From the
The mechanism for the reaction of cytochrome c with
t-butyl hydroperoxide and cumene hydroperoxide was
investigated. ESR spin trapping studies using 5,5-dimethyl-1-pyrroline
N-oxide were performed to demonstrate the presence of
hydroperoxide-derived peroxyl, alkoxyl, and methyl radicals. Computer
simulation of the experimental data obtained at various
5,5-dimethyl-1-pyrroline N-oxide concentrations was used to
determine the relative contributions of each radical adduct to each
composite ESR spectrum. From these analyses, it was concluded that the
alkoxyl radical of the hydroperoxide was the initial radical produced,
presumably by homolytic scission of the O-O bond by ferric
cytochrome c. This was in contrast to a previous ESR study
that proposed a heterolytic peroxidase-type mechanism for the reaction
of cytochrome c with organic hydroperoxides. Methyl radicals
were produced from the
Cytochrome c is an intermembrane mitochondrial heme
protein that functions in electron transport by shuttling electrons
from cytochrome c reductase to the cytochrome c oxidase complex. There is one protoporphyrin IX heme unit/molecule
of cytochrome c, and the iron cycles between the ferric and
ferrous oxidation states in the native form
(1) . A histidine
nitrogen provides the fifth ligand for the heme iron, while the sixth
position is occupied by a sulfur ligand from
methionine
(2, 3, 4) . Thus, in the native form,
cytochrome c is hexacoordinate. However, the methionine ligand
is readily displaced by the presence of an alternate ligand or even
subtle changes in temperature, pH, or ionic strength (3). It has been
proposed that the loss of the methionine ligand results in the loss of
the electron transport capability of cytochrome c(4) .
In addition to electron transport, it has been suggested for several
decades that cytochrome c might be involved in the
hydroperoxide-dependent initiation of free radical-mediated lipid
peroxidation and protein
damage
(5, 6, 7, 8, 9, 10) .
Recently, ESR spectroscopy has been used to demonstrate that several
heme proteins, including cytochrome c, can react with organic
hydroperoxides to produce free
radicals
(11, 12, 13, 14, 15) .
Kalyanaraman et al.(11) reported the direct ESR
detection of peroxyl radicals from the reaction of hematin with
t-butyl hydroperoxide and cumene hydroperoxide. In separate
studies
(13, 14) , the ESR spin trapping technique was
employed to demonstrate that peroxyl, alkoxyl, and carbon-centered
radicals are produced from the reaction of various heme proteins
(including cytochrome c) with organic hydroperoxides. In such
studies, a spin trap such as 5,5-dimethyl-1-pyrroline N-oxide
(DMPO)
Based on ESR spectra and other data, several mechanisms
have been postulated for radical production involving the reaction of
heme proteins with organic hydroperoxides. A peroxidase-type mechanism
is one possibility
(11, 14) . In this case, the ferric
form of cytochrome c would heterolytically cleave the peroxide
O-O bond, and the organic hydroperoxide would be reduced to its
corresponding alcohol. Also, a ferryl radical form of cytochrome c (i.e. compound I) would result that could abstract a
hydrogen from another molecule of hydroperoxide, thus producing a
peroxyl radical. Alternatively, the ferric cytochrome c could
cleave the peroxide O-O bond homolytically, which would produce
an alkoxyl radical. Tappel
(16) has suggested that this might
occur without a valence change of the heme iron. Instead, an iron-bound
hydroxyl radical may be formed
(16) . Yet another explanation
invokes a cytochrome c-catalyzed Haber-Weiss reaction, in
which the organic hydroperoxide replaces hydrogen
peroxide
(17, 18, 19) .
It has been concluded
from recent ESR investigations that production of radicals by the
cytochrome c/organic hydroperoxide system occurs via the
peroxidase-type mechanism
(13) . The results reported here do not
support these previous conclusions. Instead, we have provided ESR spin
trapping evidence that indicates that the alkoxyl radical is the
primary radical released into solution by the reaction of cytochrome
c with an organic hydroperoxide. The implications of this
finding with regard to the aforementioned mechanisms and the free
radical-mediated lipid peroxidation and protein damage of the
cytochrome c/organic hydroperoxide system are discussed.
The ESR spectrum recorded from a reaction mixture containing
cytochrome c, t-butyl hydroperoxide, and DMPO
revealed a mixture of four DMPO radical adducts (Fig. 1) and was
similar to what has been reported previously
(13) . The computer
simulation of the spectrum obtained using 22.5 mM DMPO was
superimposed on the experimental spectrum (Fig. 1A).
From their respective hyperfine coupling constants, the adducts were
assigned as peroxyl (methyl peroxyl; see below) (a
The relative contributions of the peroxyl and alkoxyl
adducts to the composite ESR spectrum were influenced by the
concentration of DMPO (Fig. 2, A, C, and
E). Qualitatively, one can see that as the DMPO concentration
was increased, the relative contribution of the peroxyl radical adduct
decreased, while that of the t-butyloxyl radical increased. In
the absence of cytochrome c, a composite spectrum with greatly
reduced intensity was observed (Fig. 2, B, D,
and F). The results were similar when cumene hydroperoxide
replaced t-butyl hydroperoxide in the DMPO spin trapping
experiments (Fig. 3). The computer simulation fit well with the
experimental data (Fig. 3A), and the presence of each
radical adduct can clearly be seen in the spectral deconvolution
(Fig. 3, B-E). In the case of cumene
hydroperoxide, the DMPO adducts were assigned as peroxyl (methyl
peroxyl; see below) (a
The reactions of the initial radical that occur
subsequent to its production can be suppressed by increasing the
concentration of the spin trap. Thus, as more spin trap is added, more
of the initial radical is trapped. This prevents the initial radical
from undergoing reactions with other species or itself that lead to
secondary radicals. By doing this, one can determine which radical is
the primary or initial radical in a multiple species ESR spectrum. This
method was used previously by Ledwith et al.(28) . We
used this strategy to determine whether the peroxyl or the alkoxyl
radical was the initial radical produced by the reaction of cytochrome
c with these organic hydroperoxides. To do this, the relative
contributions of the DMPO adducts were measured at various DMPO
concentrations. In these experiments, the relative concentrations of
the radical adducts as represented by the low-field lines were
calculated using computer simulation. To eliminate the possible
contribution of non-cytochrome c-catalyzed radical production
(e.g. seen in Fig. 2, B, D, and
F), parallel experiments were performed in the absence of
cytochrome c at each DMPO concentration. Spectral subtractions
were then performed, thus removing the contribution of cytochrome
c-independent radical production. It can clearly be seen that
as the DMPO concentration was increased between 22.5 and 360
mM, the contribution of the peroxyl radical to the composite
ESR spectrum decreased significantly (i.e. from
The same experiment was performed with cumene hydroperoxide, and a
similar result was found (Fig. 7). As the DMPO concentration was
increased between 45 mM and 720 mM, the peroxyl
radical adduct decreased from 86 to 0%, while the cumyloxyl radical
adduct increased from 10 to >90%. The contributions of the methyl
radical and DMPOX adducts remained minimal (i.e. <10%)
throughout the range of DMPO concentrations.
The peroxyl radical
adduct detected in the preceding experiments could have arisen from one
or all of the following reactions.
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy Because of the spectroscopic ambiguity in the ESR of peroxyl
radical adducts, it is not possible to differentiate between the methyl
peroxyl radical adduct and tertiary peroxyl radical adducts. Thus,
Reactions 1-3 are all valid possibilities. To determine if the
peroxyl radical was being produced by Reaction 1, experiments were
performed in the absence of oxygen (Fig. 8). A low DMPO
concentration was used to maximize the intensity of the peroxyl radical
adduct. It can be seen with both hydroperoxides that when oxygen was
removed from the system, the signal intensity of the peroxyl radical
adduct diminished almost completely. In addition, the intensity of the
methyl radical adduct increased significantly. It is possible to detect
tertiary peroxyl radicals using direct ESR
(11) . However, under
our experimental conditions, peroxyl radicals were not detected via
direct ESR.
We also studied the spectral changes that occurred in
the Soret and visible absorption bands of cytochrome c during
its reaction with t-butyl hydroperoxide and cumene
hydroperoxide. Ferricytochrome c has a maximum Soret
absorbance at 410 nm as well as another maximum absorbance at 528 nm
(29). When cytochrome c is converted to the ferrous form, the
Soret band is slightly red-shifted (i.e. 416 nm), and
prominent
The mechanism by which radicals are produced from the
reaction of organic hydroperoxides with heme proteins has been the
subject of considerable debate. The complexity in interpreting these
data arises from the fact that a multitude of reactions can occur
subsequent to the reaction of the heme iron with the hydroperoxide. For
example, the peroxidase mechanism predicts that the peroxyl radical
would be produced by the reaction of t-butyl hydroperoxide
with cytochrome c (Cytc).
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy Thus, in this case, the hydroperoxide would undergo a 2-electron
reduction, in a heterolytic manner, to its corresponding alcohol, and
the cytochrome c would be oxidized to a compound I-type
species (Reaction 4). The resulting compound I could then abstract
hydrogen from the organic hydroperoxide, producing a peroxyl radical
and the ferryl form of cytochrome c (Reaction 5). The well
established self-reaction of tertiary peroxyl radicals
(30, 31) would result in the formation of two alkoxyl radicals and
molecular oxygen (Reaction 6). Subsequent unimolecular
To explain
how all three of these radical adducts were observed in the ESR spin
trapping experiments, we have considered the following reaction
sequence (t-butyl hydroperoxide has been used for
demonstration here, but an analogous reaction sequence could be
inferred for cumene hydroperoxide).
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy In this reaction sequence, cytochrome c would catalyze the
homolytic scission of the peroxide O-O bond (Reaction 8),
possibly forming a ferryl form of cytochrome c. The
The spin trapping reaction,
On-line formulae not verified for accuracy (which has a rate constant of 4
Cadenas et
al.(7) demonstrated singlet oxygen production from the
reaction of cytochrome c with organic hydroperoxides. They
concluded that cytochrome c catalyzed the homolytic scission
of the hydroperoxide O-O bond and proposed that the
chemiluminescence observed was due to the self-terminating reaction of
primary peroxyl radicals that were derived from t-butyl
alkoxyl radicals. It is likely that the methyl peroxyl radical would
undergo a self-terminating reaction, producing singlet oxygen.
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy The problem with making a mechanistic conclusion based only on
chemiluminescence data is that the methyl peroxyl radicals could also
have been derived from the peroxidase mechanism (i.e. Reactions 4-7). However, the spin trapping data presented
here seem to rule out the possibility that the peroxidase mechanism is
occurring with cytochrome c.
Another possible mechanism put
forth for the reaction of cytochrome c with hydroperoxides
involves a Haber-Weiss-type reaction
sequence
(17, 18, 19) .
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy This implies that the hydroperoxide is first oxidized by 1 electron
to the peroxyl radical, with the concomitant reduction of cytochrome
c. Ferrous cytochrome c can then react with another
molecule of hydroperoxide in a Fenton-type reaction (i.e. Reaction 14). Once again, Reactions 13 and 14 would dictate that
the peroxyl radical be produced initially, followed by the formation of
the alkoxyl radical. Thus, if Reactions 13 and 14 had occurred, one
would predict that as the DMPO concentration was increased, both the
peroxyl and alkoxyl radical adducts would have increased simultaneously
(which was not the case). In addition, no spectrophotometric evidence
for ferrous cytochrome c was obtained. Therefore, we concluded
that radical production via Reactions 13 and 14 was unlikely. It has
also been reported that iron release from cytochrome c might
be the cause for radical production (18). However, the absence of an
effect of EDTA or DTPA on the observed ESR signals in our cytochrome
c/hydroperoxide reaction systems seemed to rule out this
possibility. Also, the addition of cyanide reduced the intensity of the
ESR signals to the level seen in the control experiments. This
suggested that radical production required the interaction of the heme
iron with the hydroperoxide.
The question as to the fate of the
ferryl cytochrome c (which was used to balance Reaction 8)
remains unanswered. One might expect that it would abstract a hydrogen
from the hydroperoxide to produce a peroxyl radical and ferric
cytochrome c.
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy In this case, the peroxyl and alkoxyl radical adducts would have
increased simultaneously as the concentration of DMPO was increased,
which was not observed. It is possible that the bleaching of cytochrome
c observed during the reaction was due to an attack of the
ferryl iron on the protein. The fact that cyanide inhibited the
bleaching indicated that an interaction of the hydroperoxide with the
heme iron (i.e. inner sphere electron transfer) was required
for protein damage to occur. In addition, the fact that DMPO did not
prevent bleaching suggested that the protein damage was not caused by
diffusible hydroperoxide-derived radicals. However, more experimental
data are required to address this question.
We believe that the ESR
data presented here provide strong evidence for the homolytic scission
of the O-O bond by cytochrome c initially producing
alkoxyl radicals. The peroxyl radicals detected in these as well as
previous ESR studies likely arise from Reaction 1 and, possibly,
Reactions 2 and 3. The results have relevance with regard to heme
protein-catalyzed lipid peroxidation and/or protein damage. Both
peroxyl and alkoxyl radicals are capable of abstracting allylic
hydrogens and thus of initiating lipid peroxidation. However, allylic
hydrogen abstraction by alkoxyl radicals is more favorable. This is
because the bond energy for the corresponding alcohol produced from
hydrogen abstraction by an alkoxyl radical (
We thank Dr. Krzysztof Reszka for helpful discussions
and for suggesting the anaerobic experiments.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-scission of the alkoxyl radical. The
peroxyl radicals are shown to be secondary products formed from the
reaction of oxygen with the methyl radical to produce the methyl
peroxyl radical. In separate experiments, visible absorption
spectroscopy revealed that the heme center was destroyed during the
reaction. Both the heme destruction and production of radical adducts
were inhibited by cyanide, presumably due to the formation of a
cyanoheme complex.
(
)
is added to a reaction mixture
containing the heme protein and hydroperoxide. The resulting ESR
spectrum reveals a mixture of radical adducts that can be identified by
their characteristic nitrogen and hydrogen hyperfine coupling
constants.
Chemicals
Horse heart cytochrome c was
purchased from Boehringer Mannheim and used as received. DMPO, DTPA,
EDTA, t-butyl hydroperoxide (t-BuOOH), and Desferal
(desferrioxamine mesylate) were all obtained from Sigma. DMPO was
vacuum-distilled at room temperature twice before use. Potassium
cyanide and cumene hydroperoxide were obtained from Aldrich. Cumene
hydroperoxide was purified by preparing the sodium salt according to
the method of Mair and Hall
(20) . The buffers and reagents were
prepared in deionized water and were treated with Chelex 100 (Bio-Rad).
ESR Spin Trapping Experiments
The ESR spectra were
recorded using a Bruker ECS-106 spectrometer operating at 9.77 GHz with
a 50-kHz modulation frequency and a TM cavity. In the
experiments shown in Fig. 1-4, the reactions were initiated
by the addition of the hydroperoxide and pipetted into the flat cell.
The time delay before the spectra were recorded was
1.5 min. In
the experiments shown in Fig. 6and Fig. 7, the reaction
mixtures were introduced to the flat cell by means of aspiration (with
a dead time of only
4 s) using a modified Gilford rapid sampling
device
(21) . This was done to minimize the possible decay of
radical adducts during the DMPO-dependence studies. All ESR experiments
were performed in 100 mM sodium phosphate, pH 7.4, at room
temperature. The reactions were initiated by the addition of the
hydroperoxide. For the anaerobic experiments shown in Fig. 8, the
oxygen was removed by nitrogen purging, as described
previously
(21) , using a bottle equipped with a rubber septum
and syringe needles. The solution was purged for 10 min, after which
glucose (10 mM) and glucose oxidase (100 units) were added.
The reactions were initiated by the simultaneous addition of cytochrome
c and DMPO. In both the aerobic and anaerobic experiments
shown in Fig. 8, the mixture was introduced to the flat cell
using the rapid sampling device.
Figure 1:
Computer simulation
and deconvolution of the ESR spectrum obtained from the reaction
mixture containing cytochrome c and t-BuOOH. A is the computer simulation (dashedline)
superimposed on the experimental spectrum obtained using 20
µM cytochrome c (Cytc), 12.5 mMt-butyl hydroperoxide, and 22.5 mM DMPO.
B-E are the individual simulations of each species in
the composite spectrum. The hyperfine values used for each species are
provided under ``Results.'' B is the simulated
spectrum for DMPO/OOR: line width, 0.70 G; line shape, 44% lorentzian,
56% gaussian; and mole ratio, 0.70. C is the simulated
spectrum for DMPO/OC(CH)
: line width, 0.75 G;
line shape, 42% lorentzian, 58% gaussian; and mole ratio, 0.22. D is the simulated spectrum for DMPO/CH
: line width,
0.70 G; line shape, 18% lorentzian, 82% gaussian; and mole ratio, 0.04.
E is the simulated spectrum for DMPOX: line width, 0.45 G;
line shape, 49% lorentzian, 56% gaussian; and mole ratio,
0.04.
Figure 6:
Effect of
the DMPO concentration on the relative contributions of each radical
adduct to the composite ESR spectrum obtained from the reaction of
cytochrome c with t-butyl hydroperoxide. All reaction
mixtures contained 20 µM cytochrome c, 12.5
mMt-butyl hydroperoxide, and 100 mM
phosphate buffer, pH 7.4. The DMPO concentrations used were 22.5, 45,
112, 135, 158, 180, and 360 mM. , DMPO/OOR;
,
DMPO/OC(CH
)
;
, DMPO/CH
;
, DMPOX.
Figure 7:
Effect of the DMPO concentration on the
relative contributions of each radical adduct to the composite ESR
spectrum obtained from the reaction of cytochrome c with
cumene hydroperoxide. All reaction mixtures contained 20
µM cytochrome c, 25 mM cumene
hydroperoxide, and 100 mM phosphate buffer, pH 7.4. The DMPO
concentrations used were 45, 90, 135, 158, 180, 270, 360, and 720
mM. , DMPO/OOR;
,
DMPO/OC(CH
)
C
H
;
,
DMPO/CH
;
, DMPOX.
Figure 8:
Effect of oxygen on the production of
radical adducts from the reaction of cytochrome c with
t-BuOOH and cumene hydroperoxide. A is the spectrum
obtained in the presence of oxygen from a reaction mixture containing
20 µM cytochrome c, 25 mM cumene
hydroperoxide (CumOOH), and 20 mM DMPO
(CumOOH/Air). B is the spectrum obtained when the
system was purged of oxygen using nitrogen bubbling and using the
conditions from A (CumOOH/N). C is the spectrum obtained in the presence of oxygen from a reaction
mixture containing 20 µM cytochrome c, 12.5
mMt-butyl hydroperoxide, and 20 mM DMPO
(t-BuOOH/Air). D is the spectrum obtained when the
system was purged of oxygen using nitrogen bubbling and using the
conditions from C (t-BuOOH/N
). The
spectrometer conditions were the same as indicated in the legend of
Fig. 2.
Computer Simulations
The computer simulations were
performed using a program developed by David Duling at NIEHS that is
available to the public through Internet. The details of this program
were the subject of a recent publication
(22) . The parameters
that were used for simulating the experimental ESR spectra are
described in the figure legends. The data points in Fig. 6and
Fig. 7
were obtained by simulating the low-field lines of each
species in spectra that were obtained using rapid sampling. The
component spectra were then double-integrated to give relative radical
adduct concentrations. Experiments using each DMPO concentration were
performed both in the presence and absence of cytochrome c.
Prior to computer simulation, the spectra obtained in the absence of
cytochrome c were subtracted from those obtained in its
presence. Thus, the contribution of the cytochrome
c-independent species was eliminated.
Visible Absorption Spectroscopy
The spectral
changes of cytochrome c were recorded using a multichannel
photodiode on a Hewlett-Packard 8415A diode array spectrophotometer.
The reactions were initiated by the addition of the hydroperoxide and
were performed in 100 mM sodium phosphate, pH 7.4, at room
temperature. Further details of these experiments are provided in the
figure legends.
= 14.45 G, a
=
10.75 G, and a
= 1.35 G),
t-butyloxyl (a
= 14.9 G and
a
= 16.2 G), methyl
(a
= 16.4 G and
a
= 23.4 G), and
5,5-dimethylpyrrolidone-2-(oxy)-(1) (DMPOX) (a
= 7.25 G and a
(2H)
= 4.05 G). The hyperfine values appeared to be consistent with
the values reported in previous literature
(14) . DMPOX is an
oxidation product of DMPO that is thought to arise from the
decomposition of DMPO peroxyl radical adducts
(17) .
Deconvolution of the composite spectrum was performed by simulating
each species separately using the relative intensities and hyperfine
values obtained from the composite computer simulation (Fig. 1,
B-E). Inclusion of the DMPO hydroxyl radical adduct in
the composite simulation did not improve the quality of the fit
(i.e. the correlation coefficient did not increase). In
addition, if t-butyl hydroperoxide was replaced by hydrogen
peroxide, only DMPOX, not the DMPO hydroxyl radical adduct, was
detected.
= 14.45 G,
a
= 10.70 G, and
a
= 1.35 G), cumyloxyl
(a
= 14.75 G and
a
= 15.84 G), methyl
(a
= 16.5 G and
a
= 23.35 G), and DMPOX
(a
= 7.25 G and
a
(2H) = 4.05 G). These
hyperfine coupling values were similar to those reported by
Davies
(13) . Once again, as DMPO was increased, the contribution
of the peroxyl radical adduct to the composite spectrum was diminished,
while the cumyloxyl radical adduct contribution increased
(Fig. 4, A, C, and E). The control
experiments in the absence of cytochrome c are shown at each
DMPO concentration (Fig. 4, B, D, and
F).
Figure 2:
Composite ESR spectrum of DMPO radical
adducts from incubations of cytochrome c and t-BuOOH
at various DMPO concentrations. A is the ESR signal obtained
from a reaction mixture containing 20 µM cytochrome c (Cytc), 12.5 mMt-butyl hydroperoxide,
and 22.5 mM DMPO (i.e. the same as seen in Fig.
1A). B is the spectrum obtained using the conditions
from A, except cytochrome c was omitted. C is the spectrum obtained when the DMPO concentration was increased
to 90 mM. D is the spectrum obtained using the
conditions from C, except cytochrome c was omitted.
E is the spectrum obtained when the DMPO concentration was
increased to 360 mM. F is the spectrum obtained using
the conditions from E, except cytochrome c was
omitted. The spectrometer conditions were as follows: modulation
amplitude, 0.5 G; microwave power, 20 milliwatts; time constant, 0.69
s; scan time, 600 s; scan range, 100 G; and receiver gain, 2.5
10
for A-D and 1.6
10
for E and F.
Figure 3:
Computer simulation and deconvolution of
the ESR spectrum obtained from the reaction mixture containing
cytochrome c and cumene hydroperoxide. A is the
computer simulation (dashedline) superimposed on the
experimental spectrum obtained using 20 µM cytochrome
c (Cytc), 25 mM cumene hydroperoxide
(CumOOH), and 45 mM DMPO. B-E are the
individual simulations of each species in the composite spectrum. The
hyperfine values used for each species are provided under
``Results.'' B is the simulated spectrum for
DMPO/OOR: line width, 0.70 G; line shape, 51% lorentzian, 49% gaussian;
and mole ratio, 0.68. C is the simulated spectrum for
DMPO/OC(CH)
C
H
: line
width, 0.75 G; line shape, 0% lorentzian, 100% gaussian; and mole
ratio, 0.13. D is the simulated spectrum for
DMPO/CH
: line width, 0.75 G; line shape, 53% lorentzian,
47% gaussian; and mole ratio, 0.06. E is the simulated
spectrum for DMPOX: line width, 0.45 G; line shape, 95% lorentzian, 5%
gaussian; and mole ratio, 0.13.
Figure 4:
Composite ESR spectrum of DMPO radical
adducts from incubations of cytochrome c and cumene
hydroperoxide at various DMPO concentrations. A is the
spectrum obtained from a reaction mixture containing 20 µM
cytochrome c (Cytc), 25 mM cumene
hydroperoxide (CumOOH), and 45 mM DMPO (i.e. the same as seen in Fig. 3A). B is the spectrum
obtained using the conditions from A, except cytochrome c was omitted. C is the spectrum obtained when DMPO was
increased to 135 mM. D is the spectrum obtained using
the conditions from C, except cytochrome c was
omitted. E is the spectrum obtained when the DMPO
concentration was increased to 360 mM. F is the
spectrum obtained using the conditions from E, except
cytochrome c was omitted. The spectrometer conditions were as
follows: modulation amplitude, 0.5 G; microwave power, 20 milliwatts;
time constant, 0.69 s; scan time, 600 s; scan range, 100 G; and
receiver gain, 2.5 10
for A-D and
1.6
10
for E and
F.
The origin of the signals observed in the absence of
cytochrome c is unknown. The reagents used in these
experiments were Chelex-treated to minimize the contribution of trace
transition metals. In addition, neither EDTA nor DTPA had any effect on
the concentrations of radical adducts in the cytochrome
c-catalyzed reaction or the control reaction mixtures (data
not shown). Desferal did decrease the intensity of the signal seen in
the absence of cytochrome c (data not shown). However, the
addition of Desferal to the cytochrome c-catalyzed reaction
resulted in the scavenging of the hydroperoxide-derived radicals and
the appearance of the Desferal nitroxide free radical (Fig. 5).
The 1-electron oxidation of Desferal to a stable nitroxide has been
demonstrated in several free radical-generating systems
(23) .
The hyperfine coupling constants used to simulate the spectra shown in
Fig. 5B (dashedline) were obtained
from a report by Morehouse et al.(23) and are as
follows: a = 7.85 G and
a
(2H) = 6.35 G. Twenty-seven percent of the
free radicals in the composite spectrum (Fig. 5B) were
due to the Desferal nitroxide radical. Although Desferal is often used
in these systems as a transition metal
chelator
(24, 25) , it is also a powerful radical
scavenger and is even capable of reacting with superoxide
(23) .
Thus, it is unclear whether Desferal was acting as a metal chelator or
a radical scavenger. The addition of 10 mM cyanide to the
cytochrome c-catalyzed reaction also decreased the radical
adduct concentrations to the levels seen in the control experiments,
giving a spectrum that was predominantly the DMPO hydroxyl radical
adduct (data not shown). Cyanide coordinates with the heme iron of
cytochrome c at the sixth axial position
(26, 27) and thereby prevents its reaction with the hydroperoxide.
Figure 5:
Effect of Desferal on the production of
radical adducts from the reaction of cytochrome c with
t-BuOOH. A is the spectrum obtained from a reaction
mixture containing 20 µM cytochrome c (Cytc), 12.5 mMt-butyl hydroperoxide,
and 90 mM DMPO. B is the spectrum obtained using the
conditions from A, except Desferal (50 µM) was
added. The dashedline superimposed on the
experimental spectrum in B is the computer simulation. The
hyperfine values used for the simulation are provided under
``Results.'' The radical adducts included in the simulation
were as follows. DMPO/OOR: line width, 0.75 G; line shape, 58%
lorentzian, 42% gaussian; and mole ratio, 0.40.
DMPO/OC(CH)
: line width, 0.65 G; line shape, 0%
lorentzian, 100% gaussian; and mole ratio, 0.31. DMPO/CH
:
line width, 0.65 G; line shape, 0% lorentzian, 100% gaussian; and mole
ratio, 0.02. Desferal nitroxide: line width, 0.65 G; line shape, 99%
lorentzian, 1% gaussian; and mole ratio, 0.27. The spectrometer
conditions were the same as indicated in the legend of Fig.
2.
The data in Fig. 2and Fig. 4made it apparent that the
proportions of the radical adducts observed in the composite spectrum
were highly dependent on the concentration of DMPO. As will be
discussed later, there are a number of reactions by which alkoxyl
radicals can be produced from peroxyl radicals and vice versa. However,
the different mechanisms proposed for the reaction of cytochrome c with these hydroperoxides dictate that the initial radical
produced is either the peroxyl radical (as predicted by the heterolytic
peroxidase mechanism) or the alkoxyl radical (via the homolytic
mechanism).
85 to
15%) (Fig. 6). The t-butyloxyl radical, on the other
hand, constituted only
10% of the composite spectrum at 22.5
mM DMPO. However, at 360 mM DMPO, the
t-butyloxyl radical accounted for 80% of the radicals that
were trapped. The contribution of the methyl radical, which arises from
the
-scission of the t-butyloxyl radical, and the DMPOX
adduct remained nearly unchanged as the DMPO concentration was varied.
- and
-absorption bands appear at 550 and 521 nm,
respectively
(29) . In addition, both the ferric and ferrous
forms of native cytochrome c have a charge transfer absorption
maximum at 695 nm that is due to the iron-sulfur bond of the
axial methionine ligand
(3) . This 695 nm band has been used as
an indicator that cytochrome c is in its native hexacoordinate
form
(3, 4) . It has been reported previously that the
Soret and visible bands of ferricytochrome c are bleached upon
the addition of hydrogen peroxide and t-butyl
hydroperoxide
(7, 18) . Also, the characteristic red
shift of the Soret band associated with compound I formation when a
ferriperoxidase reacts with H
O
was not observed
for cytochrome c(18) . Based on these results, it has
been previously concluded that a compound I form of cytochrome c is not formed from the reaction with hydroperoxides. It has also
been suggested that the heme iron of cytochrome c cannot
coordinate with the peroxide oxygen because it is already
hexacoordinate
(18) . However, separate studies using the 695 nm
absorption band as well as proton NMR have shown that even minor
perturbations in pH, temperature, or ionic strength result in the loss
of the sixth methionine ligand
(3, 4) . We found that the
addition of either t-butyl or cumene hydroperoxide to
cytochrome c (data not shown for t-butyl
hydroperoxide) resulted in the rapid bleaching of the Soret band as
well as the 528 nm absorbance maximum (Fig. 9A). The
- and
-absorption bands characteristic of the ferrous form of
cytochrome c were not observed at any time during the
reaction. The 695 nm band was observed prior to the addition of the
hydroperoxides (which indicated that the cytochrome c was
initially hexacoordinate under our experimental conditions), but
disappeared with time after the hydroperoxides were added (data not
shown). Consistent with the ESR data, the addition of cyanide prevented
the bleaching (Fig. 9B). Instead, a cytochrome
c-cyanide complex was observed that was similar to that
demonstrated in previous reports
(26, 27) . On the other
hand, the addition of DMPO did not affect the bleaching when either
t-butyl or cumene hydroperoxide was added (data not shown).
Figure 9:
Visible absorption spectra of cytochrome
c following the addition of cumene hydroperoxide. In
A, the reaction mixture contained 20 µM
cytochrome c and 25 mM cumene hydroperoxide. B was obtained using the conditions from A, except 10
mM cyanide was added just prior to the addition of cumene
hydroperoxide. Absorption spectra were recorded every 45 s. The
arrows indicate the initial scan and the direction of the
absorbance change with time.
-scission
of the alkoxyl radical would form the methyl radical (Reaction
7)
(32) . Therefore, this mechanism can account for all the
radicals that are observed in the spin trapping studies. In fact,
Davies
(13) invoked the peroxidase mechanism to explain his ESR
results from the reaction of organic hydroperoxides with various heme
proteins including cytochrome c. However, the peroxidase
mechanism would dictate that the peroxyl radical be produced initially
and that the alkoxyl and methyl radicals be formed as products of its
breakdown. If this were true, one would expect that as the DMPO
concentration was increased, more peroxyl radical would be trapped and
less of the DMPO alkoxyl radical adduct would be detected. In fact, the
exact opposite was observed in the present study. It was clearly
demonstrated that the alkoxyl radical adduct dominated at the highest
DMPO concentrations. In addition, the peroxyl radical adduct decreased
to a very nominal proportion of the total radical adduct as the
concentration of spin trap was increased. Using the plots shown in
Fig. 6
and Fig. 7, one could extrapolate that as the DMPO
concentration was raised infinitely, the percent of the ESR spectrum
resulting from the peroxyl radical adduct would approach zero, while
that of the alkoxyl radical adduct would approach 100.
-scission of the tertiary alkoxyl radical (Reaction 9) would yield
the methyl radical that was observed. The reaction of the methyl
radical with oxygen results in the formation of the methyl peroxyl
radical (Reaction 10). The peroxyl radical adduct signals observed were
primarily due to the methyl peroxyl radical. Evidence for this
conclusion was obtained when oxygen was omitted from the reaction
mixture. Under anaerobic conditions, the peroxyl radical adduct signal
disappeared and was almost completely replaced by the methyl radical
adduct signal. It should be noted that the hyperfine coupling constants
for the DMPO methyloxyl radical adduct are typical of DMPO peroxyl
radical adducts (33). Thus, the signals we are assigning to the methyl
peroxyl adduct might be due to the methyloxyl radical adduct. In either
case, the radical adduct would be derived from the methyl radical and
thus would not change our mechanistic conclusions (i.e. Reactions 8-10).
10
M
s
)
(12) competes with
-scission. Therefore, as more alkoxyl
radical is trapped (i.e. more spin trap is added), less
peroxyl radical can be formed via Reactions 9 and 10. The DMPOX
observed in either case is probably due to the breakdown of the peroxyl
radical and/or the oxidation of DMPO by the ferryl species. The methyl
radical adduct remained relatively unchanged as the DMPO concentration
was increased. Apparently, as DMPO was increased, the rate of the
methyl radical trapping increased to compensate for the decrease in
methyl radical production via the
-scission Reaction 7. Thus, even
though less methyl radical was being formed due to the increased
trapping of t-BuO (Reaction 11), the amount of methyl radical
adduct detected remained approximately the same.
104 kcal/mol) is
considerably higher than that of the hydroperoxide resulting from
hydrogen abstraction by a peroxyl radical (
90
kcal/mol)
(34) . In fact, pulse radiolysis studies have shown
that the t-butyloxyl radical abstracts hydrogens from several
polyunsaturated fatty acids at a rate of
2.0
10
M
s
(35) ,
while the rate constant for hydrogen abstraction by peroxyl radicals is
only 5
10
M
s
(31). Thus, the finding that alkoxyl
radicals are formed initially in the cytochrome c-catalyzed
degradation of hydroperoxides may have significance when considering
the relative roles of alkoxyl and peroxyl radicals in heme
protein-catalyzed lipid peroxidation and/or protein damage.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.