Bicarbonate Is Required for the Peroxidase Function of
Cu,Zn-Superoxide Dismutase at Physiological pH*
Sornampillai
Sankarapandi and
Jay L.
Zweier
From the Molecular and Cellular Biophysics Laboratories, Department
of Medicine, Division of Cardiology and the Electron Paramagnetic
Resonance Center, The Johns Hopkins Medical Institutions,
Baltimore, Maryland 21224
 |
ABSTRACT |
Cu,Zn-superoxide dismutase (SOD1) acts as a
peroxidase in the presence of H2O2 at
high pH (pH > 9). The high pH species of H2O2,
HO2
, was
previously implicated as the reactive species. However, recent EPR
studies of the enzyme performed in the physiological pH range 7.4-7.6
with the spin trap 5,5'-dimethyl-1-pyrolline-N-oxide attributed the intense EPR signal of
5,5'-dimethyl-1-pyrolline-N-oxide-OH obtained from SOD1 and
H2O2 to the peroxidase activity of the enzyme.
The present study establishes that this intense signal is obtained only
in the presence of bicarbonate. To explore the critical role of
HCO3
, a
comprehensive EPR investigation of the radical production and redox
state of the active site copper was performed. The results indicate
that HCO3
competes with other anions for the anion-binding site of SOD1 (Arg141) but does not bind directly to the copper.
Structurally different anions that bind to Arg141 did not
stimulate, but rather blocked, peroxidase function, ruling out an
effect due to mere anion binding. However, the structurally similar
anions HSeO3
and HSO3
mimic HCO3
in stimulating peroxidase function. These data suggest that
HCO3
bound
to Arg141 anchors the neutral H2O2
molecule at the active site copper, enabling its redox cleavage. Thus,
SOD1 acquires peroxidase activity at physiological pH only in the
presence of
HCO3
or
structurally similar anions. Alterations in pH that shift the
HCO3
/CO2
equilibrium as occur in disease processes such as ischemia, sepsis, or
shock would modulate the peroxidase function of SOD1.
 |
INTRODUCTION |
Superoxide dismutases
(SODs1; EC 1.15.1.1) are a
ubiquitous family of metalloenzymes that catalyze the dismutation of
superoxide anion, O
2, to form O2 and
H2O2. These enzymes are highly efficient in
catalyzing the dismutation at a rate close to the diffusion limit, over
the entire pH range from 5 to 10 (1, 2). Three distinct isozymes are
present in mammalian cells: the cytosolic, homodimeric Cu,Zn-enzyme
(Cu,Zn-SOD or SOD1), the manganese-containing mitochondrial SOD (Mn-SOD
or SOD2) and the extracellular form of CuZn-SOD (or SOD3) (2). Several
lines of evidence indicate that CuZn-SOD, but not Mn-SOD, undergoes
free radical damage by its own product H2O2
(3), resulting in inactivation (4-7) and fragmentation of the enzyme
(8, 9) and production of the highly reactive oxidant ·OH (5,
9-12). The inactivation of CuZn-SOD is reported to be caused by the
oxidation of the active site histidine, His118 (4-6, 13),
but the mechanism of this reaction is not fully understood. Hodgson and
Fridovich (5, 14) proposed a mechanism in which
H2O2 first reduces the Cu(II) and then reacts
with the Cu(I) to give a potent oxidant, most likely a
Cu2+-bound ·OH, which can attack an adjacent
histidine and destroy the integrity of the catalytic site. Alternately,
exogenous reductants such as xanthine, urate, formate, and azide can
protect the enzyme when they serve as sacrificial substances and spare
the essential histidines (5, 14, 15). Thus, CuZn-SOD, in addition to its SOD activity, can exert a peroxidase function toward these exogenous reductants at rates competitive with its own oxidative inactivation (5, 14).
Prior studies reported that inactivation of CuZn-SOD by
H2O2 proceeds rapidly only at pH values above
9.0 (5-8, 12, 14, 16-18). HO2
,
rather than H2O2, was implicated as the active
species, due to its resemblance to O
2 in structure and charge
(6-8, 12, 16-18). Although H2O2 was able to
reduce the active site Cu2+ to Cu+ at high
concentrations, the reaction of H2O2 to form
the bound ·OH was favored only when the pH was raised above 8, since the pKa for H2O2 is
11.9 (12). However, more recently, EPR spin trapping studies
demonstrated that CuZn-SOD and H2O2 at normal
physiological pH values in the presence of the spin trap, DMPO, yield
large amounts of the hydroxyl adduct, DMPO-OH, which was attributed to
free ·OH (10, 11). Subsequently, two related spin trapping
studies (19, 20), also performed at pH 7.4, implicated this phenomenon in the gain-of-function of CuZn-SOD mutants associated with the familial form of amyotrophic lateral sclerosis, a progressive degenerative disorder of motor neurons leading to paralysis. In general, it is difficult to explain these results based on the alkaline-based HO2
-mediated reaction
mechanism. While H2O2 with its
pKa of 11.9 gives rise to trace amounts of
HO2
at pH 7.4 and this might react
with Cu+ at the active site, the rate constant of this
reaction is quite low at neutral pH (kobs ~ 0.1 M
1 s
1) (12). Therefore, one
would expect little if any detectable DMPO-OH generation at pH 7.4. Hence, the high magnitude EPR signals obtained (10, 11, 20) cannot be
accounted for by this reaction at neutral pH.
While most of the in vitro spin trapping studies in the
physiological pH range are carried out in a variety of buffers, all of
the above spin trapping studies with CuZn-SOD (10, 19, 20) and
H2O2 were performed only in bicarbonate buffer.
However, there has been no prior explanation for the importance of
HCO3
in this system. Therefore, in the
present study, we evaluate the role of
HCO3
in the peroxidase function of
CuZn-SOD. We observe that large magnitude DMPO-OH generation occurs
only in bicarbonate buffer with no significant signal obtained in other
buffers. The critical role of HCO3
in
producing these signals is characterized. Structural changes of the
active site copper in the presence of
HCO3
are studied by EPR measurements
of the Cu(II). These studies suggest that at physiological pH,
HCO3
binds to the anion-binding site
of CuZn-SOD and facilitates the approach of
H2O2 to the active site, enabling its redox cleavage.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Bovine erythrocyte CuZn-SOD was purchased from
Sigma (98% enzyme, 4000-5800 units/mg) or Worthington (100% enzyme,
2273 units/mg). Activity was assayed by the method of Beauchamp and
Fridovich (21). Human CuZn-SOD, Mn-SOD, sodium formate, sodium azide, sodium bicarbonate, sodium bisulfite, sodium hydrogen selenite, hydrogen peroxide (30% w/w), Hepes, Tris, sodium hydrogen phosphate, disodium hydrogen phosphate, CAPS, potassium thiocyanate, potassium sulfate, sodium nitrate, borate,
2,2,6,6-tetramethyl-1-piperidinyl-1-oxy (TEMPO),
diethylenetriaminepentaacetic acid (DTPA), Me2SO, and ethanol were obtained from Sigma. Deferoxamine mesylate was obtained from Ciba Pharmaceuticals, Inc., and
Fe3+-(nitrilotriacetate)2 (Fe-NTA) was prepared
as described previously (22).
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was
obtained from Boehringer Mannheim. Purified DMPO was purchased from
Oklahoma Medical Research Foundation Spin Trap Source. Phosphate buffer
(pH 7.0), containing 100 µM DTPA and purged with dry
nitrogen gas, was used to prepare stock solutions of DMPO.
EPR and Optical Spectroscopy--
EPR spectra were recorded in
quartz flat cells at room temperature with a Bruker ER 300 or ESP 300E
spectrometer operating at X-band with 100-kHz modulation frequency and
a TM 110 cavity. The microwave frequency and magnetic field were
precisely measured using an EIP 575 microwave frequency counter and a
Bruker ER035M NMR gaussmeter. EPR spectral simulations were performed
using computer programs as described previously (23). Quantitation of
the free radical signals was performed by computer simulation of the
spectra and by comparison of the double integral of the observed signal
with that of a TEMPO standard (10 mM) measured under
identical conditions (24). EPR spectra of the active site Cu2+ of CuZn-SOD were recorded in 3-mm quartz tubes at 77 K
using a liquid nitrogen dewar. A Perkin-Elmer Lambda-6 UV-VIS
spectrophotometer was used for optical studies.
 |
RESULTS |
Either human or bovine CuZn-SOD (1.25 µM), dissolved
in 23.5 mM bicarbonate buffer (pH 7.4) equilibrated with
5% CO2 and 95% N2, gave rise to a large EPR
signal when treated with H2O2 (1 mM) in the presence of DMPO (50 mM) (Fig.
1A). The quartet signal with
typical intensity ratio, 1:2:2:1, and hyperfine couplings, aH = aN = 14.9 G,
confirmed by computer simulation, corresponds to DMPO-OH (24, 25). The
intense signal was only obtained in bicarbonate buffer and was largely
insensitive to changes in pH in the range 7.0-8.0. With similar
conditions of enzyme and H2O2 concentrations
but in other buffers including Hepes, PBS, and Tris (Fig.
1B), no signal was observed. Previously, the reaction between H2O2 and the reduced copper of the
active site was shown to increase at high pH, due to the ionization of
H2O2 to HO2
(pKa = 11.9) (6, 12). Such an enhanced interaction was suggested to promote the formation of the copper-bound ·OH
and rapid inactivation of the enzyme (6, 12). Anticipating a parallel
increase in the DMPO-OH formation, experiments were carried out at pH
10.5. While no signal was observed in CAPS buffer (Fig. 1C),
a small DMPO-OH signal was obtained in Na2CO3
buffer (Fig. 1D). Thus, the presence of
HCO3
rather than a high pH is critical
for the DMPO-OH formation. To exclude the possibility of trapping some
bicarbonate-derived radicals obtained from a reaction between
·OH and HCO3
, a Fenton
·OH generating system of Fe-NTA (10 µM) and
H2O2 (1 mM) was carried out in
phosphate buffer (pH 7.0) with DMPO (50 mM) in the presence or absence of HCO3
(20 mM). Except for the first 5 min, the DMPO-OH signal
declined in the presence of HCO3
compared with that observed in the absence of
HCO3
(Fig.
2). In similar experiments with the
Fenton ·OH generating system at pH 10.5, similar DMPO-OH spectra
were observed, demonstrating that the decrease in signal from SOD at pH
10.5 shown in Fig. 1D was not simply due to a loss of
efficacy of the trap.

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Fig. 1.
EPR spectra of DMPO-OH generated by CuZn-SOD
and H2O2 in different buffers. Spectra
observed from CuZn-SOD (1.25 µM) and
H2O2 (1 mM) in 23.5 mM
NaHCO3 buffer (pH 7.4) equilibrated with 5%
CO2 and 95% N2 (A) or 50 mM Hepes (pH 7.4) (B). Note that similar spectra
were obtained with other buffers such as 50 mM phosphate
(pH 7.4) and 50 mM Tris·HCl (pH 7.4); C, 50 mM CAPS buffer (pH 10.5); D, 50 mM
Na2CO3 buffer (pH 10.5). Spectra were recorded
in the presence of 50 mM DMPO at a microwave frequency of
9.78 GHz, a microwave power of 20 milliwatts, and a modulation
amplitude of 0.5 G. Each spectrum consists of the sum of 10 30-s scans
recorded 10 min after the addition of
H2O2.
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Fig. 2.
DMPO-OH formation from a Fenton hydroxyl
radical generating system in bicarbonate buffer. EPR spectra were
recorded with parameters as described in the legend to Fig. 1 from
Fe-NTA (10 µM) and H2O2 (1 mM) in 50 mM phosphate buffer (pH 7.4)
containing 100 µM DTPA and no NaHCO3
(A) or 23.5 mM NaHCO3
(B). Concentration of DMPO-OH was obtained as described
under "Experimental Procedures."
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The vital role played by HCO3
in the
peroxidative function of CuZn-SOD toward DMPO in producing DMPO-OH was
further investigated in the context of the enzyme's affinity for
anions (2, 26). The substrate for SOD itself is an anion, and several
investigations have reported the effects of anion binding to CuZn-SOD
with some of them inhibiting the enzymatic activity (2, 26-35).
Production of DMPO-OH by CuZn-SOD and H2O2 was
examined in the presence of a variety of anions, such as azide,
nitrate, sulfate, borate, thiocyanate and formate. These anions were
reported to have affinity for the anion site of the enzyme. EPR spectra
were measured every 5 min for more than 1 h following the addition
of H2O2 (1 mM) to CuZn-SOD (1.25 µM), DMPO (50 mM), and the anion (20 mM) at pH 7.0 (Fig. 3). As
shown in Fig. 3, these anions did not cause significant DMPO-OH
generation in comparison with HCO3
.
However, these anions when taken together with
HCO3
, diminished the signal normally
observed with HCO3
. As an example, the
effect of NaH2PO4 and NaCl are shown in Fig. 4, A and B. The
decrease in the signal ranged from 20 to 90%, depending on the type of
anion and its concentration.

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Fig. 3.
Role of anion binding in DMPO-OH generation
from CuZn-SOD and H2O2. Spectra were
observed from CuZn-SOD (1.25 µM),
H2O2 (1 mM), and DMPO (50 mM) in Hepes buffer (50 mM, pH 7.4) containing
different anions at 20 mM concentrations. EPR spectral
parameters were as described in Fig. 1. The concentration of DMPO-OH
was obtained as described under "Experimental Procedures."
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Fig. 4.
Bicarbonate-mediated DMPO-OH generation in
the presence of competing anions. Spectra were observed from
CuZn-SOD (1.25 µM), H2O2 (1 mM), and DMPO (50 mM) in 23.5 mM
NaHCO3 buffer (pH 7.4) equilibrated with 5%
CO2 and 95% N2 containing different
concentrations of NaH2PO4 (A) and
NaCl (B). EPR spectral parameters were as described in Fig.
1. Concentration of DMPO-OH was obtained as described under
"Experimental Procedures."
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In the past, x-ray crystallography and EPR investigations of CuZn-SOD
indicated the proximity of anion binding residues to the copper site
(2, 27-30, 33-37). Only certain anions that directly coordinate the
copper cause significant changes in the EPR spectrum of the
Cu2+ (2, 27, 29-31, 36). Thiocyanate
(Kd ~ 200 mM) and azide
(Kd ~ 10-16 mM) are typical examples
that coordinate the Cu2+ and induce distinct changes in the
copper hyperfine splitting parameters (27-30). We compared the effects
of thiocyanate (200 mM), azide (20 mM), and
bicarbonate (200 mM) at pH 7.0 on the Cu2+-EPR
at 77K of CuZn-SOD (0.5 mM) (Fig.
5). The anisotropic hyperfine coupling
constants (A
) of SCN-SOD (148 G; Fig.
5B) and N3-SOD (156 G; Fig. 5C) were
considerably larger than that of the native enzyme (135 G, Fig.
5A), in agreement with the previous reports (27-30). In
contrast, the A
value for CuZn-SOD with
HCO3
(Fig. 5D) remained the
same as the native enzyme, indicating that
HCO3
does not bind directly to the
copper.

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Fig. 5.
EPR spectra of the active site
Cu2+ of SOD. Spectra were observed from 0.5 mM CuZn-SOD in phosphate buffer (50 mM, pH 7.4)
at 77 K. A, control with no anions; B, with 200 mM KSCN; C, with 20 mM
NaN3; D, with 200 mM
NaHCO3. Spectra were recorded at a microwave frequency of
9.35 GHz, a microwave power of 20 milliwatts, and a modulation
amplitude of 4 G.
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To further understand the unique mechanism in which
HCO3
facilitates the access of the
active site by the neutral H2O2, several anions
were studied based on their structural similarity to
HCO3
. Indeed, bisulfite
(HSO3
) and biselenite
(HSeO3
), which are isostructural to
HCO3
, effected the generation of
DMPO-OH in a similar fashion as HCO3
.
While the sample containing 1.25 µM CuZn-SOD, and 1 mM H2O2, in the presence of DMPO
did not give rise to any signal in the absence of these anions or
HCO3
(Fig.
6A), it generated intense EPR
signals when supplemented with 20 mM
HCO3
(Fig. 6B),
HSO3
(Fig. 6C), or
HSeO3
(Fig. 6D). The signal
obtained with HSO3
(Fig.
6C) actually corresponds to the
·SO3
radical adduct of DMPO,
DMPO-·SO3
, as confirmed by the
characteristic hyperfine splittings, aH = 16.0 G
and aN = 14.7 G (38). Prior studies reported
that ·SO3
radicals might also
be generated by the autooxidation of the bisulfite anion, which could
be prevented by 1 mM DTPA (38). In addition,
H2O2 has been reported to react directly with
bisulfite to produce ·SO3
(38).
Hence, we included 1 mM DTPA in our experiments to prevent spontaneous ·SO3
generation,
and control measurements in the absence of CuZn-SOD were performed to
subtract the contribution from the direct reaction of bisulfite with
H2O2 for the signal of
DMPO-·SO3
in Fig.
6C.

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Fig. 6.
Mimicking of bicarbonate-mediated DMPO-OH
generation by isostructural anions. EPR spectra were observed from
CuZn-SOD (1.25 µM) and H2O2 (1 mM), DTPA (1 mM) in Hepes buffer (50 mM, pH 7.4) containing no anions (A), 20 mM NaHCO3 (B), 20 mM
NaHSO3 (C), or 20 mM
NaHSeO3 (D). EPR spectral parameters were as
described in the legend to Fig. 1. Each spectrum consists of the sum of
10 30-s scans recorded immediately after the addition of
H2O2.
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Two distinct experiments were performed to assess the effect of
concentrations of H2O2 and
HCO3
on the DMPO-OH signal generation.
In the first one, DMPO-OH production was evaluated from a mixture of
1.25 µM CuZn-SOD, 50 mM DMPO, and 20 mM HCO3
with varying
concentrations of H2O2 (0-50 mM).
In the second one, [H2O2] was kept fixed at 1 mM, while [HCO3
] was
varied from 0 to 10 mM. In both experiments, the EPR signal was accumulated for 10 min, immediately after the addition of H2O2. As shown in Fig.
7A, the signal increased
linearly when [H2O2] was increased from 0 to
2 mM and reached a plateau at about 10 mM.
However, at [H2O2] higher than 30 mM, there was a slow decline in the signal generation.
Similarly, with respect to HCO3
, the
signal increased linearly from 0 to 2 mM and reached a
plateau at about 6 mM (Fig. 7B).

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Fig. 7.
Dependence of the DMPO-OH signal amplitude on
the concentration of HCO3 and
H2O2. EPR spectra were recorded as
described in the legend to Fig. 1 from CuZn-SOD (1.25 µM)
and DMPO (50 mM) with varying amounts of
H2O2 in 23.5 mM NaHCO3
buffer (pH 7.4) equilibrated with 5% CO2 and 95%
N2 (A) or CuZn-SOD (1.25 µM), DMPO
(50 mM), and H2O2 (1 mM) with varying amounts of NaHCO3 in
deaerated, double distilled and deionized water (B). The
final pH of the samples was from 5.0 to 7.0; the concentration of
DMPO-OH was obtained as described under "Experimental
Procedures."
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In order to confirm that the peroxidase function of CuZn-SOD and its
dependence on bicarbonate is a generalized phenomenon observed with
substrates other than DMPO, experiments were performed with ABTS, a
chromogenic substrate very widely used for measuring peroxidase
activity (39). ABTS has an absorption maximum at 340 nm (
= 3.6 × 104 M
1·cm
1),
and it is oxidized by peroxidases to its radical cation, which has an
absorption maximum at 415 nm (
= 3.6 × 104
M
1·cm
1) together with lesser
maxima at 395, 640, and 720 nm (39). The decrease in the absorbance at
340 nm and the increase at 415 nm is usually monitored for peroxidase
activity measurements. The top panel of Fig.
8 depicts the spectral changes of ABTS
observed with SOD (4 µM), H2O2 (6 mM), and ABTS (10 µM) in the absence (A) and presence (B) of 23.5 mM
bicarbonate (pH 7.4). Since the ABTS cation radical can also be
detected by EPR, its generation in the absence or presence of
bicarbonate was also followed by EPR measurements. EPR spectra in the
bottom panel of Fig. 8 were also obtained with
SOD (4 µM), H2O2 (10 mM) and ABTS (200 µM) in the absence
(A) and presence (B) of 23.5 mM
bicarbonate (pH 7.4). The fine splittings arise from the
hyperfine interaction from four nitrogens and 10 hydrogens of the ethyl
groups. With both types of measurements, it was observed that the
peroxidase activity as evidenced by the formation of the radical cation
is bicarbonate-dependent.

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Fig. 8.
Bicarbonate dependent peroxidation of
ABTS. Top panel, optical absorption spectra of ABTS (10 µM) with SOD (4 µM) and
H2O2 (6 mM) in the absence
(A) as well as in the presence (B) of 23.5 mM bicarbonate (pH 7.4). Bottom panel, EPR
spectra were obtained with ABTS (200 µM), SOD (4 µM), and H2O2 (10 mM)
in the absence (A) as well as in the presence (B)
of 23.5 mM bicarbonate (pH 7.4). EPR spectral parameters
were as described in the legend to Fig. 1. Each spectrum consists of
the sum of 10 30-s scans recorded immediately after the addition of
H2O2.
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 |
DISCUSSION |
Superoxide, a normal by-product of aerobic metabolism, is
generated in diverse physiological and pathological processes such as
oxidative phosphorylation and the respiratory burst of activated phagocytes (15, 40). O
2 has a very short half-life and can lead to the formation of various reactive oxygen species including ·OH, H2O2, and peroxynitrite
(ONOO
), which can damage cellular macromolecules and
contribute to tissue injury (15). The fundamental necessity for SOD as
a first defense against O
2, is documented by its presence in
all aerobic organisms, and its deficiency has been reported to cause
abnormalities in various organisms (15). SOD has been intensively
studied as a therapeutic agent in pathological conditions related to
oxidative stress, tissue damage, and inflammation (40, 41). Contrary to
this, there have been reports implicating CuZn-SOD in the pathogenesis of certain disorders through impaired function or overexpression (42,
43). For instance, overexpression of the SOD1 gene on chromosome 21 of
Down syndrome patients was reported to cause oxidative damage to
biomacromolecules (42). Recent studies demonstrated point mutations in
the SOD1 gene in some cases of familial ALS (44, 45). Initial studies
of the familial amyotrophic lateral sclerosis-associated mutants
predicted that the mutations destabilize the protein structure, leading
to a less active enzyme (45). Subsequent studies with transgenic mice
overexpressing familial amyotrophic lateral sclerosis-linked mutations
suggested that motor neuron degeneration was caused by some toxic
function gained by the mutant SOD1 (45). The nature of this cytotoxic
gain-of-function is yet to be identified, and current studies of the
mechanisms have focused on the non-SOD activity of the enzyme involving
H2O2 (19, 20, 45).
Hodgson and Fridovich (5, 14) showed that in addition to the major SOD
activity, CuZn-SOD possesses a peroxidase activity that utilizes its
own dismutation product, H2O2, as a substrate according to the following mechanism.
The oxidant ·OH generated in Reaction 2 was assumed to
remain bound to the copper because it did not react with known
scavengers for free ·OH like alcohols or benzoate. This oxidant
attacks the imidazole (ImH) of an adjacent histidine (at the active
site) as in Reaction 3 and inactivates the enzyme (5). Exogenous
electron donors such as formate, azide, and urate were found to serve
as sacrificial substrates and prevent the inactivation (5, 14).
However, early studies reported that the interaction between CuZn-SOD
and H2O2 was an affinity reaction increasing
with increasing pH, with rapid enzyme inactivation at pH > 9.0 (5-8, 12, 14, 16-18). Blech and Borders (6) proposed that the
reactive species was HO2
. The
pKa for H2O2 is 11.9, so an
increase in pH would lead to an increase in the concentration of
HO2
. It was proposed that
HO2
coordinated directly to the copper
to form the reactive complex (6). They proposed a kinetic model for the
inactivation process as follows (6).
The apparent dissociation constant for the enzyme-peroxide
complex, decreased progressively with increasing pH, from 15.5 mM at pH 9.0 to 1.11 mM at pH 11.5 (6). This
was justified in view of the enzyme's high electrostatic affinity for
anionic substances including its substrate, O
2 (12). The x-ray
structure of CuZn-SOD illustrated the existence of a positively charged channel with amino acid residues, Lys120 and
Lys134 at the top and Arg141 inside the channel
positioned close (~4-5 Å) to the active site copper (37). This
channel is responsible for the electrostatic guidance of the anionic
substrate to the active site (37). Arg141 with a
pKa of 12 has been frequently implicated as
essential in anchoring the superoxide anion in a suitable position (2, 37). Other small anions such as cyanide, azide, halides, phosphate, borate, formate, cyanate, and thiocyanate were also known to have easy
access to the channel and bind to the active site copper or
Arg141 (26, 28). Since HO2
is a small negatively charged species, differing from O
2 by only a hydrogen atom, the electrostatic effects are presumed to be
similar (12). This explanation was further validated by the kinetic
study of the reactions between H2O2 and
CuZn-SOD, which indicated that the electrostatic control of the access
of the peroxide to the active site was the rate-determining step of the two redox reactions (Reactions 1 and 2), and that
HO2
was the reactive species in both
processes. Furthermore, two independent groups (8, 18) studied the
effect of exposure of CuZn-SOD to O
2 and
H2O2 at physiological pH (7.4-7.8) and temperature (37 °C). The first report (18) demonstrated that enzyme
inactivation by H2O2 occurred only in the
presence of O
2, since H2O2 alone was
unable to cause significant reduction of Cu2+ to
Cu+. The second report (8) indicated that CuZn-SOD
inactivation and oxidative protein degradation in red blood cells was
greater when cells were exposed to a flux of O
2 and
H2O2 than a bolus of
H2O2 alone. Both reports implicate the role of
HO2
.
Therefore, it is clear that the large amounts of DMPO-OH generated by
CuZn-SOD and H2O2 at pH 7.4 in the present
study as well as prior reports cannot be explained by a simple
interaction between the enzyme and H2O2. It is
important to note that all these spin-trapping studies were performed
only in bicarbonate buffer. In the present study, we observed that no
significant DMPO-OH was obtained in other buffers. Hence, the aim of
the present work was to explain the interaction between the enzyme and
H2O2 in the generation of DMPO-OH at
physiological pH. This report delineates the critical role played by
HCO3
in the peroxidase function of
CuZn-SOD at physiological pH, and in turn, it establishes that the
HCO3
/CO2 equilibrium may
influence free radical metabolism in normal pathophysiology and disease.
We demonstrate that the large amounts of DMPO-OH obtained from
CuZn-SOD, H2O2, and DMPO are formed by a rapid
bicarbonate-assisted one-electron oxidation and hydroxylation of
DMPO (Fig. 1). The fact that high pH alone could not trigger this
signal generation indicates that this mechanism is quite distinct from
that of high pH inactivation by H2O2. The large
signal decrease observed in the Na2CO3 buffer
at pH 10.5 (Fig. 1D) can be explained by a combination of
factors. First, based on the pK of free
HCO3
of 10.25, its concentration would
be more than 2-fold decreased at pH 10.5. Furthermore the pK
of the HCO3
bound to the arginine of
the anion-binding site, Arg141, might be further shifted
downward. In addition, compared with pH 7.4, some deprotonation of
Arg141 would occur at pH 10.5 (6). It has also been
reported that at high pH, OH
competes for the anion site
(2). At this highly alkaline pH, changes in protein conformation could
also occur, altering the structure of the enzyme and the relationship
of the anion and copper sites. All of these factors would contribute to
decreasing bicarbonate binding and explain the decrease in
bicarbonate-dependent peroxidase function at high pH.
However, even at pH 10.5, a small signal was seen as shown in Fig.
1D in contrast to the absence of signal in CAPS buffer at
this pH (Fig. 1C).
Other probable reactions such as trapping of a fast decaying
HCO3
radical intermediate obtained
from ·OH or improvement of the spin trapping efficiency in
bicarbonate buffer was ruled out by the control experiments with the
Fenton system, where the HCO3
greatly
decreased the DMPO-OH formation (Fig. 2). In the Fenton system
HCO3
(23.5 mM) resulted in
more than a 5-fold decrease in the DMPO-OH signal, while in the
CuZn-SOD system HCO3
induced more than
a 100-fold increase in DMPO-OH. Further evidence that adduct formation
occurs at the active site of the enzyme is provided by the fact that
DMPO alkyl adducts are not observed even in the presence of
Me2SO or ethanol (10). The generation of the ABTS radical
cation (Fig. 8, bottom panel) and changes in the
optical absorption spec-trum of ABTS (Fig. 8, top
panel) confirm that the bicarbonate-induced peroxidase
function of CuZn-SOD at physiological pH is a generalized phenomenon
occurring with substrates other than DMPO.
Experiments performed with different anions that bind to the anion site
demonstrate that mere binding of any anion was not sufficient for
DMPO-OH generation (Fig. 3). However, the ability of these anions to
decrease the signal amplitude observed with HCO3
indicates that
HCO3
indeed binds to the anion site
(Arg141) for which other anions compete (Fig. 4). In the
past, techniques including x-ray crystallography (28), NMR (46), and
EPR (27, 29) have been used to identify anions that bind directly to the copper. For instance, the crystal structure of azide-inhibited CuZn-SOD suggested that azide mimicked O
2 binding, with a
direct coordination to the Cu2+ at the place of the
copper-bound water molecule and formation of an ion pair with the
active site residue Arg141 (28). The coordination sphere of
Cu2+ was partly transformed from the rhombic symmetry of
the native enzyme to axial symmetry (27-29). This resulted in a change
of the EPR spectrum of Cu2+ at 77 K, which was observed
here (Fig. 5B) as well as by other researchers (27, 29, 30).
Similarly, SCN
was shown to replace the bridging
histidine between the copper and zinc without inhibiting the SOD
activity (30), which resulted in changes in the EPR spectrum of
Cu2+ (as shown in Fig. 5C). Unlike azide and
SCN
, HCO3
did not induce
any changes in the EPR spectrum of Cu2+ at 77 K (Fig. 5),
suggesting that it did not bind directly to the Cu2+.
From the evidence presented here, it can be presumed that
HCO3
binds to Arg141 but
not to the copper. The species HO2
was
previously suggested to mimic superoxide binding at high pH. At
physiological pH, H2O2 predominantly exists as
a neutral molecule rather than the deprotonated species. Hence,
H2O2 can be suggested to bind at the same place
as HO2
but would require hydrogen
bonding to the Arg141-bound
HCO3
as shown in Scheme
1.
Evidence for Scheme 1 is contributed by the experiments with the
isostructural anions, HSeO3
and
HSO3
(Fig. 6). The structure and
charge of these anions is similar to
HCO3
, where selenium or sulfur
replaces the carbon atom. Kinetics of DMPO-OH production studied by
varying concentrations of H2O2 and
HCO3
(Fig. 7) provide further evidence
for this hypothesis. These kinetics can be modeled as an enzyme
(E)-activator (A)-substrate (S) complex (47)-based reaction
in which both SA and A, but not S alone, bind to the enzyme to yield
ESA, where E is CuZn-SOD, S is
H2O2, A is
HCO3
, and P is the
product.

Reaction 5
View larger version (6K)
The velocity equation for this system is given by the following
expression (47).
|
(Eq. 1)
|
According to this equation, at a constant [S]t,
increasing [A]t will result in an increasing rate up to the
point where all of the S is converted to SA (this will happen when
[S]t = [A]t, when K0 is very
small compared with [A]t) (47). Increasing [A]t
further will decrease the rate, as A competes with SA for the enzyme
(47). Fig. 7 reflects a similar phenomenon, suggesting the validity of
the Scheme I for this system. It would be interesting to ascertain the
precise arrangement of these molecules in the active site by structural
methods. Unfortunately, the reaction between CuZn-SOD and
H2O2 in the presence of
HCO3
proceeds rapidly, suggesting that
the ternary complex is quite labile.
Interestingly, there is a precedent for an obligatory role of
HCO3
binding for metalloprotein
function. Over 20 years ago, it was shown that metal binding to the
iron transport protein transferrin is
HCO3
-dependent, and
subsequently arginine was identified as the anion binding ligand
(48-50). However, in transferrin,
HCO3
also binds to the metal. Thus,
there are both similarities and differences in the process of anion
binding by transferrin and CuZn-SOD.
It is possible that the peroxidase function of CuZn-SOD is a mechanism
by which the enzyme degrades H2O2 released at
the active site following superoxide dismutation. This could serve the
important biological role of preventing
H2O2-mediated injury under conditions where
other peroxidases are not present. Identifying intrinsic substrates,
which are as potent as DMPO in accepting one electron, would be
important in determining the role of this process in normal physiology
and disease. Similarly, understanding the role of
HCO3
in this system is very important
for elucidating the larger physiological implications of the effects of
alterations in pH and the
HCO3
/CO2 equilibrium on
this process. In a number of important disease processes such as
ischemia, sepsis, or shock, marked metabolic acidosis occurs with
decreases in tissue pH to values as low as 5.5 (51). Under these
conditions, HCO3
concentration would
decrease by more than 1 order of magnitude, resulting in decreased
CuZn-SOD peroxidase function.
In summary, CuZn-SOD acquires a peroxidase function at physiological pH
in the presence of HCO3
, and this
activity is responsible for the decomposition of
H2O2 and the oxidation and hydroxylation of
DMPO. This function is distinct from the high pH peroxidase activity
and inactivation of the enzyme. Bicarbonate does not bind directly to
the active site copper but binds to the adjacent anion-binding site of
the enzyme. These data support the existence of a peroxidase mechanism in which HCO3
bound at the anion
binding site anchors the neutral H2O2 molecule at the active site copper, enabling its redox cleavage. Bicarbonate anion thus plays a critical role in this peroxidase activity. Therefore, alterations in pH and the
HCO3
/CO2 equilibrium that
occur in a number of disease processes could modulate the peroxidase
function of the enzyme.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Periannan Kuppusamy for helpful
discussions and assistance with EPR spectral simulation and
quantitation. We also thank Dr. David Borchelt and Dr. Irwin Fridovich
for helpful advice.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants HL-38324 and HL-52315 and a grant-in-aid from the American Heart
Association (to J. L. Z.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Electron Paramagnetic
Resonance Center, Johns Hopkins Asthma and Allergy Center, Rm. LA-14,
5501 Hopkins Bayview Circle, Baltimore, MD 21224. Tel.: 410-550-0339;
Fax: 410-550-2448.
The abbreviations used are:
SOD, superoxide
dismutase; SOD1 or CuZn-SOD, Cu,Zn-superoxide dismutase; O
2, superoxide; DMPO, 5,5'-dimethyl-1-pyrroline-N-oxide; TEMPO, 2,2,6,6-tetramethyl-1-piperidinyl-1-oxy; DTPA, diethylenetriaminepentaacetic acid; Fe-NTA, Fe3+-(nitrilotriacetate)2; CAPS, 3-(cyclohexylamino)propanesulfonic acid; ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid.
 |
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