH 6.5 or 7.8. The complete removal of the zinc and copper ions was verified
by metal content analysis of the apo-SOD samples, performed with a
Perkin-Elmer 3030 atomic absorption spectrometer equipped with a
graphite furnace. The zinc-containing derivatives were prepared by the
addition of substoichiometric amounts of zinc to an apo-SOD dissolved
in 100 mM potassium phosphate buffer, pH 6.5.
Differential Scanning Calorimetry--
A MicroCal MC-2
ultrasensitive differential calorimeter (MicroCal Inc. Northampton, Ma)
interfaced to a personal computer was used. Protein samples were
dissolved at 1-3 mg/ml concentration, dialyzed against 0.1 M potassium phosphate buffer at the appropriate pH, and
deaerated under mild vacuum for 10 min before loading in the sample
cell. The reference cell was filled with deaerated dialysis buffer. A
scan rate of 60 °C/h was used in all the experiments. At each pH, a
buffer versus buffer base line run was first obtained and
then subtracted from the sample curves. The reversibility of thermal
transitions was checked by a second heating cycle of the same sample
immediately after ending and cooling the previous scan. Data analysis
was performed with the software package (Origin), also supplied by
MicroCal, after subtracting a progress line or (in the case of the
zinc-reconstituted proteins) a straight line connecting the initial and
final temperatures of the overall transition. Thermodynamic data at all
pHs were fitted assuming the calorimetric transition to be two-state.
The validity of this assumption was confirmed by the good agreement
between the experimental and the calculated curves for the native
holoenzyme and for the apoenzyme at all pHs. Zinc-reconstituted enzymes
were instead fitted assuming non-two-state thermal transitions. For
each peak Tm (temperature of maximum heat capacity),
Hc (calorimetric enthalpy of denaturation),
HvH (van't Hoff enthalpy of denaturation, equal
to
Hc for a two-state transition), and
Cp (difference in heat capacity between the
denatured and the native state) were obtained by deconvolution. Two
different Origin software options of deconvolutions were used. Native
Cu,Zn-SOD and apoprotein were deconvoluted according to a "two-state
with
Cp" model, which fits the curve after
creating an appropriate base line. Zinc-reconstituted proteins were
deconvoluted according to a "non-two-state" model as described
under "Results." Errors are estimated to be ±0.3 °C for
Tm and ±10% for
H. The residual
activity of scanned samples was measured at 30 °C using the
pyrogallol method (25).
EPR Spectroscopy--
EPR spectra were recorded at room
temperature on a Bruker ESP 300 spectrometer operating at 9 GHz with
100-kHz field modulation. The pH titration was carried out adding small
aliquots of diluted NaOH to a protein sample at 2 mM copper
concentration. The pH of the sample solution was checked both after the
addition of NaOH and at the end of the measurements. The pH of the
alkaline-denatured protein was lowered by the addition of small amounts
of diluted HCl.
Heat Stability of E. coli Cu,Zn-SOD Activity--
Cu,Zn-SOD
samples at a concentration of 0.04 mg/ml were incubated at 37 °C in
20 mM Tris-HCl buffers, pH 6.8, 8.0, and 8.8. Aliquots were
withdrawn at different times and immediately assayed for residual
activity by the pyrogallol method (25). The effect of metal chelators
on the enzyme activity upon incubation at 37 °C was analyzed by
incubating the enzyme in 20 mM Tris-HCl buffers containing
0.1 mM EDTA. The reversibility of pH-dependent
loss of enzyme activity was checked by measuring the activity of the enzyme after the addition of small amounts of a diluted HCl
solution.
Urea-induced Active-site Modifications--
Experiments were
carried out by diluting protein samples (at 0.3 mM copper
concentration) in potassium phosphate buffer, pH 7.0, containing
variable amounts of urea. Optical spectra of the E. coli
enzyme were recorded 20 min after incubation at room temperature and
checked after 2 h of incubation. The visible spectra did not appreciably change during this incubation period. UV spectra were checked at the end of the experiments. The optical and UV spectra of
bovine Cu,Zn-SOD were found to be unaffected by incubation with 8 M urea. Spectrophotometric measurements were carried out with a
2 Perkin-Elmer spectrophotometer.
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RESULTS |
Thermal Unfolding of E. coli Cu,Zn-SOD at pH 7.8--
To compare
the stability of the E. coli enzyme to that of dimeric
Cu,Zn-SODs, we have initially performed DSC experiments under
conditions (100 mM phosphate buffer, pH 7.8, scan rate = 60 °C/h) that have already been used to study the unfolding of several SODs (10, 19-21). The DSC profile of E. coli
Cu,Zn-SOD under the above-mentioned conditions is shown in Fig.
1A. No aggregates were
observed after the heating cycle, and a second scan of the same sample
showed that, at variance with all the other Cu,Zn-SODs, the
denaturation of the enzyme is highly reversible.

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Fig. 1.
DSC scan of E. coli Cu,Zn-SOD of
E. coli in 100 mM phosphate buffer, pH7.8.
All calorimetric traces were corrected by a buffer-buffer base line.
Panel A, first heating scan (solid line);
reheating (dotted line). Panel B, first scan
(solid line); progress baseline (dotted line).
Dashed line, theoretical curve fitted with the data in Table
I. Panel C, fitted transition curves.
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Deconvolution of the thermogram shows that it may be described by the
sum of two independent two-state transitions (Table I). The first scan profile, its
calculated progress base line, and the theoretical curve fitted with
the data reported in Table I are shown in Fig. 1B, whereas
the two deconvoluted transitions, characterized by melting temperature
(Tm) values of 52.6 and 65.9 °C, respectively,
are shown in Fig. 1C. The presence of two peaks in the
differential scanning calorimetry profile could be indicative of a
two-step denaturation process or of the presence of two species with
different stability. Previous studies carried out in different
experimental conditions have provided evidence that in the case of the
bovine Cu,Zn-SOD, the thermal denaturation of the enzyme is
characterized by two partially resolved transitions, representing the
oxidized and reduced forms of the enzyme (18, 19). However, we have
previously found that under the experimental conditions used in this
work, the denaturation of the bovine enzyme as well as of the enzymes
from sheep, shark, yeast, and of the two variants from Xenopus
laevis, is characterized by a single endotherm (21). As the copper
content of the protein samples used for DSC studies was found to be
about 0.72 mol of copper/mol of protein and this metal amount was
consistent with the ratio of the
Hc of the second
peak to the total
Hc, we analyzed the thermal
stability of a protein sample completely devoid of both copper and
zinc. Fig. 2B shows that the
DSC profile of apo-SOD at pH 7.8 is characterized by a single transition with a Tm of 53.6 °C (Table I), very
close to that of the less stable species observed in Fig. 1. This
finding suggests that the Cu,Zn-SOD purified from E. coli
consists of a mixture of a holo- (Tm 66 °C) and
of an apoenzyme (Tm 53 °C).
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Table I
Thermodynamic parameters for holo- and apo-Cu,ZnSOD from E. coli
reversible unfolding
The data represent the best fit obtained after subtraction of a base
line calculated from the progress of the reaction according to a
two-state model ( Hc = HvH)
with Cp. 1 and 2 refer to the low and high temperature transitions,
respectively.
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Fig. 2.
pH sensitivity of Cu,Zn-SOD unfolding.
All calorimetric traces were corrected by a buffer-buffer baseline.
Panel A, native holoenzyme thermal profile in 100 mM phosphate buffer pH 7.8 ( ), 7.4 (·· - ··), 7.0 (· - · - ·), 6.5 (- - - ), 6.0 (····). Panel
B, apoenzyme in 100 mM phosphate buffer, pH 7.8 ( ),
and 6.5 (- - -). Thermodynamic data obtained by deconvolution are reported in Table I.
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pH-dependence of E. coli Cu,Zn-SOD Denaturation--
Previous
preliminary studies on the E. coli Cu,Zn-SOD have suggested
that the catalytic and structural properties of the enzyme may be
negatively affected by alkaline pH. In fact, the enzyme is inactivated
in native gels at pH 8.8 (13, 14), and gel filtration chromatography
indicates an increase in the hydrodynamic volume with increasing pH
values (14). Therefore, DSC scans were carried out in the
physiologically relevant pH range of 6.0-7.8, and the results are
shown in Fig. 2A and in Table I. At variance with the bovine
enzyme whose Tm is stable in this pH interval (18),
the stability of both peaks that characterize the thermal unfolding of
the E. coli enzyme progressively increases at pH values
closer to the pI (5.6), reaching at pH 6.0 the values of 58.8 and
79.3 °C, respectively. The reversibility of denaturation was also
analyzed at pH 6.5, with results similar to those obtained at pH 7.8. The Tm of the apoenzyme at pH 6.5 (see Fig. 2B and Table I) is nearly superimposable to that of the
first peak at this pH, further confirming the hypothesis that this is due to a molecular species with an incomplete metal content.
Interestingly, the denaturation of apo-SOD at pH 6.5 is more than 90%
reversible (Table I).
At all pH values, a sharp drop in heat capacity (Cp) was observed after
the high temperature transition (Fig. 2A).
Cp values obtained by deconvolution (which are always affected by large errors,
depending on the drawing of the progress base line) revealed that
although the
Cp is always positive for the apoprotein peak, it is
always negative for the holoenzyme peak (Table I). It appears that an
exotherm is superimposed on the unfolding endotherm, probably due to
protonation of basic residues of the protein. This phenomenon prevents
accurate calculations of the unfolding
Cps and of other related
thermodynamic data.
Zinc Contribution to Cu,Zn-SOD Thermal Stability--
The above
reported results show that metal ions contribute to the thermal
stability of the monomeric Cu,Zn-SOD from E. coli to a much
lower extent than in the eukaryotic enzymes. In fact, although metal
presence increases the Tm of bovine Cu,Zn-SOD nearly
35-40 °C (17, 18, 21), the presence of the two metals increases the
Tm of the E. coli enzyme only 12-20
degrees, depending on the pH value. The contribution of the zinc ion to the stability of the bacterial enzyme has been further studied by DSC
scans on apoprotein solutions treated with variable amounts of zinc at
pH 6.5. Upon substoichiometric addition of zinc (0.45 equivalents/mol),
the thermal profile (Fig. 3) shows an
endotherm centered approximately at 69 °C, which is not
substantially changed in the presence of 0.9 equivalents of zinc.
Deconvolution revealed the presence of two transitions under the peak
(Table II), probably corresponding to the
apo and the zinc-containing enzyme. The finding that the
zinc-containing protein is more stable than the apoenzyme confirms the
hypothesis that the recombinant enzyme purified from the periplasm of
E. coli cells is a mixture of holo- and apo- (devoid of both
metals) enzyme. Unlike the bovine enzyme, the addition of 1.35 mol of
zinc/mol did not further increase the stability of the enzyme but
rather caused the appearance of a new species showing an irreversible
melting transition at low temperature (43.7 °C). Such a novel peak
could be due to the binding of zinc to a spurious site or to a
zinc-induced distortion of the copper binding site. Analysis of the
thermal profiles obtained from the zinc-reconstituted protein was
complicated by the difficulty of drawing appropriate base lines.
Indicative data, shown in Table II, were obtained by subtracting from
the thermograms a linear base line connecting the initial and final
temperatures of the whole transition. Deconvolution data show that
Hc and
HvH reach equal
values (a necessary condition for a two-state transition) only when
more than 1 zinc atom is bound/mol of protein.

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Fig. 3.
Zinc-induced stabilization of E. coli
apo-Cu,Zn-SOD in 100 mM buffer pH 6.5. All
calorimetric traces were corrected by a buffer-buffer base line. Zinc
ions/mol of protein: 0.45 (dotted line); 0.9 (solid
line); 1.35, (dashed line). Thermodynamic data obtained
by deconvolution are reported in Table II.
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Table II
Thermodynamic parameters for the zinc-reconstituted protein
unfolding
The data represent the best fit obtained by deconvolution according to
a non-two-state model ( Hc HvH). 1 and 2 refer to the low and high
temperature transitions, respectively.
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Recovery of SOD Activity after DSC Scans and Effect of Incubation
at Different pH on the Enzyme Activity--
At the end of DSC scans at
pH 6.5 and 7.8, the enzyme was diluted to 40 µg/ml with the same
buffers and assayed for residual activity. The enzyme scanned at pH 6.5 regained approximately 85% activity, whereas the sample scanned at pH
7.8 recovered only 24% of its initial activity. Analysis of the scan
profiles of the two samples indicated that the lower recovery of
activity at pH 7.8 was not explained by a lower reversibility of
unfolding at this pH (Table I) but suggested that at alkaline pH the
enzyme refolded to a less active conformation. This was confirmed by the observation that the activity of the sample scanned at pH 7.8 increased more than twice upon lowering the pH to 6.5 by the addition
of a few drops of a diluted HCl solution. This result prompted us to
further investigate the effect of pH on the enzyme activity. Samples of
the enzyme were incubated at 37 °C up to 3 h in different
buffer conditions, and the activity of the enzyme at different times of
incubation was assayed by the pyrogallol method. As shown in Fig.
4A, the enzyme underwent a
progressive, pH-dependent loss of activity, leveling out at
38% of the starting activity value in Tris-HCl, pH 8.8. The
pH-dependent loss of activity of the E. coli
enzyme incubated at alkaline pH was more than 90% reversible. The
bovine enzyme did not display any loss of activity in all conditions
tested. When incubation of the enzyme was carried out in the presence
of EDTA, the decrease of activity was much faster and completely
irreversible (Fig. 4B).

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Fig. 4.
Decrease of E. coli Cu,Zn-SOD
activity as a function of pH. The enzyme was incubated at 37 °C
in 20 mM Tris-HCl buffer (A) or in 20 mM Tris-HCl, 0.1 mM EDTA buffer (B).
Aliquots were withdrawn at the indicated times and immediately assayed
by the pirogallol method to measure residual activity. , pH 6.8;
, pH 8.0, , pH 8.8, , bovine enzyme in the 6.8-8.8 pH
range.
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Solvent-induced Perturbations of the Active-site Region--
The
reversibility of the pH-dependent denaturation of the
enzyme was also studied at room temperature by EPR spectroscopy (Fig.
5). The EPR spectrum of the E. coli Cu,Zn-SOD is slightly more axial (g
= 2.260, g
=2.064) than the corresponding spectrum of the
eukaryotic enzymes (26). It did not change in the 5.5-10 pH range, but
at pH 10.5 a copper-biuret-type EPR spectrum appeared, suggesting
a regular square-planar coordination of the copper ion, as it is bound
by four peptide nitrogens in a denatured protein (26). However, this
pH-induced modification of the EPR spectrum, which occurs at much lower
pH values with respect to other Cu,Zn-SODs (26, 27), is completely
reversible by lowering the pH to neutrality, at variance with the cases
of alkaline denaturation of other copper proteins. Such a change of the
EPR spectrum was paralleled by alteration of the enzyme activity, which
at pH 10.5 was below the detection limit of the assay method used and
was fully recovered by adjusting the solution pH to neutral values.

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Fig. 5.
EPR spectra at room temperature of 2 mM E. coli Cu,Zn-SOD as a function of pH.
A, pH 7.0; B, pH 8.5; C, pH 9.5;
D, pH 10.5; E, sample D lowered to pH
7.0. Setting conditions: 20 milliwatt microwave power, 9.83 GHz
microwave power; 1.0 millitesla, modulation amplitude.
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The lower rhombicity of copper geometry of the E. coli
enzyme is reflected by the blue shift of the peak of the copper
absorption band to 663 nm (Fig. 6,
spectrum a), instead of 680 nm, typical of all the
eukaryotic Cu,Zn-SODs. At variance with the well established stability
of eukaryotic Cu,Zn-SODs in the presence of high concentrations of urea
(28), the copper absorption band of the E. coli enzyme was
further blue-shifted as a function of the urea concentration, reaching
630 nm in 6 M urea (Fig. 6). The UV spectrum of the enzyme was not substantially affected by urea and displayed minor
modifications only in 8 M urea (not shown). All the
modifications were fully reversible, as the original spectrum of the
E. coli enzyme was completely restored by dilution of the
samples to urea concentrations lower than 4 M (not
shown).

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Fig. 6.
Urea-induced perturbation of the E. coli Cu,Zn-SOD active site. The enzyme (0.3 mM
copper concentration) was incubated at the following urea molar
concentrations: a, 0; b, 2; c, 4; d, 5; e, 5.5; f, 6; g, 8. A, absorbance.
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DISCUSSION |
The DSC thermograms of the E. coli Cu,Zn-SOD purified
from cells overexpressing the enzyme consist of two well resolved
components whose transition temperatures are both highly influenced by
pH. According to the denaturation profiles of the apo and of the
copper-free zinc-containing derivatives and to the different
Cp
values of the two forms (negative for the first component and positive
for the second one), we assign the first peak to the presence of a metal-free form and the second one to holo-Cu,Zn-SOD. The presence of a
consistent amount of apoprotein is not usual in Cu,Zn-SOD preparations
as the eukaryotic enzymes are generally purified with a nearly complete
zinc and copper complement comprised of between 1.5 and 2.0 atoms of
metal/protein dimer. However, the results reported in Fig. 4 indicate
that, at variance with eukaryotic Cu,Zn-SODs, the E. coli
enzyme has a lower affinity for the active-site metals, both of which
can be removed by EDTA at pH > 6 (Fig. 4B). This
finding is in agreement with a recent report showing that the metals
are easily lost by the enzyme also at room temperature and that the
addition of zinc or copper to the enzyme solution increases the
recovery of activity of the heat-inactivated enzyme (29).
Several DSC studies have been focused on the thermal stability of
different Cu,Zn-SODs (10, 17-21), but the results obtained by
different authors are not always directly comparable to each other, as
the unfolding of the enzyme is significantly influenced by solvent
composition and scan rate (18). Therefore, to investigate the
contribution of the quaternary structure to the stability of the
enzyme, we have performed DSC experiments under a condition that has
been used previously to study the thermal stability of a number of
Cu,Zn-SODs. In Table III, the
Tm values of some Cu,Zn-SODs variants are reported,
showing that the E. coli enzyme is significantly less stable
than all the other variants so far studied. The difference is
particularly evident with respect to the bovine, ovine, and human
enzymes, which possess a Tm about 20 °C higher,
whereas the Tm of the monomeric enzyme is less than
8 degrees lower than that found for the Cu,Zn-SOD from yeast, X. laevis, and the prokaryote P. leiognathi. This finding
suggests that subunit interactions may provide a contribution to the
thermostability of this class of enzymes, but also that the compactness
of the
-barrel fold is sufficient to confer a remarkable
conformational stability to the monomeric Cu,Zn-SOD. This statement is
reinforced by DSC experiments carried out at lower pH, which showed
that the stability of the Cu,Zn-SOD from E. coli is heavily
influenced by pH in the relatively narrow interval roughly
corresponding to the physiological pH range of an enteric bacterium. In
fact, we have found that the Tm of the holoprotein
at pH 6.0 is 79.3 °C, about 14 °C higher than the Tm observed at pH 7.8, whereas the difference for
the apoenzyme is 6 °C between this two pH values. Such a
pH-dependent stability is a peculiar feature of the
E. coli enzyme, as the Tm of the bovine
enzyme is stable in the 6.0-8.0 pH range (18), and it is well
established that the spectroscopic and catalytic properties of all the
eukaryotic Cu,Zn-SODs are stable in this pH range (26, 30, 31). It is
worth noting that the Tm at pH 7.8 of the apoenzyme
from E. coli is very similar to that of the bovine enzyme,
and that metals increase the Tm of the protein only
13 °C compared with the 35 °C increase reported for the bovine
enzyme under the same conditions (21). Moreover, it was previously
shown that metal-induced reorganization of the active site is critical
for the pH stability of eukaryotic Cu,Zn-SODs, as the stability of the
bovine apoenzyme is pH-dependent, reaching its maximum
Tm value near pH 6.0 (18). These observations suggest that differences in the active-site structure of eukaryotic and
prokaryotic Cu,Zn-SODs are major determinants of their different thermal stability.
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Table III
Tm of different Cu,Zn-SODs
With the exception of the Tm value of the Cu,Zn-SOD
from E. coli, all the other Tm values
reported here are taken from literature data (10, 20, 21) and refer to Cu,Zn-SODs scanned under identical experimental conditions (100 mM phosphate buffer, pH 7.8; scan rate = 60 °C/h).
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The high pH sensitivity of the monomeric enzyme is confirmed by a
titration of the EPR spectrum as a function of pH, showing that the
protein-bound copper assumes a biuret type of conformation at much
lower pH values (10.5 versus 12.5) than the Cu,Zn-SODs from
ox (26) and P. leiognathi (27). These findings, together with the previously reported inactivation in native polyacrylamide gel
electrophoresis at pH 8.8 (13, 14), clearly demonstrate that the enzyme
from E. coli is more sensitive to alkaline denaturation than
the highly homologous dimeric protein from the bacterium P. leiognathi. Moreover, we have also observed that the enzyme loses
its catalytic activity upon incubation at 37 °C, and that the extent
of this inactivation is pH- and buffer-dependent. The enzyme activity is stable at pH 6.0, but at higher pH values, the
activity significantly drops to reach 38% of its initial value in
Tris-HCl buffer, pH 8.8. Also the eukaryotic Cu,Zn-SODs are inhibited
at alkaline pH, but this inhibition, due to protonic equilibria of
basic residues near the active site of the enzyme (31), occurs at
higher pH values. Therefore, the time-dependent loss of
activity of E. coli Cu,Zn-SOD at much lower pH values is
rather indicative of a conformational change involving the active-site
structure. Inspection of E. coli Cu,Zn-SOD amino acid sequence (8, 9) and of the recently solved three-dimensional structure
of the dimeric enzyme from P. leiognathi (10) may provide
some clues in this regard. Bacterial enzymes share with the monomer of
eukaryotic Cu,Zn-SODs a conserved
-barrel topology (7-10) but
display differences in the organization of the major loops. In
particular, the architecture of the active site is largely modified by
a 4-amino acid deletion in loop 7,8 and a 7-amino acid insertion in the
"S-S" subloop that creates a very long and solvent-exposed loop,
containing a cluster of charged residues that are strictly conserved in
all the bacterial Cu,Zn-SODs. These charged residues have been proposed
to be involved in substrate steering to the active site (10) but could
also play an important structural role. The lack of dimeric structure
in the E. coli enzyme, which is characterized by a highly
polar molecular surface (9), could increase flexibility of this loop
and explain its unusual active-site accessibility. Alterations in the
active-site structure of the monomeric enzyme may be inferred by the
EPR and optical spectra that are clearly different with respect to that of the eukaryotic and P. leiognathi enzymes (26, 27). All these observations suggest that the copper environment of E. coli Cu,Zn-SOD may be more susceptible to solvent modifications
than that of the dimeric enzymes. This is confirmed by the urea-induced alteration of the copper site of the E. coli enzyme, as
deduced by the changes of its optical spectrum as a function of urea
concentration. It is interesting to note that the monomeric derivatives
of human and wheat germ Cu,Zn-SODs display a more axial coordination of the copper chromophore (11, 12) and an increased distance of the
water-coordinated molecule (12), thus suggesting that the dimeric
structure may be important to reduce flexibility of the active site
also in Cu,Zn-SODs lacking the 7-residue insertion found in the
bacterial enzymes.
One of the most intriguing features of the E. coli enzyme
evidenced by DSC experiments, pH titration of the EPR spectrum, and
recovery of activity of the enzyme incubated at alkaline pH is its
efficient reversibility of unfolding. All dimeric Cu,Zn-SODs have been
shown to undergo irreversible aggregation after exposure to
temperatures higher than Tm, with the only exception being the yeast enzyme, which partially refolds to a new conformation of lower stability than the native one (10, 17-21). At least two
factors are known to be involved in this phenomenon: metal ions and
free cysteines. It is well established that incorrect disulfide bond
formation, concomitant with cysteine oxidation probably enhanced by
metals, is a cause of aggregation of heat-denatured proteins (32). In
the case of Cu,Zn-SODs it has been observed that the denaturation of
the bovine apoenzyme is partially reversible (18) and that the
substitution by site-directed mutagenesis of Cys residues not involved
in disulfide bonds, although producing moderate effects on
conformational stability (19, 20, 33), greatly increases the
reversibility of thermal denaturation of human and bovine Cu,Zn-SODs
thermal denaturation. Moreover, the only dimeric Cu,Zn-SOD, which shows
a partial reversibility after a DSC scan is the yeast enzyme, which
does not contain free cysteines (17). The Cu,Zn-SOD from E. coli does not contains cysteines that could cause formation of
incorrect disulfide bonds, and this undoubtedly contributes to its
efficient refolding. However, the dimeric enzyme from P. leiognathi also denatures irreversibly (10) despite lack of free
cysteines. Probably, solvent-induced distortion of the hydrophobic
subunit interface plays a role in the aggregation of heat-denatured
dimeric Cu,Zn-SODs, whereas the polar nature of the E. coli
Cu,Zn-SOD surface favors its efficient refolding to the native
structure.