From the Departamento de Bioquímica, Facultad
de Medicina, Universidad de la República,
11800 Montevideo, Uruguay, the ¶ Webb-Waring Institute,
University of Colorado, Denver, Colorado 80262, and
the ** Department of Anesthesiology and Center for Free Radical
Research, University of Alabama, Birmingham, Alabama 35233
Received for publication, October 16, 2000, and in revised form, December 27, 2000
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ABSTRACT |
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Manganese superoxide dismutase (Mn-SOD), a
critical mitochondrial antioxidant enzyme, becomes inactivated and
nitrated in vitro and potentially in vivo by
peroxynitrite. Since peroxynitrite readily reacts with transition metal
centers, we assessed the role of the manganese ion in the reaction
between peroxynitrite and Mn-SOD. Peroxynitrite reacts with human
recombinant and Escherichia coli Mn-SOD with a second order
rate constant of 1.0 ± 0.2 × 105 and 1.4 ± 0.2 × 105 M Manganese-superoxide dismutase (Mn-SOD) is the SOD isoform
1found in the mitochondrial
matrix of eukaryotes and in a variety of prokaryotes (1-3). Mn-SODs
from different organisms are homologous and have a manganese ion in the
active site. Whereas the human mitochondrial enzyme is a homotetramer
(~88 kDa) (4), Escherichia coli Mn-SOD (45.8 kDa) is a
dimer (3). Mitochondria are essential organelles where most of the cell
superoxide (O Nitric oxide (NO·) is a relatively unreactive free radical
formed by nitric oxide synthase (12). However, fast reaction of nitric
oxide with superoxide gives rise to peroxynitrite anion (ONOO Mn-SOD inhibits peroxynitrite formation in mitochondria, but it may be
oxidatively inactivated by excess peroxynitrite. Indeed, peroxynitrite-mediated nitration of tyrosine 34 of human Mn-SOD results
in enzyme inactivation in vitro (23, 24). The presence of a
nitrated and dysfunctional enzyme in rejected human renal allografts
(25) strongly supports the relevance of this process in
vivo. Mn-SOD inactivation, due to tyrosine 34 nitration, would lead to an increase in peroxynitrite formation that would in turn impair mitochondrial energy metabolism (26) and signal apoptotic cell
death (27).
Peroxynitrite decomposition is catalyzed by a variety of Lewis acids
(28). These are electron-accepting compounds like H+ (29,
30), carbon dioxide (31), and transition metals (29) that favor the
cleavage of the O-O bond and lead to the formation of nitrating
species. In the case of H+, peroxynitrous acid (ONOOH)
undergoes homolysis to hydroxyl radical and nitrogen dioxide with
yields up to 30% (28, 32), resulting in nitration yields in the range
of 6-10% (29, 30). In the case of transition metal-containing
compounds, such as metalloproteins and Mn-porphyrin SOD mimetics,
higher nitration yields are obtained (29, 33-35). These facts led us
to consider that the manganese-ion should play an important role in the
reaction of peroxynitrite with Mn-SOD. In addition, manganese may
facilitate the formation of nitrating species at the active site, which
could react with the critical tyrosine 34.
In this work, we have studied the role of the manganese metal center in
the decomposition kinetics of peroxynitrite and in peroxynitrite-dependent nitration of the enzyme and
non-protein aromatic residues. These studies shed light on the nature
of peroxynitrite reaction with Mn-SOD and provide further rationale to
account for the toxic actions that peroxynitrite may promote in
vivo.
Chemicals--
Manganese-superoxide dismutase of E. coli, potassium phosphate (mono- and di-basic),
diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic
acid (EDTA), sodium nitrite, hydrogen peroxide, manganese dioxide
(MnO2), xanthine, cytochrome c,
para-hydroxyphenylacetic acid (HPA), glutathione, sodium
bicarbonate, desferrioxamine, dimethyl sulfoxide, Trizma (Tris base),
8-hydroxyquinoline, guanidinium hydrochloride, manganese chloride, zinc
chloride, sucrose, bovine serum albumin (BSA), Tween 20, sodium
chloride, bicinchoninic acid reagents, and all electrophoretic reagents
were purchased from Sigma. HPLC-grade methanol was obtained from
J. T. Baker Inc. Bovine milk xanthine oxidase was obtained from
Calbiochem-Novabiochem. Mn(III) and Zn(II) tetrakis-(4-benzoic acid)
porphyrins, (Mn(tbap)) and Zn(tbap)), were synthesized and generously
supplied by Ines Batini
A rabbit polyclonal antibody against nitrotyrosine was raised with
nitrated keyhole limpet hemocyanin and purified in our laboratory by
affinity chromatography as described elsewhere (36). The rabbit
polyclonal antibody against human Mn-SOD was a kind gift from
Dr. Ling-Yi Chang (University of Colorado). The donkey monoclonal
antibody against rabbit IgG, linked to horseradish peroxidase,
nitrocellulose (0.45 µm pore size, Hybond C extra), and
luminol-enhanced chemiluminescence detection kit (ECL) were obtained
from Amersham Pharmacia Biotech.
Peroxynitrite was synthesized in a quenched flow reactor as described
previously (37), and excess hydrogen peroxide was removed by treatment
with MnO2. Peroxynitrite concentrations were determined
spectrophotometrically at 302 nm (
The reagents for Mn-SOD expression and purification were the following:
tryptone and yeast extract for LB medium were obtained from Difco, and
methyl viologen (paraquat), ampicillin, Tris-HCl, cytochrome
c, CaCl2, DNase, RNase, Sephadex G-25 and
CM-Sepharose were purchased from Sigma. KCl and potassium phosphate
were from Mallinckrodt, and MnCl2 was from Matheson,
Coleman, and Bell. (NH4)2SO4
(enzyme-grade) was the highest grade commercially available.
Expression and Purification of Human Recombinant
Mn-SOD--
E. coli sodAsodB strain QC774 lacking Mn-SOD
and Fe-SOD was transformed with the pGB1 expression vector (39)
containing the coding sequence of wild type human recombinant Mn-SOD
(hrMn-SOD), minus that encoding residues 2-24 (i.e. minus
the mitochondrial targeting sequence). The cells were grown in LB
medium supplemented with 0.2 mM MnCl2, 50 µg/ml ampicillin, and 30 µM paraquat to induce the
overexpression of pGB1. The wild type pGB1-hrMn-SOD-transformed cell
cultures were incubated overnight at 37 °C. The cultures were
incubated with orbital shaking at 200 rpm. The cells were harvested and
broken by ultrasonication on ice. The crude extracts were treated with
100 units/ml DNase, 10 mg/ml RNase, 10 mM
MgCl2, 10 mM CaCl2, 0.15 M NaCl in 10 mM K2PO4,
pH 7.0, at room temperature for 1 h. Mn-SOD was precipitated by
(NH4)2SO4 fractionation between 65 and 80% saturation. The precipitate obtained at 80% saturation was
desalted by gel filtration chromatography on Sephadex G-25 in 20 mM potassium acetate buffer, pH 6.6. The proteins were then applied to a CM-Sepharose column and eluted in a gradient of 0-100 mM KCl. The yield of purified hrMn-SOD was 72%.
SOD Activity--
SOD activity was determined measuring the
inhibition of the reduction of cytochrome c by the
xanthine-xanthine oxidase system (40). The concentrations of E. coli Mn-SOD and hrMn-SOD were measured by absorbance at 282 nm
( Removal of Manganese from the Active Site, Reconstitution and
Substitution by Zinc--
E. coli Mn-SOD was dialyzed for
16-20 h against 20 mM 8-hydroxyquinoline, 2.5 M guanidinium hydrochloride, 5 mM Tris
chloride, and 0.1 mM EDTA at pH 3.8 and 4 °C (43). This
caused a loss of manganese-from the active site and of enzyme activity.
This enzyme was then dialyzed for 16 h at 4 °C and pH 7.8 against (a) 50 mM Tris chloride (44),
(b) 5 mM Tris chloride, 0.01 mM
MnCl2 or (c) 5 mM Tris chloride, 1 mM ZnCl2 (43), obtaining the apo, the apo/Mn,
or the apo/Zn, respectively. Finally, the three preparations were
extensively dialyzed against 100 mM potassium phosphate
buffer, 0.1 mM DTPA, pH 7.4. The reconstitution of the
apoenzyme with Mn(II) rendered apo/Mn enzyme preparations with specific
activities ranging between 40 and 80% of that of the native enzyme,
which are reported in each of the experiments performed.
Removal of Manganese from the Active Site of Human Recombinant
Mn-SOD--
25 ml of hrMn-SOD (200 µg/ml) were dialyzed 48 h at
4 °C against 400 ml of 20 mM Tris-HCl buffer, pH 3.8, containing 1.5 M guanidinium hydrochloride, 20 mM 8-hydroxyquinoline, and 0.5 M sucrose. The
apoenzyme produced was then dialyzed 48 h against 200 ml of 20 mM Tris-HCl, pH 8.8, containing 0.5 M sucrose
and either 0.6 M MnCl2 or 0.6 M
ZnCl2, with a change of buffer after 24 h. Finally,
the samples were again dialyzed 48 h against 200 ml of 20 mM Tris-HCl, pH 8.8, containing 0.5 M sucrose,
changing the buffer each 12 h. The apoenzyme and the reconstituted
enzymes were assayed for SOD activity and protein content as described above. Recovered specific activities as percentages of original activity were-apoenzyme 0%, apo/Mn enzyme 21%, and apo/Zn enzyme 1%.
Metal Analyses--
Zinc and manganese content of the enzymes
were determined with a graphite furnace atomic absorption spectrometer
(Spectra 20, Varian Instruments, Victoria, Australia). Calibration
curves of each element were made from dilutions in deionized water of atomic absorption standards. The enzyme samples were diluted with water
and metal ion content calibrated against standards.
Kinetic Studies--
The kinetics of peroxynitrite decomposition
in absence and presence of enzyme were studied in a stopped-flow
spectrophotometer (Applied Photophysics, SF.17MV) with a mixing time of
less than 2 ms, at 302 nm. An initial rate approach was used to analyze the data; the first 0.1 s were fit to a linear plot, and the rate constant was determined as the ratio between the slope and the difference between the initial and final absorbance
(Ao
Kinetics of Mn(tbap) and Zn(tbap) reaction with peroxynitrite were
studied under pseudo-first order conditions with peroxynitrite in
excess over the porphyrin, following absorbance changes on the
porphyrin, at 468 and 421 nm, respectively, as described previously (34). Data obtained in the first 0.03-0.2 s were fit to single exponential and pseudo-first order rate constants determined. Reactions
were performed at 37.0 ± 0.1 °C, and the final pH of the
mixture was measured at the outlet.
Kinetics of HPA nitration by peroxynitrite, in the absence and in
presence of Zn(tbap), were studied using an initial rate approach, at
430 nm.
Nitration of HPA--
Nitration of HPA by peroxynitrite was
assessed spectrophotometrically. After the reaction had taken place,
the pH of the solution was adjusted to 10-11 with 6 N
NaOH, and absorbance was recorded at 430 nm. Absorbance of a control
containing everything except peroxynitrite was subtracted before
determining-the nitro-HPA concentration (
Nitration of HPA was also studied by high performance liquid
chromatography (HPLC)-based techniques. Standards and samples of HPA
and nitro-HPA were separated using a Gilson 306 pump (Wilson Medical
Electronics, Inc.) and a C18-derivatized silica column. Samples were eluted with 50 mM potassium phosphate, pH 3, and HPLC-grade methanol. Elution conditions consisted of 5 min at 10%
(v/v) methanol, followed by a 10-min linear gradient from 5 to 45%
(v/v) methanol and finally 15 min at 45% (v/v) methanol. Absorbance
was monitored at 280 and 365 nm. Nitro-HPA concentration was calculated
from the peak area using a calibration curve performed with a standard.
Samples that contained protein were filtered using a cellulose membrane
with a 10,000 Da cut-off, previous to injection.
Western Blot Analyses--
SDS-polyacrylamide gel
electrophoresis of protein samples was performed on 13% polyacrylamide
gels, and proteins were transferred electrophoretically (20 mA, 16 h) to nitrocellulose membranes. Membranes were blocked with 5% bovine
serum albumin (BSA) in 50 mM Tris chloride, pH 7.4, 150 mM NaCl (TBS), 0.6% Tween 20 (blocking buffer). For
detection of nitrotyrosine nitrocellulose membranes were incubated (1 h
at 25 °C) with 0.2 mg/ml anti-nitrotyrosine antibody (1/1000
dilution) in blocking buffer. After extensive washing in TBS, 0.6%
Tween 20, the immunocomplexed membranes were probed (1 h at 25 °C)
with horseradish peroxidase-linked secondary antibody (1/6000 dilution)
in TBS, 0.1% BSA, 0.3% Tween 20. Probed membranes were washed with
TBS, 0.3% Tween 20, and immunoreactive proteins were visualized with a
luminol-enhanced chemiluminescence detection kit (ECL). Photographs of
the Western blots were then scanned, and relative nitration was
determined by densitometric techniques using the data analyzer program
Scion Image (Scion Corp.). Images were set to gray scale, and
average pixel intensity (sum of all the gray values of all the pixels
divided by the number of pixels) of each band was determined. Band area
was also determined. The product of the band area and the average pixel
intensity is proportional with the amount of nitrotyrosine in the blot.
Detection of Mn-SOD dimers was carried out in the same way, probing
with a 1/6000 dilution of the antibody against human Mn-SOD.
Electrospray-Mass Spectrometry Studies--
All electrospray
ionization-mass spectrometry experiments were performed on a PE-Sciex
(Concord, Ontario, Canada) API-III triple-quadrupole mass spectrometer
equipped with an atmospheric pressure ion source. Positive ion mass
spectra were acquired for capillary LC-MS. For the capillary LC-MS
analysis, the effluent from a 300-µm inner diameter × 15 cm
capillary Vydac C18 column (LC-Packings, San Francisco, California) was
introduced directly into the ionization needle of the mass
spectrometer. The column was equilibrated in 0.1% formic acid, and the
flow rate was maintained at 5.7 µl/min by splitting the 0.4 ml/min
flow from a Hewlett-Packard model 1050 HPLC system with an Accurate
brand (1:70) stream splitter and the capillary column. The protein was
desalted by dialysis against deionized water, concentrated using spin
filters (Fisher, 5000 Mr cut off), and loaded in
0.1% formic acid that was maintained for 3-5 min of the run, followed
by a linear gradient of 0-80% acetonitrile in 0.1% formic acid over
the next 10 min.
Mn-SOD Structure Analysis--
E. coli Mn-SOD
structure analysis was performed using the Swiss PDB Viewer program
(Glaxo Wellcome Experimental Research).
General Conditions--
All experiments involving peroxynitrite
reactions were carried out in 100 mM potassium phosphate
buffer, 0.1 mM DTPA at 37 °C and pH 7.4 ± 0.1, unless otherwise specified. These buffers were prepared daily to
minimize carbon dioxide contamination. Experiments reported herein were
performed a minimum of three times with similar results obtained.
Results are expressed as means ± S.D. or by a representative
example. Graphics and curve fitting were generated in Slide-Write 2.1 for Windows (Advanced Graphic Software Inc.).
Kinetics of Peroxynitrite Reaction with Mn-SOD--
The decay of
peroxynitrite (0.1 mM) was followed in absence and in
presence of hrMn-SOD (10 µM) (Fig.
1). At these concentrations pseudo-first
order conditions are not achieved, and the kinetic traces of
peroxynitrite decay in the presence of the enzyme did not follow a
single exponential function (Fig. 1, inset). Indeed, the
rate of peroxynitrite decomposition in the presence of hrMn-SOD was
initially faster, reflecting the reaction of peroxynitrite with the
enzyme (Fig. 1, inset).
To obtain the rate constant of peroxynitrite reaction with hrMn-SOD,
the decay of peroxynitrite (0.2 mM) was followed in the absence and in presence of different concentrations (2.5-15
µM) of hrMn-SOD tetramer, obtaining plots such as
presented in Fig. 1. The apparent rate constant of peroxynitrite
decomposition (kobs) was determined by measuring
the initial rate of peroxynitrite decay (i.e. during the
first 100 ms), as reported recently (45). The plot of the apparent rate
constants of peroxynitrite decomposition as a function of Mn-SOD
concentrations was linear (Fig. 2). The slope of such plot rendered a second order rate constant for the reaction of hrMn-SOD with peroxynitrite of 1.0 ± 0.04 × 105 M
The fast reaction of peroxynitrite with the enzyme suggested that the
manganese ion in the active site should be the primary target of the
oxidant. Considering this and the fact that other compounds that
contain manganese ions (33-35) have been reported to catalyze the
nitration of phenols, it seemed reasonable that the reaction of
peroxynitrite with the manganese ion would lead to the formation of a
nitrating species that could be responsible for the nitration of
tyrosine 34. These considerations led us to assess the role of the
metal center in peroxynitrite decomposition kinetics and
peroxynitrite-mediated nitration of the enzyme.
Removal of Manganese from the Active Site and Reconstitution with
Manganese or Zinc Ions--
To study the role of the metal center in
peroxynitrite reaction with Mn-SOD, the metal was removed from the
active site of the E. coli enzyme, and the apoenzyme (apo)
was obtained. The apoenzyme was then reconstituted with manganese
(apo/Mn) or substituted with zinc (apo/Zn). The activity and metal
content of these enzymes is shown in Table
I. The native enzyme presented 0.7 manganese-atoms per monomer, in agreement with that reported for the
E. coli enzyme (3). The manganese-content of the holoenzyme,
apo, apo/Mn, and apo/Zn enzymes correlated with the activity, in
agreement with the literature (51). The apo/Zn enzyme presented
slightly more than one atom of zinc per monomer, implying the
existence of unspecific binding of the metal to the enzyme.
Zinc was considered to be a good candidate for the substitution of
manganese and evaluation of peroxynitrite reactions with Mn-SOD. On one
hand, the Zn(II) and Mn(II) ions have similar sizes (ionic radius of
0.74 and 0.80 Å, respectively (52)), and zinc is reported to bind to
Mn-SOD in a stoichiometric amount displacing manganese-from the active
site (51), so the apo/Zn conformation would probably be similar to the
native one. On the other hand, zinc is not capable of rendering high
oxidation states (53), such as those proposed to participate in
tyrosine nitration in the case of manganese and iron (i.e.
oxo-manganese (O=Mn(IV)) and oxo-iron (O=Fe(IV))) (34, 35), so the
apo/Zn enzyme would not be an efficient promoter of nitration reactions.
The apo, apo/Mn, and apo/Zn forms of hrMn-SOD were obtained in 0.5 M sucrose. When the disaccharide was extracted by dialysis, these enzymes largely precipitated (60-70%). The different stability of the human and E. coli preparations may be due to their
different quaternary structures, tetrameric and dimeric, respectively.
Role of the Metal Center in Peroxynitrite Decomposition
Kinetics--
Peroxynitrite decomposition kinetics in the presence of
the apo, apo/Mn, and apo/Zn forms of the enzyme was assessed as
described in Fig. 1. Table II reports the
second order rate constants determined for the decomposition of
peroxynitrite in the presence and absence of 2.5 µM
enzyme. The apoenzyme did not affect peroxynitrite decomposition kinetics in a detectable way, so the product of its second order rate
constant and the concentration of enzyme is less than 10% of that
determined for peroxynitrite alone (kapo × [apo]< 0.1 × kONOO Role of the Metal Center in Enzyme Nitration--
E.
coli Mn-SOD, apo, apo/Mn, and apo/Zn (5 µM) were
incubated with peroxynitrite (0.1 mM). Tyrosine nitration
was assessed by immunoblot techniques using a highly specific
anti-nitrotyrosine antibody (Fig. 3). The
apo/Mn enzyme was more nitrated than the apo/Zn enzyme, suggesting that
the metal center could be involved in protein-tyrosine nitration.
Although the apo/Mn was more nitrated than the native enzyme, the
latter presented larger inactivation with respect to the untreated
enzyme, consistent with subtle changes in protein conformation on
apo/Mn. Surprisingly, the apoenzyme was more nitrated than the native,
apo/Mn, and apo/Zn enzymes, which must be related to a larger surface
exposure and accessibility to tyrosine residues in the apoenzyme, due
to the denaturalization and metal extraction process.
Mass spectrometry studies of the native enzyme and the apoenzyme showed
that incubation with peroxynitrite resulted in the formation of a
species with a molecular mass 47 Da higher than the control
enzyme (22,944 to 22,991 Da), consistent with the addition of a single
nitro group to both enzymes (Fig. 4).
Integration of the area below the peaks showed that the nitration
yields with respect to the protein were higher in the apoenzyme than in
the holoenzyme, in agreement with the results obtained by immunoblot techniques.
The exposure of the holoenzyme to peroxynitrite under these conditions
resulted not only in nitration but also in a small degree of
dimerization of the enzyme. Densitometric analysis of Western blots,
using an antibody against human Mn-SOD, revealed that ~3-5% of the
native enzyme was present as dimer after the exposure to peroxynitrite
(not shown).
Mn-SOD and Peroxynitrite-dependent Nitration of
Phenols--
4-Hydroxyphenylacetic acid (5 mM) was exposed
to peroxynitrite (1 mM) in the absence and in the presence
of increasing concentrations of E. coli Mn-SOD (Fig.
5A). While in absence of
enzyme 10.3% of the added peroxynitrite was recovered as nitro-HPA,
and in presence of the E. coli Mn-SOD, nitration yields
increased with the concentration of enzyme, fitting a hyperbolic
profile. A maximum of 12.5% nitration yield could be predicted from
these data. Considering the maximum nitration yield
(%Rmax) and that obtained in absence of enzyme
(%R0) (see Equation 1) the maximum increase in
nitration yield was calculated to be 21%.
Then the role of the metal center, in promoting
peroxynitrite-dependent nitration of HPA, was assessed. HPA
(5 mM) was exposed to peroxynitrite (1 mM) in
the presence of holo, apo, apo/Mn, and apo/Zn enzymes (5 µM) (Fig. 5B), and percentage increases in
nitration yields were determined to be 14.3, 0, 10.6, and 0.6%, respectively. These results unambiguously show that the manganese-ion is responsible for the increase in HPA nitration yields observed in
presence of Mn-SOD.
Kinetics of Peroxynitrite Decomposition in the Presence of Mn(tbap)
and Zn(tbap)--
Due to the surprisingly high rate constant obtained
for the reaction of peroxynitrite with the apo/Zn, compared with that obtained for the native and apo/Mn, and to obtain a better
comprehension of peroxynitrite reactivity with these metals, we studied
the reaction of peroxynitrite with low molecular weight complexes of
manganese and zinc with a substituted porphyrin (tbap).
The reaction rate of the Mn(tbap) and Zn(tbap) (8 µM)
with peroxynitrite was followed at 468 and 421 nm, the respective Soret peak wavelengths of these porphyrins. Peroxynitrite was present in
5-50-fold excess (0.04-4 mM) over the porphyrin achieving
pseudo-first order conditions. The kinetic traces of the Mn(tbap)
reaction with excess peroxynitrite displayed a biphasic pattern as
follows: a first order descent (Fig.
6A), followed by a slow
recovery of the absorbance values. A total absorbance recovery was
observed for Mn(tbap), similar to that described for the reaction of
other manganese-porphyrins (34). In the case of Zn(tbap), the kinetics were more complex. An initial rapid descent (Fig 6A, inset)
was followed by a slower one. This second descent was independent of
peroxynitrite concentration and had a kobs value
similar to that of the proton-catalyzed decomposition of peroxynitrite,
suggesting reactions between peroxynitrite-derived radicals and the
porphyrin moiety. This latter idea is consistent with the fact that
Zn(tbap) recovered its initial absorbance only partially. The observed rate constants determined from the exponential fit in the first 30-200
ms (Fig. 6A, inset) were plotted in function of
peroxynitrite concentration (Fig. 6B). From the slope of
these plots, second order rate constants for the reactions of Mn(tbap)
and Zn(tbap) with peroxynitrite of 6.8 ± 0.1 × 104 and 4.9 ± 0.1 × 105
M Mn(tbap) and Zn(tbap) Catalysis of
Peroxynitrite-dependent Nitration of HPA--
Mn(tbap)
(1-20 µM) increased peroxynitrite (1 mM)-dependent yield of nitration of HPA (5 mM) in a dose-dependent fashion. These results
fit a hyperbolic plot, and a maximum increase in nitration yields of
350% was determined, in agreement with previous reports (33). On the
other hand, Zn(tbap) did not promote
peroxynitrite-dependent nitration of HPA (Fig.
7), and in fact at high concentrations of
Zn(tbap) (20 µM) a small decrease in nitration yields was
observed, in agreement with recent reports (35). These results are in agreement with those obtained with the native enzyme and
zinc-substituted apoenzyme, supporting the role for the active site
manganese ion in promoting the peroxynitrite-dependent
nitration of low molecular weight phenols. At the same time it is clear
that either the environment of the manganese ion in the enzyme or the
poor accessibility of HPA to the active site makes the enzyme a less
efficient promoter of peroxynitrite-dependent nitration of
phenols, compared with the manganese-porphyrin.
The fast reaction of Zn(tbap) with peroxynitrite in conjunction with a
marginal effect on nitration yields supports the idea that zinc is
behaving like a Lewis acid, resulting in nitration yields similar to
those of H+-catalyzed nitration. This hypothesis was
further evaluated by experiments showing that Zn(tbap) (10-50
µM) accelerated peroxynitrite (1 mM)-dependent HPA (5 mM) nitration
and diminished peroxynitrite nitration yields in presence of carbon
dioxide (0.2 mM) (not shown).
Effect of Scavengers on Mn-SOD Nitration and
Inactivation--
Different compounds known to interact either with
peroxynitrite or with hydroxyl radical or nitrogen dioxide were
assessed for their ability to inhibit or increase the nitration and
inactivation of E. coli Mn-SOD (5 µM) by
peroxynitrite (0.5 mM) (Fig.
8A). Coincubation of Mn-SOD
with 1 mM glutathione (GSH) and HPA largely prevented both
nitration and inactivation. Dimethyl sulfoxide (Me2SO) (10 mM), a well known hydroxyl
radical scavenger, was a weak inhibitor of inactivation and nitration,
implying a modest role for the radical pathway in the nitration of the
holoenzyme. Most interestingly, bicarbonate
(HCO
At higher concentrations of Mn-SOD,
HCO Peroxynitrite reacts with human recombinant and E. coli
Mn-SOD in a direct reaction with second order rate constants of
1.0 ± 0.2 × 105 and 1.4 ± 0.2 × 105 M The surprisingly high rate constant determined for the reaction between
peroxynitrite and the zinc-substituted apoenzyme suggested that the
decomposition kinetics of peroxynitrite could also be affected by the
nonredox metal zinc (53). Indeed, kinetic rate determinations for the
reaction of peroxynitrite with Mn(tbap) and Zn(tbap), revealed that the
zinc-substituted porphyrin reacts with peroxynitrite faster than its
manganese counterpart. The decomposition of peroxynitrite in the
presence of Mn-SOD and Mn(tbap) is attributed to a redox reaction which
involves the oxidation of the metal ion (34, 54). However, in the
presence of the zinc-substituted apoenzyme or Zn(tbap), peroxynitrite
decomposition must proceed through a different pathway that may involve
the utilization of the zinc ion as a Lewis acid.
Peroxynitrite reaction with Mn-SOD leads to the formation of nitrating
species, capable of modifying low molecular weight aromatic compounds
in a manganese-dependent process (Fig. 5). In addition, and
as reported for the human recombinant Mn-SOD (23, 24), peroxynitrite
promoted the inactivation of the E. coli enzyme mainly by
the nitration of one tyrosine residue. While in the apoenzyme
peroxynitrite-derived hydroxyl radical and nitrogen dioxide-mediated
tyrosine nitration, the manganese ion played an important role in
tyrosine nitration in the holoenzyme (Fig. 3). The nitrating species
could be either a nitronium ion (NO E. coli Mn-SOD tyrosine residues have different
degrees of solvent accessibility, as revealed by the analysis of the
native structure using the Swiss PDB Viewer program. Tyrosine 34 is
less accessible to the solvent than Tyr-2, Tyr-9, and Tyr-11 and
equally accessible as Tyr-173, Tyr-174, and Tyr-184, but
tyrosine 34 is the residue located closest to the active site, only at
5 Å from the manganese ion. The attraction of peroxynitrite to the
active site, probably by the basic residues in the channel entrance, and its reaction with the manganese ion leading to the formation of
nitrating species provide a reasonable explanation to the fact that
tyrosine 34 is the tyrosine residue most susceptible to nitration by
peroxynitrite (23, 24).
Considering the specific activity of purified Mn-SOD (4000 ± 1000 units/mg (1, 4, 58, 59)) and that observed in mitochondria
(10 ± 2 units/mg, in heart and liver
mitochondria),2 as well as
the enzyme molecular mass (88 kDa) and mitochondrial volume (1.2 µl/mg), a concentration of Mn-SOD inside the mitochondria of 20 ± 10 µM (80 µM subunits) was estimated. In
concentrations similar to those found in the mitochondria, GSH
protected Mn-SOD from nitration and inactivation both in the absence
and presence of carbon dioxide (Fig. 8). These data underscore the role
of GSH as a mitochondrial antioxidant and raise the question on the mechanism of nitration and inactivation of the enzyme in
vivo. In this sense, Mn-SOD reported to copurify with
QH2:cytocrome c reductase (complex III) (60) may
be partially associated to the sites of mitochondrial superoxide
production, hence of peroxynitrite formation, constituting a
preferential target for the oxidant.
Since Mn-SOD is a critical mitochondrial antioxidant, its nitration
represents a severe hazard that will promote oxidative damage and may
ultimately signal cell death. Strategies directed to attenuate
nitration of Mn-SOD tyrosine 34 should result in a better mitochondrial
and cellular outcome under conditions of excess peroxynitrite formation.
1
s
1 at pH 7.47 and 37 °C, respectively. The
E. coli apoenzyme, obtained by removing the manganese ion
from the active site, presents a rate constant <104
M
1 s
1
for the reaction with peroxynitrite, whereas that of the
manganese-reconstituted apoenzyme (apo/Mn) was comparable to that of
the holoenzyme. Peroxynitrite-dependent nitration of
4-hydroxyphenylacetic acid was increased 21% by Mn-SOD. The apo/Mn
also promoted nitration, but the apo and the zinc-substituted apoenzyme
(apo/Zn) enzymes did not. The extent of tyrosine nitration in the
enzyme was also affected by the presence and nature (i.e. manganese or zinc) of the metal center in the active site. For comparative purposes, we also studied the reaction of peroxynitrite with low molecular weight complexes of manganese and zinc with tetrakis-(4-benzoic acid) porphyrin (tbap). Mn(tbap) reacts with peroxynitrite with a rate constant of 6.8 ± 0.1 × 104 M
1
s
1 and maximally increases nitration yields
by 350%. Zn(tbap), on the other hand, affords protection against
nitration. Our results indicate that the manganese ion in Mn-SOD plays
an important role in the decomposition kinetics of peroxynitrite and in
peroxynitrite-dependent nitration of self and remote
tyrosine residues.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(8), induce Mn-SOD expression. Experiments
with knock-out mice shed further light on the relevance of this enzyme,
with Mn-SOD-deficient mice surviving only up to 3 weeks of age (9, 10)
and presenting many features of mitochondrial disease associated with
reactive oxygen species toxicity (11).
), a potent oxidant (13-15). Peroxynitrite is
formed during sepsis, inflammation, excitotoxicity, and
ischemia-reperfusion of tissues, conditions under which the cellular
production of nitric oxide and superoxide increase (12, 16-18), and
participates in reactions related with the pathological expression of
these processes. Recent reports regarding the presence of nitric oxide
synthase in the mitochondria (19-21), along with the easy diffusion of
nitric oxide through membranes (22), make the intramitochondrial
formation of peroxynitrite possible and highlight the relevance of its
interactions with intramitochondrial targets.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Haberle and Irwin Fridovich (Duke University).
302 = 1670 M
1 cm
1)
(38).
282 = 8.67 × 104
M
1 cm
1
(3)) and 280 nm (
280 = 1.81 × 105
M
1 cm
1
(41, 42)) respectively, and by the bicinchoninic acid method obtaining
concordant results. Enzyme preparations of hrMn-SOD and E. coli Mn-SOD, used in the different assays, typically had specific
activities of 2500 and 3200 units/mg, respectively.
Af). To ensure the
accuracy of the rate constant determinations, 200 absorbance
measurements were acquired during the initial part of the reaction
(first 0.2 s) and 200 further points were acquired until more than
99.9% peroxynitrite had decomposed (0.2-10 s) (45).
430 = 4400 M
1 cm
1)
(29). Percent yield was calculated with respect to initial peroxynitrite concentration.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (13K):
[in a new window]
Fig. 1.
Kinetic traces of peroxynitrite reaction with
hrMn-SOD. Time course of the decomposition of peroxynitrite (0.1 mM) in the absence (solid line) and presence
(dashed line) of hrMn-SOD (10 µM) in 200 mM KPi, at 37 °C and pH 7.46 ± 0.01, made measuring the decrease in absorbance at 302 nm. Absorbance
(A) was normalized subtracting the absorbance at final time
(Af) and dividing by the amplitude
(Ao Af). Inset,
logarithmic plot of the data in the first 1.5 s and linear fit of
the first 100 ms.
1
s
1 per tetramer and 2.5 × 104 M
1
s
1 per monomer, at pH 7.47 and 37 °C. This
constant correlates well with that obtained for other metalloproteins
(46-50) and strongly suggests a role for the manganese ion in the
decomposition kinetics of peroxynitrite. The second order rate constant
for the reaction of E. coli Mn-SOD (2.5 µM)
with peroxynitrite (0.2 mM) was determined, considering the
kobs and the enzyme concentration, to be
1.4 ± 0.2 × 105
M
1 s
1
per dimer at pH 7.4 and 37 °C.
View larger version (10K):
[in a new window]
Fig. 2.
Second order plot for the reaction of
peroxynitrite with hrMn-SOD. Peroxynitrite (0.2 mM)
was mixed with hrMn-SOD (2.5-15 µM), and peroxynitrite
decay was measured as the decrease in absorbance at 302 nm, under the
conditions described in Fig. 1. The apparent rate of peroxynitrite
decomposition (kobs) was determined by measuring
the slope in the first 100 ms.
Extraction of the manganese ion from E. coli Mn-SOD active site,
reconstitution or substitution by zinc ion
). These
considerations indicate that the second order rate constant of the
apoenzyme must be smaller than 4 × 104
M
1 s
1
and probably reflects the reaction rate of peroxynitrite with the
enzyme amino acids (45). The apo/Mn rate constant was 37% that of the
holoenzyme, in agreement with the data obtained for the SOD-specific
activity, which is in turn proportional to the manganese content (51).
Thus it is reasonable to assume that apo/Mn preparations with the same
manganese content than the holoenzyme will recover 100% of the rate
constant value. The apo/Zn presented an unexpectedly high rate
constant, approximately twice that obtained for the holoenzyme. These
results support the idea that metal center plays a central role in
peroxynitrite decomposition kinetics.
Role of the metal center in peroxynitrite decomposition kinetics
View larger version (17K):
[in a new window]
Fig. 3.
Role of the metal center in enzyme
nitration. Immunochemical detection of nitrotyrosine was performed
after peroxynitrite (0.1 mM) exposure to either E. coli Mn-SOD, apoenzyme, apo/Mn, or apo/Zn enzyme (5 µM). The blot was scanned and analyzed by densitometric
techniques. Nitration is expressed as relative to the native enzyme
exposed to peroxynitrite. Activity is expressed relative to the native
enzyme incubated in absence of peroxynitrite.
View larger version (17K):
[in a new window]
Fig. 4.
Electrospray-mass spectrometry of
peroxynitrite-treated Mn-SOD and apoenzyme. A, Mn-SOD
(5 µM); B, Mn-SOD incubated with peroxynitrite
(0.1 mM); C, apoenzyme (5 µM);
D, apoenzyme incubated with peroxynitrite (0.1 mM). Samples were prepared and analyzed as described under
"Experimental Procedures."
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Fig. 5.
Mn-SOD enhances
peroxynitrite-dependent nitration of HPA.
A, HPA (5 mM) was incubated in absence and in
presence of different concentrations of E. coli Mn-SOD.
Yields of nitro-HPA were assessed, spectrophotometrically at 430 nm,
after the addition of peroxynitrite (1 mM). B,
increase in nitration yields of HPA was determined after the exposure
of HPA (5 mM) to peroxynitrite (1 mM) in the
presence of E. coli Mn-SOD, apoenzyme, apo/Mn, and apo/Zn (5 µM).
The Mn-SOD-promoted increase in nitration yields was also assessed
by HPLC techniques. HPA (5 mM) was incubated with
peroxynitrite (1 mM) in the absence and presence of
E. coli Mn-SOD (5 µM). Mn-SOD was extracted
from the samples by filtration, and HPA and nitro-HPA were separated
using a reverse phase chromatography column, presenting elution times
of 19.7 and 24.1 min, respectively. Nitro-HPA was quantified measuring
the peak area obtained at 360 nm. In the absence of the enzyme a
nitration yield of 10.9 ± 0.7% was obtained, whereas in the
presence of Mn-SOD this increased to 12.6 ± 0.4% (not shown), in
complete agreement with the data obtained by spectrophotometric techniques.
(Eq. 1)
1 s
1
at pH 7.2 and 37 °C were obtained, respectively. The ratio between the rate constants obtained from the Mn- and Zn-porphyrins is in good
agreement with that obtained with the native enzyme and zinc-substituted apoenzyme.
View larger version (11K):
[in a new window]
Fig. 6.
Kinetic traces and rate constant of
peroxynitrite reaction with Mn- and Zn(tbap). A, time
course of the reaction of 8 µM Mn(tbap) (solid
line) and Zn(tbap) (dashed line), with peroxynitrite
(0.32 mM) in 100 mM KPi, at
37 °C and pH 7.25 ± 0.01. Inset, plot of the first
0.05 s. B, second order plot for the initial reaction
of Mn(tbap) (circle) and Zn(tbap) (triangle) with
peroxynitrite.
View larger version (11K):
[in a new window]
Fig. 7.
Effect of Mn(tbap) and Zn(tbap) on
peroxynitrite-dependent nitration yields. HPA (5 mM) was incubated in absence and in presence of different
concentrations of Mn(tbap) (circle) and Zn(tbap)
(triangle). Yields of nitro-HPA were assessed
spectrophotometrically after the addition of peroxynitrite (1 mM).
View larger version (42K):
[in a new window]
Fig. 8.
Compounds that protect Mn-SOD from nitration
and inactivation. Immunochemical detection of nitrotyrosine in the
enzyme. A, E. coli Mn-SOD (5 µM)
was incubated with peroxynitrite (0.5 mM) in the presence
of 1 mM HPA, 20 mM
HCO
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
s
1 at pH 7.47 and 37 °C, respectively. The
rate constant for the reaction with the E. coli apoenzyme
was at least 1 order of magnitude smaller, whereas that of the apo/Mn
was comparable with that of the holoenzyme, confirming that the
reaction between Mn-SOD and peroxynitrite largely depends on the
presence of the metal in the active site. The reactions of
peroxynitrite with metalloproteins are typically fast; therefore, these
are likely to be major targets in vivo. In this context, the
reactivity of peroxynitrite with Mn-SOD is similar to that previously
reported for mitochondrial aconitase (47), cytochrome
c2+ (48), alcohol dehydrogenase (46), and
peroxidases (49).
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ACKNOWLEDGEMENTS |
---|
We thank Marion Kirk and Stanley Digerness for their assistance in the mass and absorption spectrometry studies, respectively.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant RO3-TW0099 (to B. A. F. and R. R.), the Swedish Agency for Research and Cooperation (Sweden), International Center for Genetic Engineering and Biotechnology (Trieste, Italy), Howard Hughes Medical Institute (to R. R.), and the Gustavus and Louise Pfeiffer Research Foundation (to D. H. S. and J. M. M.).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.
§ Supported in part by a fellowship from Programa de Desarrollo de las Ciencias Básicas (Uruguay).
Investigador Nacional I (Mexico).
International Research Scholar of Howard Hughes Medical
Institutes. To whom correspondence should be addressed: Departamento de
Bioquímica, Facultad de Medicina, Av. General Flores 2125, 11800 Montevideo, Uruguay. Fax: 5982-9249563; E-mail:
rradi@fmed.edu.uy.
Published, JBC Papers in Press, January 4, 2001, DOI 10.1074/jbc.M009429200
2 C. Quijano, and R. Radi, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: Mn-SOD, manganese-superoxide dismutase; SOD, superoxide dismutase; apo, apoenzyme; apo/Mn, manganese-reconstituted apoenzyme; apo/Zn, zinc-substituted apoenzyme; BSA, bovine serum albumin; HPA, para-hydroxyphenylacetic acid; tbap, tetrakis-(4-benzoic acid) porphyrin; HPLC, high performance liquid chromatography; DTPA, diethylenetriaminepentaacetic acid; EDTA, ethylenediaminetetraacetic acid; Me2SO, dimethylsulfoxide; GSH, glutathione; hrMn-SOD, human recombinant Mn-SOD.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Weisiger, R. A.,
and Fridovich, I.
(1973)
J. Biol. Chem.
248,
3582-3592 |
2. |
Keele, B. B., Jr.,
McCord, J.,
and Fridovich, I.
(1970)
J. Biol. Chem.
245,
6176-6181 |
3. | Beyer, W., Imlay, J., and Fridovich, I. (1991) Prog. Nucleic Acids Res. Mol. Biol. 40, 221-253[Medline] [Order article via Infotrieve] |
4. | Matsuda, Y., Higashiyama, S., Kijma, Y., Suzuki, Y., Kawano, K., Akiyama, M., Kawata, S., Tarui, S., Deutsch, H. F., and Taniguchi, N. (1990) Eur. J. Biochem. 194, 713-720[Abstract] |
5. | Turrens, J. F., Freeman, B. A., Levitt, J. G., and Crapo, J. D. (1982) Arch. Biochem. Biophys. 217, 401-410[Medline] [Order article via Infotrieve] |
6. | Boveris, A. (1984) Methods Enzymol. 105, 429-435[Medline] [Order article via Infotrieve] |
7. |
Krall, J.,
Bagley, A. C.,
Mullenbach, G. T.,
Hallewell, R. A.,
and Lynch, R. E.
(1988)
J. Biol. Chem.
263,
1910-1914 |
8. | Liu, R., Buettner, G. R., and Oberley, L. (2000) Free Radic. Biol. Med. 28, 1197-1205[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Lebovitz, R. M.,
Zhang, H.,
Vogel, H.,
Cartwright, J., Jr.,
Dionne, L.,
Lu, N.,
Huang, S.,
and Matzuk, M. M.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
9782-9787 |
10. | Li, Y., Huang, T.-T., Carlson, E. J., Melov, S., Ursell, P. C., Olson, J. L., Noble, L. J., Yoshimura, M. P., Berger, C., Chan, P. H., Wallace, D. C., and Epstein, C. J. (1995) Nat. Genet. 11, 376-381[Medline] [Order article via Infotrieve] |
11. |
Melov, S.,
Coskun, P.,
Patel, M.,
Tuinstra, R.,
Cottrell, B.,
Jun, A. S.,
Zastawny, T. H.,
Dizdaroglu, M.,
Goodman, S. I.,
Huang, T.,
Miziorko, H.,
Epstein, C. J.,
and Wallace, D. C.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
846-851 |
12. | Gross, S. S., and Wolin, M. S. (1995) Annu. Rev. Physiol. 57, 737-769[CrossRef][Medline] [Order article via Infotrieve] |
13. | Huie, R. E., and Padmaja, S. (1993) Free Radic. Res. Commun. 18, 195-199[Medline] [Order article via Infotrieve] |
14. | Goldstein, S., and Czapski, G. (1995) Free Radic. Biol. Med. 19, 505-510[CrossRef][Medline] [Order article via Infotrieve] |
15. | Kissner, R., Nauser, T., Bugnonm, P., Lyem, P. G., and Koppenol, W. H. (1997) Chem. Res. Toxicol. 10, 1285-1292[CrossRef][Medline] [Order article via Infotrieve] |
16. | Omar, B., McCord, J., and Downey, J. (1991) in Oxidative Stress: Oxidants and Antioxidants (Sies, H., ed) , pp. 493-527, Academic Press Inc, San Diego |
17. | Wendel, A., Niehörster, M., and Tiegs, G. (1991) in Oxidative Stress Oxidants and Antioxidants (Sies, H., ed) , pp. 585-593, Academic Press Inc, San Diego |
18. |
Eliasson, M. J. L.,
Huang, Z.,
Ferrante, R. J.,
Sasamata, M.,
Mollive, R. M. E.,
Snyder, S. H.,
and Moskowitz, M. A.
(1999)
J. Neurosci.
19,
5910-5918 |
19. | Ghafourifar, P., and Richter, C. (1997) FEBS Lett. 418, 291-296[CrossRef][Medline] [Order article via Infotrieve] |
20. |
Guilivi, C.,
Poderoso, J. P.,
and Boveris, A.
(1998)
J. Biol. Chem.
273,
11038-11043 |
21. |
Tayotan, A.,
and Giulivi, C.
(1998)
J. Biol. Chem.
273,
11044-11048 |
22. | Denicola, A., Souza, J. M., Radi, R., and Lissi, E. (1996) 328, 208-212 |
23. | MacMillan-Crow, L. A., Crow, J. P., and Thompson, J. A. (1998) Biochemistry 37, 1613-1622[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Yamakura, F.,
Taka, H.,
Fujimura, T.,
and Murayama, K.
(1998)
J. Biol. Chem.
273,
14085-14089 |
25. | MacMillan-Crow, L. A., Crow, J. P., Kerby, D. J., Beckman, J. S., and Thompson, J. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 244, 11853-11858[CrossRef] |
26. | Radi, R., Rodriguez, M., Castro, L., and Telleri, R. (1994) Arch. Biochem. Biophys. 308, 89-95[CrossRef][Medline] [Order article via Infotrieve] |
27. | Ghafourifar, P., Schenk, U., Klein, S. D., and Richter, C. (1999) J. Biol. Chem. 14, 31185-31188[CrossRef] |
28. | Radi, R., Denicola, A., Alvarez, B., Ferrer-Sueta, G., and Rubbo, H. (2000) in Nitric Oxide (Ignarro, L. J., ed) , pp. 57-82, Academic Press, San Diego |
29. | Beckman, J. S., Ischiropoulos, H., Zhu, L., van der Woerd, M., Smith, C., Chen, J., Harrison, J., Martin, J. C., and Tsai, M. (1992) Arch. Biochem. Biophys. 298, 438-445[Medline] [Order article via Infotrieve] |
30. | Ramezanian, M. S., Padmaja, S., and Koppenol, W. H. (1996) Chem. Res. Toxicol. 9, 232-239[CrossRef][Medline] [Order article via Infotrieve] |
31. | Denicola, A., Freeman, B., Trujillo, M., and Radi, R. (1996) Arch. Biochem. Biophys. 333, 49-58[CrossRef][Medline] [Order article via Infotrieve] |
32. | Beckman, J. S., Beckman, T. W., Chen, J., Marshall, P. A., and Freeman, B. A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1620-1624[Abstract] |
33. | Ferrer-Sueta, G., Ruiz-Ramirez, L., and Radi, R. (1997) Chem. Res. Toxicol. 10, 1338-1344[CrossRef][Medline] [Order article via Infotrieve] |
34. |
Ferrer-Sueta, G.,
Batini![]() ![]() |
35. | Crow, J. P. (1999) Arch. Biochem. Biophys. 371, 41-52[CrossRef][Medline] [Order article via Infotrieve] |
36. | Brito, C., Navillat, M., Tiscornia, A., Vuilier, F., Gualco, G., Dighiero, G., Radi, R., and Cayota, A. (1998) J. Immunol. 62, 3356-3366 |
37. |
Radi, R.,
Beckman, J. S.,
Bush, K. M.,
and Freeman, B. A.
(1991)
J. Biol. Chem.
266,
4244-4250 |
38. | Hughes, M. N., and Nicklin, H. G. (1968) J. Chem. Soc. (Lond.) 450-456 |
39. | Gao, B., Flores, S. C., Bose, S. K., and McCord, J. M. (1996) Gene (Amst.) 176, 269-272[CrossRef][Medline] [Order article via Infotrieve] |
40. | Flohé, L., and Ötting, F. (1987) Methods Enzymol. 105, 93-104 |
41. | Beck, Y., Oren, R., Levanon, A., Gorecki, M., and Hartman, J. R. (1987) Nucleic Acids Res. 15, 9076[Medline] [Order article via Infotrieve] |
42. | Ho, Y. S., and Crapo, J. D. (1988) FEBS Lett. 229, 256-260[CrossRef][Medline] [Order article via Infotrieve] |
43. | Ose, D. E., and Fridovich, I. (1976) J. Biol. Chem. 251, 1217-1218[Abstract] |
44. | Beyer, W. F., Reynolds, J. A., Jr., and Fridovich, I. (1989) Biochemistry 28, 4403-4409[Medline] [Order article via Infotrieve] |
45. |
Alvarez, B.,
Ferrer-Sueta, G.,
Freeman, B. A.,
and Radi, R.
(1999)
J. Biol. Chem.
274,
842-848 |
46. | Crow, J. P., Beckman, J. S., and McCord, J. M. (1995) Biochemistry 34, 3544-3552[Medline] [Order article via Infotrieve] |
47. |
Castro, L.,
Rodriguez, M.,
and Radi, R.
(1994)
J. Biol. Chem.
269,
29409-29415 |
48. | Thomson, L., Trujillo, M., Telleri, R., and Radi, R. (1995) Arch. Biochem. Biophys. 319, 491-497[CrossRef][Medline] [Order article via Infotrieve] |
49. | Floris, R., Piersma, S. R., Yang, G., Jones, P., and Wever, R. (1993) Eur. J. Biochem. 215, 767-775[Abstract] |
50. | Zou, M. H., Daiber, A., Peterson, J. A., Shoun, H., and Ullrich, V. (2000) Arch. Biochem. Biophys. 376, 149-155[CrossRef][Medline] [Order article via Infotrieve] |
51. | Ose, D. E., and Fridovich, I. (1979) Arch. Biochem. Biophys. 194, 360-364[Medline] [Order article via Infotrieve] |
52. | Cotton, F. A., and Wilkinson, G. (1988) Química Inorgánica Avanzada (Spanish version of Advanced Inorganic Chemistry, 4th Ed, John Wiley & Sons, Inc.) , p. 31, Editorial Limusa S. A., México DF, México |
53. | Christianson, D. W., and Cox, J. D. (1999) Annu. Rev. Biochem. 68, 33-57[CrossRef][Medline] [Order article via Infotrieve] |
54. | Groves, J. T., and Sudhakar, S. M. (1995) J. Am. Chem. Soc. 117, 9578-9579 |
55. | Ischiropoulos, H., Zhu, L., Chen, J., Tsai, M., Martin, J. C., Smith, C. D., and Beckman, J. S. (1992) Arch. Biochem. Biophys. 298, 431-437[Medline] [Order article via Infotrieve] |
56. | Zou, M. H., and Ullrich, V. (1996) FEBS Lett. 382, 101-104[CrossRef][Medline] [Order article via Infotrieve] |
57. | Zou, M., Martin, C., and Ullrich, V. (1997) Biol. Chem. Hoppe-Seyler 378, 707-713 |
58. | Marklund, S. (1978) Int. J. Biochem. 9, 299-306[Medline] [Order article via Infotrieve] |
59. | Crapo, J. D., McCord, J. M., and Fridovich, I. (1978) Methods Enzymol. 53, 382-393[Medline] [Order article via Infotrieve] |
60. | Marres, C. A. M., Van Loon, A. P. G. M., Oudshoorn, P., Van Steeg, H., Grivel, L. A., and Slater, E. C. (1985) Eur. J. Biochem. 147, 153-161[Abstract] |