An Electron Spin Resonance Spin-trapping Investigation of the
Free Radicals Formed by the Reaction of Mitochondrial Cytochrome
c Oxidase with H2O2*
Yeong-Renn
Chen
,
Michael R.
Gunther, and
Ronald P.
Mason
From the Laboratory of Pharmacology and Chemistry, NIEHS, National
Institutes of Health, Research Triangle Park, North Carolina 27709
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ABSTRACT |
The reaction of purified bovine mitochondrial
cytochrome c oxidase (CcO) and hydrogen peroxide was
studied using the ESR spin-trapping technique. A protein-centered
radical adduct was trapped by 5,5-dimethyl-1-pyrroline N-oxide and was assigned to a thiyl radical adduct based on
its hyperfine coupling constants of aN = 14.7 G
and a
H = 15.7 G. The
ESR spectra obtained using the nitroso spin traps 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS) and
2-methyl-2-nitrosopropane (MNP) indicated that both DBNBS/·CcO
and MNP/·CcO radical adducts are immobilized nitroxides formed
by the trapping of protein-derived radicals. Alkylation of the free
thiols on the enzyme with N-ethylmaleimide (NEM) prevented
5,5-dimethyl-1-pyrroline N-oxide adduct formation and
changed the spectra of the MNP and DBNBS radical adducts. Nonspecific
protease treatment of MNP-d9/·NEM-CcO
converted its spectrum from that of an immobilized nitroxide to an
isotropic three-line spectrum characteristic of rapid molecular motion.
Super-hyperfine couplings were detected in this spectrum and assigned
to the MNP/·tyrosyl adduct(s). The inhibition of either CcO or
NEM-CcO with potassium cyanide prevented detectable MNP adduct
formation, indicating heme involvement in the reaction. The results
indicate that one or more cysteine residues are the preferred reductant
of the presumed ferryl porphyrin cation radical residue intermediate.
When the cysteine residues are blocked with NEM, one or more tyrosine
residues become the preferred reductant, forming the tyrosyl radical.
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INTRODUCTION |
Oxidative stress in mitochondria has been widely recognized as an
important cause of a variety of diseases (1) including aging (2, 3),
neurodegeneration (3, 4), and cancer (5, 6). Under certain conditions
such as incomplete oxygen reduction during oxidative phosphorylation,
ischemia-reperfusion, and inflammation, the production of reactive
oxygen species by the mitochondrial electron transport chain can
increase severalfold (7, 8). It is generally thought that several
events of oxidative stress, such as calcium imbalance, lipid
peroxidation, glutathione depletion, and DNA and protein damage, arise
from excess production of reactive oxygen species in mitochondria
(8-11).
Several reports have demonstrated that reactive oxygen species can
cause oxidative inactivation of the components of the mitochondrial electron transport chain during exposure of submitochondrial particles to fluxes of O
2, ·OH, or H2O2
(12-15). However, the molecular mechanism regarding the effect of
reactive oxygen species on the mitochondrial bioenergetic enzyme
complex still remains unknown. Recently we employed the ESR
spin-trapping technique to investigate the oxidative damage of horse
heart cytochrome c (16, 17). It was clearly shown that
protein-centered radicals are formed on cytochrome c
following its reaction with hydrogen peroxide, and the protein-derived
radicals are expected to become sites of permanent oxidative damage.
The cytochrome c-derived radicals were able to oxidize
residues on peptides other than the cytochrome c (18).
Cytochrome c oxidase
(CcO),1 the terminal enzyme
of the mitochondrial respiratory chain, is a multicomponent membrane
protein with a molecular weight of 200,000, comprising 13 different
polypeptide subunits. The enzyme functions to reduce dioxygen to
H2O at the active center of heme
a3-CuB and couples proton pumping
across the mitochondrial inner membrane to provide the driving energy for ATP synthesis (19). It has been reported that a peroxidase-like reaction can occur at the heme
a3-CuB center of CcO with a very high Km for H2O2 (20, 21).
The addition of excess H2O2 can oxidize CcO to
yield a peroxy intermediate (or "compound P"), and a one-electron
reduction of compound P with either ferrocyanide or cytochrome
c yields the ferryl-oxo species (or "compound F"). Finally, a one-electron reduction of compound F returns the CcO to the
oxidized state and completes the catalytic cycle. The structure of
compound P remains unclear and is under debate (22-24). Compound F was
proposed to be structurally related to a horseradish
peroxidase-compound II-like species (25-27). One would expect that
both compounds P and F are highly reactive if the chemistry of the
H2O2/CcO system is similar to that of
peroxidases. However, no direct evidence indicates that the compound P
or compound F species of CcO can oxidize the protein and initiate free
radical-mediated lipid peroxidation as seen with metmyoglobin and
cytochrome c. Herein we report that protein-centered radical
formation on CcO was detected using the ESR spin-trapping technique.
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MATERIALS AND METHODS |
Reagents--
Ascorbic acid, ammonium sulfate, ceric ammonium
sulfate (dihydrate), diethylenetriaminepentaacetic acid, horse heart
cytochrome c, phospholipase D (EC 3.1.4.3), and sodium
cholate were purchased from Sigma and used as received. Hydrogen
peroxide (H2O2) was purchased from Fisher. The
H2O2 concentration was verified using the UV
absorption at 240 nm (
= 43.6 M
1
cm
1). Pronase (from Streptomyces griseus) was
purchased from Boehringer Mannheim and used as received. The DMPO, MNP,
NEM, potassium cyanide, and sodium dithionite were purchased from
Aldrich. DMPO was vacuum-distilled twice and stored under nitrogen at
70 °C until needed. DBNBS was synthesized using the method of Kaur
et al. (28). Prepacked Sephadex G-25 (PD-10) gel filtration
cartridges were purchased from (Amersham Pharmacia Biotech).
Preparations of Mitochondrial Cytochrome c Oxidase--
Highly
purified beef heart mitochondrial cytochrome c oxidase was
prepared and assayed according to the methods reported by Yu et
al. (29). Submitochondrial particles were used as the starting
material and subjected to ammonium sulfate precipitation in the
presence of 1.5% sodium cholate. The cytochrome c oxidase as prepared is essentially in the delipidated form and
spectrophotometrically free of complex III (cytochrome
bc1 complex). The cytochrome c oxidase contains 10-12 nmol of heme a/mg of protein
and 7-8 µg of phospholipid/mg of protein. Oxidase activity was
confirmed by measuring the oxidation of ferrous cytochrome c
and by oxygen consumption in the presence of ascorbate and ferric
cytochrome c (29).
ESR Spin-trapping Measurements--
All ESR spectra were
recorded with a Bruker ESP 300 spectrometer, using a quartz flat cell
and operating at 9.8 GHz with a modulation frequency of 100 kHz and a
TM110 cavity. The reactions were initiated by the addition
of hydrogen peroxide to the mixture of cytochrome c oxidase
and spin trap in 50 mM sodium/potassium phosphate buffer
(pH 7.4) containing 200 µM diethylenetriaminepentaacetic acid. For the preparation of thiol-blocked cytochrome c
oxidase, the NEM (0.25 M in H2O) was mixed with
the enzyme at a molar ratio of 50:1, and the reaction was allowed to
proceed for 10 min at room temperature before the addition of spin
traps and H2O2. The spectral simulations were
done using the WinSim program of the NIEHS public ESR software package
that is available on the World Wide Web (http://epr.niehs.nih.gov).
Proteolytic Digestion of Cytochrome c Oxidase--
Enzymatic
digestion of cytochrome c oxidase with Pronase was carried
out at room temperature, and phospholipase D was included to a final
concentration of 50 units/ml for 20 min before proteolysis. The Pronase
(100 mg/ml), suspended in 50 mM phosphate buffer, pH 7.4, was then added to the reaction mixture at a final concentration of 10 mg/ml.
Oxygen Consumption Experiments--
Oxygen consumption
measurements were made using a Clark-type oxygen electrode fitted to a
1.8-ml Gilson sample cell and monitored by a Yellow Springs Instrument
Company (Yellow Springs, OH) model 53 oxygen monitor. The reagents were
added in the following order: 20 µM cytochrome
c in 50 mM sodium/potassium phosphate buffer (pH
7.4) containing 1 mM ascorbate. Then, after establishing
the measurement of a 1-min base line, an appropriate amount of
cytochrome c oxidase was added to initiate oxygen uptake.
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RESULTS |
DMPO Spin Trapping of CcO Thiyl Radial Formed by
H2O2--
When the purified CcO was reacted
with 5 eq (based on the amount of heme a and heme
a3 in CcO) of hydrogen peroxide in the presence
of DMPO, a broad four-line spectrum was obtained (Fig. 1A). The spectrum was totally
dependent on protein (Fig. 1E). However, the removal by
dialysis of the excess DMPO and any small radical adducts did not
affect the spectrum (data not shown), indicating that a protein-derived
radical(s) had been detected. To identify the DMPO adduct formed in
H2O2-treated CcO, the spectrum of Fig.
1A was analyzed by computerized simulation. Fig.
1B shows the simulated spectrum of a partially immobilized
nitroxide. This DMPO adduct can be assigned to a thiyl radical adduct
based on its hyperfine coupling constants (aN = 14.7 and a
H =15.7 G)
(30). In addition, the ESR spectrum from the reaction of
H2O2 with NEM-pretreated CcO was clearly
different from and weaker than that of native protein (Fig.
1C), indicating that sulfhydryl group(s) are necessary for
the radical adduct formation. A spectrum similar to that shown in Fig.
1E was obtained when either NEM,
H2O2, and DMPO or NEM-CcO and DMPO were present
in the reaction mixture (data not shown). The addition of NEM to the
DMPO/CcO thiyl radical adduct did not affect the spectral characteristics of Fig. 1A, thus ensuring that NEM does not
react with the CcO thiyl radical adduct (data not shown). To further confirm that the Fig. 1A spectrum is of a radical adduct of
a sulfur-centered radical, Ce4+ was used to selectively
oxidize the thiol residues of CcO, which were subsequently spin-trapped
with DMPO (31). The resulting partially immobilized ESR spectrum with
hyperfine coupling constants aN = 14.7 and
a
H = 15.7 G (Fig.
2A) is nearly identical to
that of native CcO treated with H2O2. The
spectrum observed in Fig. 2A was completely abolished when
the NEM-pretreated CcO was treated with Ce4+ (Fig.
2E).

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Fig. 1.
ESR spectrum obtained from the
reaction of cytochrome c oxidase with hydrogen
peroxide in the presence of DMPO. A, the purified CcO
(0.15 mM protein or 0.30 mM heme a)
in 50 mM sodium/potassium phosphate, pH 7.4, was mixed with
DMPO (100 mM). The reaction was initiated by adding
H2O2 (1.5 mM) to the mixture at
room temperature. 0.3 ml of the sample was withdrawn and submitted to
ESR measurement. B, computer simulation of the spectrum in
A with hyperfine constants aN = 14.7 and a H = 15.7 G. C, same as A, except the CcO was pretreated with
50 eq of NEM before H2O2 addition.
D, same as A, except the
H2O2 was omitted from the system. E,
same as A, except the enzyme was substituted with buffer.
F, same as A, except the DMPO was omitted from
the reaction mixture. Instrumental parameters were as follows:
modulation amplitude, 1 G; time constant, 1.3 s for A
and C, 327 ms for D-F; gain, 1 × 105; modulation frequency, 100 kHz; microwave frequency,
9.8 GHz; microwave power, 20 milliwatts.
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Fig. 2.
ESR spectrum detected from the reaction of
cytochrome c oxidase with Ce4+ in the
presence of DMPO. A, the initial reaction mixture
contained 3 mM ceric ammonium sulfate and 0.30 mM CcO. The ceric oxidation was allowed to proceed for 5 min at room temperature before the addition of DMPO. B,
computer simulation of the spectrum in A with hyperfine
constants aN = 14.7 and
a H = 15.7 G. C, the reaction of 3 mM ceric ammonium sulfate
with DMPO. D, the reaction of CcO with DMPO. E,
same as A, except CcO was pretreated with NEM before ceric
oxidation. The instrumental parameters were exactly the same as those
in Fig. 1, except the modulation amplitude was 2 G and the time
constant was 327 ms.
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Immobilized Protein-derived Radicals Formed by
CcO/H2O2 as Detected with MNP and
DBNBS--
An immobilized nitroxide was detected when the reaction of
CcO/H2O2 was performed in the presence of
either DBNBS or MNP (Figs. 3A
and 4A). As indicated in the spectra obtained from control experiments (Figs. 3, B and C, and
4, B and C), both
the MNP/·CcO and the DBNBS/·CcO adducts were dependent on
protein and H2O2. In the absence of
H2O2, the CcO reacted with MNP to yield an ESR
spectrum of a free radical with a coupling constant of
aN = aH = 14.4 G, which
is assigned to MNP/·H, a product of MNP reduction (Fig.
3B) (32). However, the addition of
H2O2 gave a broad ESR spectrum typical of an
immobilized nitroxide. The signal persisted after dialysis to remove
excess H2O2, MNP, and small radical adducts
(data not shown), demonstrating that a protein-bound radical adduct was
formed. When the MNP/·CcO-derived adduct was subjected to
nonspecific proteolysis, the intensity of immobilized nitroxide signal
decreased, and an isotropic three-line spectrum was observed with a
nitrogen hyperfine constant of 17.1 G, which is assigned to
di-t-butylnitroxide, a decomposition product of MNP (data
not shown).

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Fig. 3.
ESR spectrum obtained from the reaction of
cytochrome c oxidase with hydrogen peroxide in the
presence of MNP. A, the reaction mixture containing
0.15 mM CcO, 0.75 mM
H2O2, and 11 mM MNP was subjected
to ESR measurement. B, same as A, except
H2O2 was eliminated from the system.
C, same as A, except the CcO was replaced with
buffer. D, CcO was incubated with 10 eq of KCN at room
temperature for 5 min and then subjected to reaction with
H2O2 in the presence of MNP. The instrumental
settings were: modulation amplitude, 1 G; time constant, 0.66 s;
scan time, 655 s; receiver gain, 1 × 105;
microwave power, 20 milliwatts.
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Fig. 4.
ESR spectrum obtained from the reaction of
cytochrome c oxidase with hydrogen peroxide in the
presence of DBNBS. A, the system containing CcO (0.28 mM), DBNBS (0.28 mM), and
H2O2 (1.4 mM) was subjected to ESR
analysis with instrumental parameters described in the legend of Fig.
3. B, same as A, except the
H2O2 was omitted from the system. C,
same as A, except the CcO was replaced with the same amount
of buffer. D, same as A, except the CcO was
pretreated with 10 eq of KCN.
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Cyanide can form a low-spin ferric complex with CcO and inhibit the
enzymatic activity detected with cytochrome c oxidation and
oxygen consumption. Accordingly, adding 10 eq of potassium cyanide/heme
before adding H2O2 inhibits the heme-catalyzed
reaction and prevents the production of MNP and DBNBS adducts (Figs.
3D and 4D). A four-line ESR spectrum with
hyperfine coupling constants aN and
aH of 14.4 G from MNP/·H was obtained
(Fig. 3D). The addition of KCN to the protein-centered radical adducts of either MNP or DBNBS did not effect the spectra of
these radical adducts in any way (data not shown).
MNP Spin Trapping of a Protein-derived Tyrosyl Radical from the
Reaction of NEM-CcO with H2O2
To evaluate
whether the MNP/·CcO and DBNBS/·CcO adducts have
properties similar to the DMPO/·CcO adduct, we pretreated the
CcO with 50 eq of NEM at room temperature to modify the cysteine
residues on the surface of CcO. As indicated in Figs.
5A and
6A, the ESR spectral
characteristics of DBNBS/·CcO and MNP/·CcO were
significantly changed when the NEM-CcO was incubated with
H2O2. The addition of NEM to preformed
MNP/·CcO or DBSBS/·CcO did not effect the spectra of
these radical adducts in any way (data not shown). These results are
consistent with the spin-trapping results obtained with DMPO, where a
sulfur-centered radical is formed in the oxidation of CcO by
H2O2. The
H2O2-dependent, persistent, immobilized nitroxides were detected in the reaction systems of NEM-CcO/H2O2/DBNBS and
NEM-CcO/H2O2/MNP (Fig. 5, A and
C, and Fig. 6, A and B). When the
sample from either Fig. 5A or 5C was submitted to
Pronase digestion, a species with a three-line ESR spectrum was
observed (Fig. 5D). The hyperfine coupling constant of this
proteolytic product is 13.6 G (as indicated by arrows in
Fig. 5D), which is similar to that reported for proteolyzed DBNBS/·metmyoglobin and DBNBS/·cytochrome c
(17, 33, 34). It should be noted that the spin adduct from the
proteolytic product of DBNBS/·NEM-CcO is relatively unstable.
The intensity of the ESR signal (Fig. 5D) decayed during the
process of scanning and Pronase digestion. Attempts to obtain a clear,
three-line spectrum by proteolysis were unsuccessful.

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Fig. 5.
ESR spectrum obtained from the reaction of
NEM-pretreated cytochrome c oxidase with hydrogen
peroxide in the presence of DBNBS. A, the purified CcO
(0.30 mM) was incubated with 50 eq of NEM at room
temperature for 10 min. The NEM-pretreated CcO was then reacted with
1.5 mM H2O2 in the presence of 300 µM DBNBS before being subjected to ESR measurement.
B, the reaction mixture was the same as in A,
except the hydrogen peroxide was omitted. C, the sample
shown in A was recovered from the flat cell and dialyzed
against 100 ml of 50 mM sodium/potassium phosphate buffer
containing 1.0% sodium cholate, pH 7.4, for 2 h before the ESR
spectrum was re-recorded. The dialysate was concentrated with a
Centricon 30 microconcentrator and brought to the same volume as in
A, prior to ESR analysis. D, same as
C, except 10 mg/ml Pronase was added to the sample. The
hyperfine coupling constant, aN = 13.6 G, is
indicated by arrows. The instrumental parameters were the
same as described in the legend to Fig. 3, except the modulation
amplitude is 2 G.
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Fig. 6.
ESR spectrum obtained from the reaction of
NEM-pretreated cytochrome c oxidase with hydrogen
peroxide in the presence of MNP. A, the CcO was
pretreated with NEM as described in the legend to Fig. 5. The sample
for ESR determination contained 0.15 mM NEM-CcO, 0.75 mM H2O2, and 11 mM MNP.
The species with broad line widths are marked with dashed
lines and the others, with sharper line shapes, are indicated by
solid lines. B, sample A was recovered
from the flat cell and dialyzed against 100 ml of 50 mM
sodium/potassium phosphate buffer containing 1.0% sodium cholate, pH
7.4, for 2 h before the ESR spectrum was re-recorded.
C, same as A, except the sample was treated with
10 mg/ml Pronase for 6 h at room temperature. D, same
as A, except the NEM-CcO was pretreated with 10 eq of KCN
for 5 min at room temperature. The instrumental parameters were the
same as described in the legend to Fig. 3, except the modulation
amplitude was 2 G for spectrum B.
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A protein-derived radical adduct was also detected in the
NEM-CcO/H2O2/MNP system (Fig. 6A).
In the ESR spectrum of Fig. 6A, two MNP adducts were
generated when the NEM-pretreated CcO reacted with
H2O2. One species (as indicated by the
solid lines in Fig. 6A) with a sharper ESR line
shape is less immobilized than the other species (as indicated by the
dashed lines) with a broad low-field extrema. However,
nonspecific proteolytic treatment (up to 6 h) of the sample from
Fig. 6A or 6B can gradually remove the species
with the broad line width and yield an isotropic three-line spectrum
with a hyperfine coupling constant of aN = 15.6 G (Fig. 6C), which is basically similar to those observed in
MNP/·metmyoglobin and MNP/·cytochrome c (17,
33, 34). The inclusion of potassium cyanide (up to 10 eq of NEM-CcO) in
the system drastically inhibited the signal of the heme-catalyzed
immobilized nitroxide, presumably because of its ability to form a
ligand at the sixth position (Fig. 6D). However, KCN did not
significantly change the spectral characteristics when the
H2O2-dependent, protein-centered
radical adduct of NEM-CcO had been previously formed (data not shown).
The isotropic three-line spectrum observed upon proteolysis of
MNP/·NEM-CcO indicates that the atoms
and
of the
nitroxide had no nuclear spin (i.e. I = 0)
and, therefore, that the protein-centered radical was located on a
tertiary carbon. In the previous investigation, Gunther et
al. (34) were able to use a low modulation amplitude to resolve
the super-hyperfine structure from the DBNBS/·metmyoglobin
adduct. Indeed, they concluded that the globin-derived radical trapped
by DBNBS was centered on the C-3 of the tryptophan residue in
metmyoglobin by comparison with the pure DBNBS/·Trp adduct. In
another report, Barr et al. (17) extended this technique and
used perdeuterated MNP (i.e. MNP-d9)
and carbon-13 labeling of the aromatic ring positions of tyrosine to
identify MNP/·cytochrome c-derived adducts (17). We
employed the same technique here, using MNP-d9
to address the structure of the MNP/·NEM-CcO adduct generated
with H2O2. Because the hyperfine structure from
the nine methyl hydrogens broadens the ESR spectrum of the MNP adduct,
it can prevent the resolution of super-hyperfine structure. The fully
deuterated MNP decreases this effect and increases the resolution of
the spectrum. A three-line spectrum with a similar hyperfine coupling
constant (aN = 15.5 G) was detected in the
MNP-d9/·NEM-CcO/H2O2
system (data not shown). To resolve the hyperfine structure, an 8-G
scan of the low-field line from this preliminary spectrum coupled with
modulation amplitude as low as 0.25 G was chosen to record a
super-hyperfine spectrum, which basically consisted of five resolved
lines. Fragmentation of the protein by proteolysis to form freely
rotating peptides gave a well resolved super-hyperfine structure (Fig.
7A). Fig. 7B is the
simulated spectrum using three nonequivalent hydrogens
(aH = 1.40, aH = 0.90, and aH = 0.69 G). The super-hyperfine structure
obtained from proteolytic MNP-d9/·NEM-CcO
was similar to that of authentic
MNP-d9/·Tyr formed by oxidation of the
free amino acid by horseradish peroxidase as reported by Gunther
et al. (33). A trace amount of another
MNP-d9 adduct was detected on the low-field side
of the major spectrum shown in Fig. 7A (as indicated by
asterisks), which was believed to be caused by incompletely
hydrolyzed peptides that remain partially immobilized. The intensity of
this more immobilized adduct gradually decreased when the incubation
time for proteolysis was prolonged. Proteases have been shown to be incapable of complete digestion of membrane proteins because of the
high hydrophobicity of the transmembrane-spanning helices (35).
Inclusion of SDS (up to 0.2%) and 2 M urea did not improve proteolysis efficiency.

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Fig. 7.
Super-hyperfine ESR spectrum obtained from
the reaction of NEM-pretreated cytochrome c oxidase
with hydrogen peroxide in the presence of
MNP-d9. A, the spectrum was
acquired using the same sample described in the legend to Fig.
6A, except 11 mM MNP-d9
was used as the spin trap. The instrumental settings were as follows:
modulation amplitude, 0.25 G; time constant, 5.3 s; scan time,
2684 s; receiver gain, 1 × 105; microwave power,
20 milliwatts. The incompletely digested adduct is identified by
asterisks. B, the simulated spectrum was
calculated using the hyperfine coupling constants
aH = 1.40, aH = 0.90, and
aH = 0.69 G.
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DISCUSSION |
In the current investigation, our ESR spin-trapping results
demonstrate that the heme-catalyzed reaction of native CcO with H2O2 gave a sulfur-centered radical that was
identified by spin trapping with DMPO. When the protein was pretreated
with thiol-blocking reagent, a carbon-centered radical was detected in
the reaction of NEM-CcO with H2O2. Presumably,
the cysteine residue of CcO was not the initial site for oxidative
damage, but a ferryl porphyrin cation radical (compound I of the
peroxidases) was probably first generated in the reaction of CcO with
H2O2. This reactive intermediate then oxidized
the thiol group(s) of CcO to form sulfur-centered radical adduct(s). As
suggested by Prütz (36, 37), cysteine can act as a "sink,"
being an important terminus for the cascading transfer of radical
centers in proteins. Similar results were also found in the hemoglobin
and bovine serum albumin when the oxidative damage of protein was
initiated with an hydroxyl radical attack (38). The protective role of
thiol group(s) in CcO can be further supported by two events observed
in this study. First, direct oxidation of CcO with Ce4+ led
to the trapping of a thiyl radical but not a carbon-centered radical,
as illustrated in Fig. 2A. Second, both Ce4+
oxidation and NEM modification had no effect on the enzymatic activity
of CcO (data not shown). As revealed by the recently determined high
resolution, three-dimensional structure of CcO, cysteine residues were
found primarily on the surface of protein and were logically involved
in this shielding effect (39). However, it may not be ruled out that
oxidative damage can be thermodynamically transferred from thiyl
radical intermediates to carbon-centered radicals as demonstrated by
Prütz et al. (40). Thiyl radicals are reactive species
capable of addition even across carbon-carbon double bonds and
possibly capable of initiating lipid peroxidation (41, 42).
The elimination of the DMPO/thiyl radical adduct with NEM gave the
spectrum shown in Fig. 1C, which is weak and apparently results from another protein-derived radical. In a like manner, NEM
also inhibited the thiyl radical formation from the direct oxidation of
CcO with Ce4+. In addition, the spectral characteristic was
also greatly changed when either DBNBS or MNP was used as the spin trap
in the reaction system of NEM-CcO/H2O2. It is
clear that one or more thiols are involved in the reaction between CcO
and H2O2. It is noteworthy that the MNP adduct
of NEM-CcO is more easily fragmented with Pronase treatment than is the
adduct of native CcO, and this observation is in line with the
sheltering effect of sulfhydryl groups in CcO, as suggested previously.
Earlier reports indicated that an immobilized nitroxide was detected in
the reaction of other heme proteins such as myoglobin and cytochrome
c with H2O2 when either DBNBS or MNP
was included in the reaction mixture (17, 34). These protein-derived,
immobilized nitroxides have been unambiguously identified as,
respectively, the tryptophanyl radical of metmyoglobin and the tyrosyl
radical of cytochrome c by resolving the low-field
super-hyperfine structures of their radical adducts. Likewise, an
analogous, immobilized nitroxide was also observed when the
NEM-pretreated CcO was reacted with H2O2 in the
presence of either of the nitroso spin traps. The trapped radical site
was located on a tertiary carbon because a three-line ESR spectrum was
detected after nonspecific proteolysis of either the DBNBS- or
MNP/·NEM-CcO-derived adducts. Tryptophan and tyrosine are two
likely amino acid residues that could form radical adducts and produce the triplet spectra observed in this study. In fact, the
aN values calculated here for the
DBNBS/·NEM-CcO-derived and MNP/·NEM-CcO-derived adducts
were very close to those reported for the corresponding adducts of
metmyoglobin and cytochrome c (17, 34).
The ESR spectrum of the DBNBS/·NEM-CcO disclosed a stable
adduct. The proteolysis of the DBNBS/·NEM-CcO-derived sample
yielded an unstable spin adduct with a three-line spectrum, raising the
possibility that DBNBS was trapping a tyrosyl radical. However, it is
unlikely for this short-lived species to be identified by resolving its
super-hyperfine structure, because of its instability.
Because both the tryptophan and tyrosine radical adducts with MNP have
similar primary aN values (17, 33, 34), the
super-hyperfine structure was necessary to identify which MNP adduct
was formed in the reaction of NEM-CcO with
H2O2. We were able to address this issue by
comparing the high resolution ESR spectrum from the
MNP-d9/·NEM-CcO-derived adduct with that
from the authentic MNP-d9/·Tyr adduct.
The fact that both resolved spectra at low field are very similar, if
not identical, served as indisputable proof that the tyrosyl radical
was trapped by MNP in the peroxidation of NEM-CcO with
H2O2. The formation of a tyrosyl radical
intermediate in the thiol-blocked CcO can be explained in a parallel
way to those in metmyoglobin and cytochrome c following
their reaction with H2O2 (17, 33). That is,
hydrogen peroxide was reduced to water, forming a compound I-like
ferryl porphyrin cation radical, which subsequently accepted an
electron from the tyrosine residue, forming a ferryl tyrosyl radical
species from NEM-CcO. It is also possible that ferryl-oxo compound II
could oxidize the amino acids in the vicinity of heme and subsequently
produce a tyrosyl radical. However, in the case of native CcO, the
cysteine residues were available and acted as a sink in an
electron-transfer process that occurred in the protein following the
reaction of heme a (or a3) with
H2O2. In fact, a ferryl-oxo species from the
reaction of dioxygen with heme a3 also had been
proposed based on resonance Raman spectroscopic studies (25, 43). It
has been suggested by Proshlyakov et al. (44-46) that the
detected ferryl-oxo intermediates correspond to the so-called compound
P and compound F, which are involved in the catalytic mechanism of CcO.
Compound P and compound F are equivalent to compound I and compound II,
respectively, of the peroxidases if the oxygen chemistry (such as
homolytic cleavage of the O-O bond of peroxide species) is similar to
that of the peroxidase (22-24). Furthermore, recent works have
demonstrated that the addition of hydrogen peroxide to the fully
oxidized CcO can result in formation of the same compound P and
compound F species (44-46). The tyrosyl radical is known to have a
role in the electron transfer events in Photosystem II (47, 48),
prostaglandin synthase (49, 50), and ribonucleotide reductase (51).
Circumstantial evidence suggests that the participation of the tyrosyl
radical in the enzyme turn-over of CcO is possible (24). As revealed by
the implication of the recently reported x-ray structure of CcO (52,
53) and by the results of mutagenesis studies (54), a highly conserved
tyrosine (Tyr-244 in bovine oxidase, Tyr-280 in
Paracoccus oxidase) is located at the active site of CcO
(24). Elucidation of the possibly catalytic role of Tyr-244 using the ESR spin-trapping technique is currently under investigation in our laboratory.
 |
FOOTNOTES |
*
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: Laboratory of
Pharmacology and Chemistry, NIEHS, NIH, P. O. Box 12233, Research Triangle Park, NC 27709. Tel.: 919-541-1501; Fax: 919-541-1043; E-mail:
chen6{at}niehs.nih.gov.
The abbreviations used are:
CcO, cytochrome
c oxidase; NEM, N-ethylmaleimide; DMPO, 5,5-dimethyl-1-pyrroline N-oxide; MNP, 2-methyl-2-nitrosopropane; DBNBS, 3,5-dibromo-4-nitrosobenzenesulfonic acid.
 |
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