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INTRODUCTION |
Mitochondria represent a primary source of
ROS1 in most aerobic
mammalian cells (1-3). The H2O2 concentration
in liver cells has been estimated to be
10
7-10
9mol/liter and that of superoxide
anion to be about 10
11 mol/liter (1, 3-6). The precursor
of H2O2 has been shown to be the superoxide
anion formed through a single electron reduction of O2 by
the electrons leaked from the substrate side of the respiratory chain
(5). The rate of H2O2 production in isolated
mitochondria under state 4 respiration is 0.6-1.0
nmol·mg
1min
1, which is about 2% of the
total oxygen uptake under physiological conditions (1, 4). Two sites in
the respiratory chain have been found to be responsible for the
generation of O
. One of these sites is located in complex I
and the other in complex III (2, 7). O
production is probably
via autooxidation of the reduced flavin mononucleotide of NADH
dehydrogenase in complex I (2). Two components, the ubisemiquinone (8)
and the reduced cytochrome b (9) have been suggested to be
the autooxidizable factors causing O
production in complex
III. Using the purified succinate-cytochrome c reductase,
Zhang et al. (10) confirmed that the exact site leaking
electrons to generate O
in complex III is in the Q-cycle.
O
is a short-lived ion that can rapidly evolve to
H2O2 along three pathways: 1) the catalysis of
superoxide dismutase; 2) chemical dismutation; and 3) HOO.,
in equilibrium with O
, reacting with membrane polyunsaturated
fatty acids to produce heat and H2O2 (11). Both
O
and H2O2 are potentially dangerous
if they are not removed in time. Therefore, a mechanism to protect
respiration enzymes from the damage of the generated ROS is needed
around the respiratory chain.
In our early studies on the purified succinate-cytochrome c
reductase, we found that the electrons transferred from succinate to
cytochrome c can be further delivered to the extra added
H2O2 (12). An alternative electron leak pathway
of respiratory chain was later suggested (13, 14). In this study, using
the cytochrome c-depleted Keilin-Hartree heart muscle
preparation (c-dHMP), we show that the alternative electron leak
pathway may acted on the scavenging of O
and
H2O2 in the mitochondrial respiratory chain.
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EXPERIMENTAL PROCEDURES |
Chemicals--
Lucigenin (bis-N-methylacridinium
nitrate), catalase, horseradish peroxidase, superoxide dismutase
from bovine erythrocytes, bovine serum albumin,
phenylmethylsulfonyl fluoride, mannitol, and EDTA were purchased from
the Sigma. Luminol (3-aminophthalhydrazide) was obtained from Acros
Organics. Cytochrome c was from Koch-Light Laboratories Ltd.
1,10-Phenanthroline anhydrous and ADP were from ICN Biomedical.
Succinic acid disodium salt was from the Aldrich Chemical Company.
HEPES was from Boehringer. NADH was from Amresco. Aprotinin was from
Roche Molecular Biochemicals. Percoll was from Amersham
Biosciences. All other reagents were of analytical grade.
Keilin-Hartree Heart Muscle Preparation and Succinate-Cytochrome
c Reductase (SCR) Preparation--
HMP was prepared according to the
method of Keilin and Hartree (15). Succinate-cytochrome c
reductase was purified from HMP according to the method of King (16).
c-dHMP was prepared according to the method of Tsou (17).
Protein concentration was determined by the Bradford method.
Measurement of the Activity of Succinate Oxidase of
HMP--
Activity was measured by the rate of oxygen consumption. A
system containing 2 ml of pH 7.4 phosphate buffer and 8 mM
succinate was warmed at 30° C, 300 mg of HMP was added, and the
oxygen consumption was measured. The mitochondria respiratory control
ratio was determined as the ratio of oxygen uptake in state 3/state 4 (18). The oxygen consumption was measured with a Clark oxygen electrode.
Assay for H2O2 Generation and
Elimination--
H2O2 generation were detected
using luminol plus horseradish peroxidase-derived chemiluminescence
with the BPCL Ultra-weak luminescence analyzer at 37° C. The
reaction mixtures contained 500 µM luminol, 2.5 units of
horseradish peroxidase, 50 mM Na-phosphate buffer pH 7.4, 4 mg/ml c-dHMP, and different cytochrome c concentrations in a
total volume of 1 ml. The luminol plus horseradish peroxidase-derived chemiluminescence was initiated by adding 300 µM NADH as
substrate. The integral of the signal peak reflects the formation of
H2O2. The relation between cytochrome
c concentration and H2O2 formation is plotted as the integrated area of the peak on the ordinate with the
cytochrome c concentration on the abscissa.
Assay for O
Generation--
O
generation was
detected via lucigenin-derived chemiluminescence (LDCL) using a BPCL
Ultra-weak luminescence analyzer at 37° C. For enzymatic systems,
the reaction mixtures contained 3 µM lucigenin, 20 mM Na-phosphate buffer pH 7.4, 3 mg/ml c-dHMP, and
different concentrations of cytochrome c in a total volume
of 1 ml. The reaction was started by adding as substrate 200 µM NADH. When 20 mM succinate was used as
substrate, the lucigenin concentration was 5 µM, and the
protein concentration of HMP was 1 mg/ml. The integral of the
chemiluminescence peak reflects the formation of O
. The relation between cytochrome c concentration and the
formation of the superoxide anion is plotted as of integrated area
of the peak on the ordinate and the cytochrome c
concentration on the abscissa.
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RESULTS |
Two Generative Sites of O
and
H2O2 in HMP--
Keilin-Hartree HMP is
the first preparation used in the study of cytochromes in the early
stage. This preparation is a suspension of physically disintegrated
mitochondrial membrane, which contains all the components of
respiratory chain. Succinate-cytochrome c reductase is
purified from HMP, which is part of respiratory chain transferring
electrons from succinate to cytochrome c. With these two
preparations we studied the generation and elimination of O
and H2O2.
Lucigenin was used as a chemiluminescent probe to monitor the
O
generation. The concentration of lucigenin used in the
experiments was 3 µM. It has been shown that at this
concentration of lucigenin, the generation of photons is proportional
to the amount of O
. Although the validity of lucigenin as a
chemiluminescent probe for detecting biological O
has been questioned based on the observation that the lucigenin may itself act
as a source of O
via autooxidation of the lucigenin cation
radical in several in vitro enzymatic systems (19, 20), 3 micromoles per liter lucigenin do not undergo redox cycling and are
useful for HMP (21, 22).
As shown in Fig. 1A, two
chemiluminescence peaks can be observed when the NADH concentration is
more than 0.7 mM, and only peak I appears when NADH is less
than 0.7 mM. With succinate as the substrate, only peak II
can be observed, as shown in Fig. 1B. With purified
succinate-cytochrome c reductase, only peak II can be
observed in the presence of succinate, as shown in Fig. 1B.
This observation indicates that peak I is formed by the O
generation of complex I and peak II is formed by complex III.

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Fig. 1.
Profiles of lucigenin-derived
chemiluminescence (CL) elicited with HMP.
A, NADH-supported chemiluminescence. The CL response was
initiated by 1 mg/ml HMP and continuously monitored at 37° C for
3000 s. B, a, succinate-supported HMP.
b, succinate-supported SCR. The assay mixture (1 ml)
contained 50 mM phosphate buffer, pH 7.4, 3 µM lucigenin, and 20 mM sodium succinate. The
CL response was initiated by 1 mg/ml HMP or SCR continuously monitored
at 37° C for 3000 s.
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O
is a short-lived ion that can rapidly evolve to
H2O2. Both O
and
H2O2 are potentially dangerous if they are not
removed in time. We addressed whether the respiratory chain has a
mechanism to protect itself from damage cause by the generated ROS.
Scavenging of O
and H2O2 by a
Cytochrome c-mediated Electron Leak Pathway--
Forman found that
O
can be oxidized by ferricytochrome c rapidly
(23), and this reaction has been used to measure the superoxide anion.
It is reasonable to consider that the direct oxidization of O
by ferricytochrome c of the respiratory chain is one of the
protective mechanisms. In yeast, there is an enzyme named cytochrome
c peroxidase that catalyzes the reduction of
H2O2 by ferrocytochrome c (24). This
reaction implies that the H2O2 could be
scavenged by the electron delivery from cytochrome c to
H2O2. The problem is that the cytochrome
c peroxidase is found only in yeast. Can
H2O2 be reduced by ferrocytochrome c
in the absence of peroxidase? The following observation, as shown in
Fig. 2 may support this possibility.

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Fig. 2.
Re-reduction of
H2O2-oxdized cytochrome
c. Reaction medium: 0.1 M phosphate
buffer, pH 7.4, 0.3 mM EDTA, 5 µg SCR, and 20 µM succinate with 8.8 mM
H2O2 added.
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A system containing mainly cytochrome c and a catalytic
amount of purified succinate-cytochrome c reductase is
employed to observe the electron delivery from cytochrome c
to H2O2. In this system, the absorption change
at 550 nm mainly reflects the redox change of cytochrome c;
the disturbance of other cytochromes in SCR was decreased to a limited
value. As shown in Fig. 2, the process of electron transfer from
succinate to cytochrome c can be observed by the increased
absorption at 550 nm when adding succinate to the system. The addition
of H2O2 during the reduction of cytochrome
c causes a decreased absorption at 550 nm. The decrease of
550 nm indicates that the reduced cytochrome c is oxidized by H2O2. With further addition of
succinate, the H2O2-oxidized cytochrome
c can be re-reduced. This result implies that the electrons transported to the cytochrome c can be further delivered to
H2O2 even in the absence of cytochrome
c peroxidase. This experiment can be performed when
H2O2 concentrations is as low as
10
7mol/liter, a value near the physiological
concentration. Thus, the electron delivery from cytochrome c
to H2O2 can happen in the absence of
peroxidase. The cytochrome c of the respiratory chain not
only transfers electrons to O2 through the
cytochrome c oxidase but also delivers electrons to
H2O2 through an electron leak path (13,
14).
Based on the above observation, an electron-leak pathway mediated by
cytochrome c can be suggested in mitochondrial respiratory chain as shown in Fig. 3.
To confirm the mechanism shown in Fig. 3, the cytochrome
c-depleted HMP was prepared according to the method
developed by Tsou (17). The effect of cytochrome c on the
generation and elimination of O
and
H2O2 was observed by the reconstitution of
cytochrome c to the c-dHMP.
Down-regulative Effect of Cytochrome c on the Generation of
O
--
The precursor of H2O2
was shown to be the O
formed through a single electron
reduction of O2 by the leaked electrons from the substrate
side of the respiratory chain. We found that O
generation in
c-dHMP is about 7 times higher than that in normal HMP. The reconstitution of cytochrome c causes the O
generation to decrease exponentially.
Fig. 4A shows the decay curve
of O
generation when cytochrome c is reconstituted
to c-dHMP. The NADH concentration used in this experiment was 200 µM. The O
generation decreased sharply when the
reconstructed cytochrome c concentration was less then 0.8 µM and became constant at about 6 µM, a
value equivalent to the content of cytochrome c in normal
HMP. Least squares analysis showed that the curve can be represented by
LDCL (× 104) = 0.82 + 11.41e
19.67x + 4.18e
0.73x. Fig. 4B shows the comparison of
the restored activity and the O
generation by the
reconstitution of cytochrome c to the c-dHMP. It can be
found that enough cytochrome c in the respiratory chain is
necessary for both maintaining the lower O
generation and
high activity of the respiratory chain.

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Fig. 4.
Decreased generation of superoxide anion with
concentration of cytochrome c reconstituted to c-dHMP
for NADH at a concentration of 200 µM. A, total O
generation for various cytochrome c concentrations. Ordinate
is the integral peak area. The exponential decay is described by LDCL
(× 104) = 0.82 + 11.41e 19.67x + 4.19e 0.73x (r = 0.997). B, the
decrease of superoxide anion generation occurs with the increase of
succinate oxidase activity when cytochrome c is added to the
reaction system. The control is the normal HMP. The protein
concentration of HMP in the system was 2.17 mg/ml and the lucigenin was
3 micromole per liter.
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The generation of O
in complex III was detected using
succinate as the substrate. The O
formation was about 8 times
higher in c-dHMP then that in normal HMP. When cytochrome c
was reconstituted, the value of O
formation decreased to the
level of normal HMP. Fig. 5A
shows that the O
formation also decayed exponentially with
the reconstituted cytochrome c. Least squares analysis of
the results showed that the curve could be represented by LDCL (× 104) = 2.02 + 12.68e
8.90x + 8.55e
1.19x. Fig. 5B shows the comparison of
the restored activity and the O
generation during the
reconstitution of cytochrome c to the c-dHMP.

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Fig. 5.
Decreased generation of superoxide anion with
concentration of cytochrome c reconstituted to c-dHMP
with succinate as substrate. A, total O generation
for various cytochrome c concentrations. Ordinate is the
integral peak area. The exponential decay is described by LDCL (× 104) = 2.02 + 12.68e 8.90x + 8.55e 1.19x (r = 0.990). B, the
decrease of superoxide anion generation occurs with the increase of
succinate oxidase activity when cytochrome c is added to the
reaction system. The control was untreated HMP. The reaction system
contained 20 mM Na-phosphate, 5 µM lucigenin,
2.17 mg c-dHMP and different concentrations of cytochrome c
reconstituted. The reaction was started by adding 20 mM
succinate.
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The above result implies that the content of cytochrome c in
the respiratory chain strongly affects the level of O
generation in the substrate side of the chain. The O
generation was markedly increased, whereas cytochrome c was
removed from the HMP. The reconstitution of cytochrome c to
c-dHMP caused the O
generation to decrease exponentially.
Down-regulative Effect of Cytochrome c on the Generation of
H2O2--
Luminol plus horseradish
peroxidase-derived chemiluminescence was used to detect
H2O2 generation when cytochrome c
was reconstituted to c-dHMP. The results are shown in Fig.
6. H2O2
generation in c-dHMP is about 7 times greater than that in the
untreated HMP. The H2O2 generation obviously
decreased, whereas cytochrome c was reconstructed to the
c-dHMP. When the reconstructed cytochrome c concentration
reached 5 µM, H2O2 generation
decreased to the same value as that in normal HMP. Least squares
analysis of the decay curve result in an formula of LDCL (× 103) = 0.20 + 49.72e
0.94x + 60.35e
0.21x (r = 0.999).

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Fig. 6.
H2O2
generation for various concentrations of cytochrome c
reconstituted to c-dHMP with NADH as substrate. Ordinate is
the integral peak area. The exponential decay is described by LDCL (× 103) = 0.20 + 49.72e 0.94x + 60.36e 0.21x (r = 0.999). The control is
untreated HMP. The c-dHMP protein concentration was 4 mg/ml.
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The above result suggests that the amount of cytochrome c in
the respiratory chain strongly affects the generation of
H2O2. Enough cytochrome c is
necessary to keep a lower physiological H2O2
concentration in mitochondria. When cytochrome c was
released from the respiratory chain, H2O2
generation increased markedly.
Comparison of the Cytochrome c-depleted HMP and the KCN-inhibited
HMP on the Generation of O
and
H2O2--
The depletion of cytochrome
c stopped the electron transfer of the respiratory chain at
complex III, whereas KCN inhibition blocked the electron transfer at
the terminal oxidase. Both of the treatments increased the electron
leak in the substrate side of the respiratory chain. It was found that
the increasing level is very different in these two treated HMP. As
shown in the Fig. 7A, the
enhancement of H2O2 caused by KCN inhibition is
only 60% of the enhancement caused by the depletion of cytochrome
c from HMP. The enhancement of O
occurred in
c-dHMP but not in KCN-inhibited HMP (Fig. 7B). The
generative levels of O
and H2O2
are essentially different in the preparations with or without
cytochrome c.

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Fig. 7.
The influence of KCN to ROS generation in HMP
system. A, reaction of H2O2
generation. B, reaction of superoxide anion
generation.
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DISCUSSION |
It is well established that mitochondria are the main source of
ROS generation in cell. An antioxidative role of cytochrome c in vivo has been suggested (25, 27). In the
living cell, a compound generated in a metabolic step is always
eliminated in the next step of the metabolic reaction chain. The oxygen
radicals also obey the rule of metabolism. It has been well accepted
that the generation of O
and H2O2 in the substrate side of respiratory chain is a result of electron leakage
of the chain. In this study, an alternative electron-leak pathway is
suggested to explain the elimination of the generated O
and H2O2. Based on the scheme shown in Fig. 3,
the level of O
and H2O2 is in a
balanced state between generation in the substrate side of respiratory
chain and elimination in the oxygen side cytochrome c. The
lower content of cytochrome c in the respiratory chain causes the higher level of O
and H2O2 accumulated.
The experimental results demonstrate that cytochrome c
strongly affects the generation and elimination of O
and
H2O2 in the mitochondrial respiratory chain.
The amount of O
and H2O2 generated in
the c-dHMP is 7-8 times higher than that in normal preparations. The
reconstitution of cytochrome c to the c-dHMP causes
O
and H2O2 generation to decrease
exponentially. This result implies that sufficient cytochrome
c in the respiratory chain is needed for both the high
activity of respiration and the low generation of O
and
H2O2. This property of cytochrome c
is facilitated to the mitochondria for keeping reactive oxygen
species at a normal physiological level. The release of cytochrome c from the respiratory chain will cause a
dramatic increase of O
and H2O2 generation.
The exponential decay curves of the O
and
H2O2 generation were all simulated as an
equation having one constant term and two exponential terms. The
following three mechanisms might be involved in the effect of
cytochrome c on the generation and elimination of
O
and H2O2. First, the reconstitution
of cytochrome c facilitates the electron transfer of the
respiratory chain, thus the leakage of electrons reduced and the
decreased generation of O
and H2O2.
This mechanism may correspond to the constant term of the simulation
formula. Second, the O
generated by the electron leakage can
be oxidized by ferricytochrome c directly based on the
reaction mentioned by Forman and Fridovich (23). Third, the
H2O2 can be scavenged by the cytochrome
c-mediated electron delivery shown in Fig. 3. The second two
mechanisms may correspond to the two exponential terms of the
simulation formula. The cytochrome c not only affects the
generation of O
and H2O2 by making
the electron transfer of the respiratory chain more fluent, but also
eliminating the generated O
and H2O2
through a cytochrome c-mediated electron-leak pathway.
The O
and H2O2 generation in
cytochrome c-depleted HMP and KCN-inhibited HMP are quite
difference. The level of H2O2 in KCN-HMP is
only 60% of that in c-dHMP. This result reflects that the cytochrome
c in KCN-HMP scavenges about 40%
H2O2 through the cytochrome
c-mediated electron-leak pathway. Once the O
is
generated in the substrate side of the respiratory chain, one portion
is dismutated to H2O2 and another portion is
rapidly scavenged by ferricytochrome c directly. This could
be the reason that O
is not accumulated in preparations
containing cytochrome c. The more cytochrome c
that is reconstituted to the c-dHMP, the less O
and
H2O2 are accumulated. These results support
that the alternative electron-leak pathway mediated by cytochrome
c is working in mitochondria.
Cytochrome c is strongly involved in cell apoptosis (26).
Two groups of cytochrome c in mitochondria are suggested:
the bound cytochrome c in the outer face of the inner
membrane of mitochondria and the free cytochrome c in the
space of inner and outer membrane (27, 28). In our experiments, the
reconstituted cytochrome c is in the loosely bound state
with the HMP membrane, therefore the loosely bound cytochrome
c may have an antioxidative effect in mitochondria. It is
revealed that a dramatic increase of O
and
H2O2 in mitochondria appears at the same time
that cytochrome c leaves the respiratory chain. Therefore,
the involvement of electron leakage in cell apoptosis is an interesting
project. The burst of O
and H2O2
happens earlier than that of cytochrome c arriving at the
precaspase-stimulating position, suggesting that ROS may act as a kind
of apoptogen in the very early stage of apoptosis.
Cytochrome c is a small, very stable hemoprotein containing
covalently bound heme c as a prosthetic group. The primary recognized function of cytochrome c is to transfer electrons from
complex III (QH2-cytochrome c reductase) to
complex IV (cytochrome c oxidase) in the respiratory chain.
The finding of cytochrome c starting cell apoptosis implies
that the cytochrome c has a different function in different
locations in the cell. The observations in this study lead us to
propose that cytochrome c may act as an antioxidative factor
to regulate the O
and H2O2 level of
mitochondria, and this is involved in programmed cell death.