Content uploaded by Florian L Muller
Author content
All content in this area was uploaded by Florian L Muller on Jan 06, 2016
Content may be subject to copyright.
Biochem. J. (2008) 409, 491–499 (Printed in Great Britain) doi:10.1042/BJ20071162
491
High rates of superoxide production in skeletal-muscle mitochondria
respiring on both complex I- and complex II-linked substrates
Florian L. MULLER*
1
, Yuhong LIU†, Muhammad A. ABDUL-GHANI‡, Michael S. LUSTGARTEN§, Arunabh BHATTACHARYA*†,
Youngmok C. JANG† and Holly VAN REMMEN*†‡§
*Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, Texas Research Park Campus, 15355 Lambda Drive, San Antonio,
TX 78229-3900, U.S.A., †Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229-3900, U.S.A.,
‡South Texas Veterans Health Care System, San Antonio, TX 78284-7762, U.S.A., and §Department of Physiology, University of Texas Health Science Center at San Antonio, San
Antonio, TX 78229-3900, U.S.A.
Despite the considerable interest in superoxide as a potential
cause of pathology, the mechanisms of its deleterious production
by mitochondria remain poorly understood. Previous studies in
purified mitochondria have found that the highest rates of super-
oxide production are observed with succinate-driven reverse-
electron transfer through complex I, although the physiological
importance of this pathway is disputed because it necessitates
high concentrations of succinate and is thought not to occur
when NAD is in the reduced state. However, very few studies
have examined the rates of superoxide production with mitochon-
dria respiring on both NADH-linked (e.g. glutamate) and
complex II-linked substrates. In the present study, we find
that the rates of superoxide production (measured indirectly as
H
2
O
2
) with glutamate + succinate (∼ 1100 pmol of H
2
O
2
· min
−1
·
mg
−1
) were unexpectedly much higher than with succinate
(∼ 400 pmol of H
2
O
2
· min
−1
· mg
−1
) or glutamate (∼ 80 pmol
of H
2
O
2
· min
−1
· mg
−1
) alone. Superoxide production with
glutamate + succinate remained high even at low substrate
concentrations (< 1 mM), was decreased by rotenone and
was completely eliminated by FCCP (carbonyl cyanide p-
trifluoromethoxyphenylhydrazone), indicating that it must in large
part originate from reverse-electron transfer through complex
I. Similar results were obtained when glutamate was replaced
with pyruvate, α-ketoglutarate or palmitoyl carnitine. In contrast,
superoxide production was consistently lowered by the addition
of malate (malate + succinate ∼ 30 pmol of H
2
O
2
· min
−1
· mg
−1
).
We propose that the inhibitory action of malate on superoxide
production can be explained by oxaloacetate inhibition of
complex II. In summary, the present results indicate that reverse-
electron transfer-mediated superoxide production can occur under
physiologically realistic substrate conditions and suggest that
oxaloacetate inhibition of complex II may be an adaptive
mechanism to minimize this.
Key words: complex I, electron transport chain, H
2
O
2
, mito-
chondria, oxaloacetate, superoxide.
INTRODUCTION
Mitochondria and ROS (reactive oxygen species) have emerged
as central players in a wide variety of pathologies. Elevation in
mitochondrial ROS production has been hypothesized to occur
in Parkinson’s disease and Alzheimer’s disease as well as aging
[1–5]. However, this belies the fact that the mechanics of mito-
chondrial superoxide production remain quite poorly understood.
While it is generally accepted that mitochondria are the main
site of cellular ROS production, studies in isolated mitochondria
have shown that the amount of H
2
O
2
released by mitochon-
dria (H
2
O
2
originates from the dismutation of O
2
•−
[6], and is
much easier to measure than O
2
•−
) under most conditions is rather
modest (< 0.1 % electron leak). The exception to this is succinate-
driven reverse-electron transfer through complex I, a condition
yielding by far the highest rates of H
2
O
2
release in isolated mi-
tochondria [7–10]. This holds true in mitochondria isolated
from diverse tissues (up to 2000 pmol of H
2
O
2
/min per mg of
mitochondrial protein [7–15]). Indeed, the frequently quoted
value of 1–5 % of electrons passing through the chain being
diverted to the formation of superoxide is only true under this
condition [16,17], and the notion that mitochondria are the
strongest source of cellular ROS largely derives from these
measurements. In isolated mitochondria, reverse-electron transfer
through complex I occurs when the ubiquinol pool is in a highly
reduced state and a strong membrane potential is present, i.e.
the energy of the membrane potential drives the ubiquinol (with
electrons provided by succinate)-dependent reduction of NAD
+
to NADH with electrons passing in the reverse direction through
complex I [18].
However, a number of investigators have questioned whether
reverse-electron transfer (and hence superoxide production
emanating from it) can occur in vivo [19–21]. Data refuting
this possibility have been presented by Hansford et al. [8].
These authors showed that superoxide production decreases
dramatically when the concentration of succinate is reduced
below 1 mM. Further, it is thought that reverse-electron transfer-
mediated superoxide production cannot occur when the final
electron acceptor in this pathway, NAD
+
, is reduced [18,22].
Hansford et al. [8] reported that succinate reverse-electron
transport-mediated superoxide production is dramatically
decreased when the NAD-linked substrates glutamate + malate
are added concomitantly with succinate [8]. In contrast, a very
recent report by Zocarato et al. [26] indicates that addition of
glutamate/malate only modestly decreases succinate-supported
superoxide production. Superoxide production is much lower
when electron transport is in the forward d irection, with substrates
like glutamate + malate or pyruvate + malate (∼ 10–90 pmol of
H
2
O
2
· min
−1
· mg
−1
[8,9,12,14,15,23], and is even reported to be
essentially undetectable by some studies [14,24]).
We undertook the present study (preliminary results of which
were presented at the 2006 Society for Free Radical Biology
Abbreviations used: FCCP, carbonyl cyanide
p
-trifluoromethoxyphenylhydrazone; HRP, horseradish peroxidase; ROS, reactive oxygen species.
1
To whom correspondence should be addressed (email aettius@aol.com).
c
The Authors Journal compilation
c
2008 Biochemical Society
492 F. L. Muller and others
and Medicine Conference [25]) to address experimentally the
argument that reverse-electron transfer-ROS production only
occurs under extreme, non-physiological substrate conditions
(contrary to what Hansford et al. [8] reported a number of years
ago, a report published while this study was in progress did indeed
conclude that high rates of ROS production could occur under
less extreme substrate conditions [26]). To this effect, we have
measured the rate of superoxide production with the complex II-
linked substrate succinate and NADH-linked substrates alone or
in combination, over a range of substrate concentrations. We note
that virtually all previous studies that have studied superoxide
production in mitochondria have employed either NADH- or
complex II-linked substrates alone, not in combination. In vivo,
both NADH and succinate are present, with greater abundance of
the former.
We observed very high rates of superoxide production when
skeletal-muscle mitochondria were respiring on both succinate
and the NADH-linked substrates, glutamate, pyruvate and
palmitoyl carnitine. We show that these rates of superoxide
production remain high even at low substrate concentrations
and originate in large part from reverse-electron transfer through
complex I, despite the fact that NAD was in the reduced state.
Only malate was found to inhibit superoxide production with
succinate, which we propose, is due to the oxaloacetate inhibition
of complex II [27–29].
Taken together, our results indicate that reverse-electron
transfer-mediated superoxide production can occur even when
NAD is in the reduced state, and at substrate concentrations that
are within the physiological range. Further, our results suggest
that oxaloacetate inhibition of complex II may be a deliberate
adaptation to minimize reverse-electron transfer-mediated super-
oxide production.
EXPERIMENTAL
Animals
Mice were maintained in the C57 B6/J background and housed
under specific pathogen-free barrier conditions. Mice were
anaesthetized and killed by cervical dislocation. All procedures
were approved by the IACUC (Institutional Animal Care and Use
Committee) at the University of Texas Health Science Center at
San Antonio. Mice used in the present study were 3–6 months of
age.
Chemicals
All chemicals were obtained from Sigma–Aldrich (St. Louis, MO,
U.S.A.) unless otherwise specified.
Isolation of skeletal-muscle mitochondria
Whole hind-limb skeletal-muscle mitochondria were purified
by the method of Chappell and Perry as modified by Ernster
and Nordenbrand [30,31]. Whole hind-limb skeletal muscle
was excised, washed with 150 mM KCl, placed in Chappell–
Perry buffer (100 mM KCl, 50 mM Tris, 1 mM EDTA, 5 mM
MgCl
2
, 1 mM ATP, pH 7.44; composition of modified Chappell–
Perry buffer: 100 mM KCl, 50 mM Tris, 0.1 mM EDTA, 5 mM
MgCl
2
, 0.2 mM ATP, pH 7.44) with protease and minced.
Homogenization was carried out by hand, in an all-glass
Potter–Elvehjem-type homogenizer. The homogenate was spun
for 10 min at 600 g and the supernatant was passed through
cheesecloth and centrifuged at 14 000 g for 10 min. T he pellet
was then washed once in modified Chappell–Perry buffer with
0.5 % BSA and once without BSA. H
2
O
2
release experiments
were conducted immediately after mitochondrial isolation. The
respiratory control ratio was ∼10 with glutamate/malate.
Preparation of respiratory substrates, inhibitors and uncouplers
Stock solutions (0.5 M) of the substrates glutamate, α-ketogluta-
rate, malate and succinate were prepared in the same reaction
buffer as above, their pH was adjusted to 7.44, and aliquots were
stored at − 80
◦
C. Stocks of pyruvate and oxaloacetate (0.5 M)
in the same buffer were prepared fresh for each experiment,
minimizing the time on ice. Palmitoyl carnitine was prepared
at 20 mM stock in 10 mM Hepes (pH 3.0) and aliquots were
stored at − 80
◦
C. Respiratory inhibitors rotenone and antimycin
A were dissolved in DMSO or ethanol and stored at − 80
◦
C.
Unless otherwise indicated, the working concentration for these
inhibitors was 5 µM. The uncoupler FCCP (carbonyl cyanide
p-trifluoromethoxyphenylhydrazone) was prepared in ethanol at
10 mM and stored at − 80
◦
C, and the final concentration, unless
stated otherwise, was 150 nM.
Measurement of mitochondrial superoxide production
Superoxide production was measured indirectly as H
2
O
2
release
from intact mitochondria. H
2
O
2
was determined with Amplex
TM
Red (Molecular Probes, Eugene, OR, U.S.A.; product no. A-
12212 [32]), as described previously [33–35]. HRP (horseradish
peroxidase; 1 unit/ml) catalyses the H
2
O
2
-dependent oxidation
of non-fluorescent Amplex
TM
Red (80 µM) to fluorescent
Resorufin Red. SOD (superoxide dismutase; Sigma) was added
at 30 units/ml, so as to convert all O
2
•−
into H
2
O
2
,anecessity
since O
2
•−
reacts very rapidly with HRP and HRP–Compound (I)
[Compound (I) is an intermediate state of peroxidases, formed
by the reaction of the enzyme with H
2
O
2
]. We monitored
Resorufin formation (Amplex
TM
Red oxidation by H
2
O
2
)ata
λ
excitation
of 545 nm and a λ
emission
of 590 nm using a Fluoroskan-FL
Ascent Type 374 multiwell plate reader (Labsystems, Helsinki,
Finland). The slope is converted into the rate of H
2
O
2
production
with a standard curve. Fluorescence remained linear with H
2
O
2
concentration from 0 to 2 µM. The assay was performed in 96-
well plates at 100 µl per well with a measuring duration of 2 0 ms,
every 2 s for ∼ 10 min. We performed all assays at 37
◦
C, in
125 mM KCl, 10 mM Hepes, 5 mM MgCl
2
and 2 mM K
2
HPO
4
(pH 7.44), with 50–20 µg of mitochondrial protein per 100 µlof
reaction buffer [24].
Measurement of the rates of ATP synthesis
ATP synthesis was measured using the luciferin/luciferase
assay from Roche. We followed luciferin chemiluminescence
using a Fluoroskan-FL Ascent type 374 multiwell plate reader
(Labsystems). Mitochondria (between 10 and 5 µg) were
incubated in 100 µl of 125 mM KCl, 10 mM Hepes, 5 mM
MgCl
2
and 2 mM K
2
HPO
4
(pH 7.44) with substrates (glutamate,
malate and succinate) and the measurement was started by the
addition of luciferin/luciferase buffer containing 0.6 mM ADP.
The initial slope was converted into nanomoles of ATP using the
standards provided in the kit.
Measurement of the membrane potential ( ) with Safranin O
Membrane potential was monitored by fluorescence of the
quench-dye Safranin O, as described by Votyakova and Reynolds
[9], based originally on the spectroscopic method of Akerman
and Wikstrom [36]. We followed Safranin O fluorescence at a
λ
ex
of 485 nm, a λ
em
of 590 nm using a Fluroskan-FL Ascent
c
The Authors Journal compilation
c
2008 Biochemical Society
Superoxide production by complex I 493
Table 1 High rates of superoxide production with glutamate/malate +
succinate
Superoxide production was measured indirectly asH
2
O
2
release, as described in the Experimental
section. The substrate concentration was 10 mM for succinate and 5 mM for glutamate and
malate. The concentration of mitochondria used was between 0.2 and 0.5 mg/ml, with the final
rates adjusted for protein concentration (pmol of H
2
O
2
/min per mg of mitochondrial protein). The
mean for eight independent mitochondrial preparations,
+
−
S.E.M., is shown. For comparison,
the rate of superoxide production with glutamate/malate only and succinate with antimycin A
are also shown.
Substrate(s) d[H
2
O
2
]/d
t
(pmol · min
−1
· mg
−1
)
Glutamate + malate + succinate 1812.23
+
−
723.23
Glutamate + malate 31.23
+
−
3.23
Succinate 425.23
+
−
250.55
Succinate + antimycin A 1121.00
+
−
181.12
Type 374 multiwell plate reader (Labsystems). Safranin O (5 µM)
was incubated with 10–5 µg of mitochondrial protein in 100 µlof
reaction buffer in 96-well black plates. We performed all assays
at 37
◦
C, in 125 mM KCl, 10 mM Hepes, 5 mM MgCl
2
and 2 mM
K
2
HPO
4
(pH 7.44).
RESULTS
High but variable rates of superoxide production with a
combination of succinate and glutamate/malate
The vast majority of studies investigating the mechanisms of
superoxide production by the electron transport chain have
employed either complex I- or complex II-linked substrates, not
both together. To better understand this phenomenon, we asked
what would happen when electrons enter the chain through both
complexes.
Results in Table 1 show the rates of superoxide production
(H
2
O
2
release) in isolated skeletal-muscle mitochondria respiring
on glutamate/malate, succinate and succinate + glutamate/malate.
In agreement with previous studies [7–9,14], we find that H
2
O
2
release with glutamate + malate is low (∼ 30 pmol of H
2
O
2
/min
per mg of mitochondrial protein) as compared with that obtained
with succinate (∼ 400 pmol of H
2
O
2
· min
−1
· mg
−1
); we note
though that the rates of H
2
O
2
release with succinate are low
as compared with the literature values (discussed below). The
rate of H
2
O
2
release with succinate + glutamate + malate was
much higher than that with succinate alone, comparable with
that observed in mitochondria inhibited with antimycin A
(which is known to dramatically increase superoxide production).
We note though that both the rates of H
2
O
2
release with
succinate alone (range: ∼ 30–900 pmol of H
2
O
2
· min
−1
· mg
−1
)
and succinate + glutamate + malate (range: ∼ 60–2600 pmol of
H
2
O
2
· min
−1
· mg
−1
) were highly variable (in contrast with that
observed with glutamate + malate alone). In other words, the rates
of superoxide production with succinate + glutamate + malate
were sometimes as low as those observed with glutamate/malate
alone, although on average, they were much higher than with
succinate. We next investigated the source of this variability.
BSA stimulates superoxide production by
succinate-reverse-electron transfer, but is inhibitory to superoxide
production with other substrate–inhibitor combinations
In our laboratory, the rates of succinate-driven reverse-electron
transfer-dependent superoxide production in skeletal-muscle
mitochondria are highly variable and generally on the low end
Figure 1 BSA stimulates the rate of superoxide production with succinate
Superoxide production (H
2
O
2
release) was measured as described in the Experimental section.
The graph shows the rate of superoxide production (
y
-axis) as a function of the amount of added
BSA (
x
-axis). The experimental preparation contained 0.25 mg/ml of mitochondria with 10 mM
succinate alone and 10 mM succinate + 10 µM rotenone. Different concentrations of BSA
(expressed as weight by volume) were added to mitochondria prior to the addition of substrate.
Each data point represents the mean
+
−
S.E.M. (
n
= 3). The rates of superoxide production with
succinate alone are represented by the open diamonds and those of succinate + rotenone
with filled squares.
(e.g. [37]) of the literature values, although still higher than those
with glutamate/malate. We identified the presence or absence of
BSA as one source of the variability in the rates of superoxide
production with succinate. In our protocol, BSA is added in the
isolation procedure, but not in the reaction buffer. Some previous
studies have added while others have omitted BSA in H
2
O
2
measurement media [7–9,12,13,38]. We discovered that omitting
the wash step after the addition of BSA in the isolation procedure
yielded mitochondria with consistently high rates of superoxide
production with succinate. We investigated the effect of BSA
on superoxide production by adding different concentrations
directly to the assay medium. Results in Figure 1 show that
superoxide production with succinate alone is considerably
increased by the addition of BSA (from ∼ 350 to 900 pmol of
H
2
O
2
· min
−1
· mg
−1
) at low concentration (0.05 %, w/v), although
high concentrations were found to be inhibitory (> 0.3 %,w/v).
Results also show that superoxide production with succinate +
rotenone is essentially unchanged, while surprisingly, that
with succinate + rotenone + antimycin A was dose-dependently
decreased (results not shown). This was not due to BSA
removing the inhibitor, because even at high concentrations
of BSA (0.6 %), antimycin A and rotenone still completely
inhibited membrane potential formation (Safranin O) and ATP
synthesis (results not shown). We performed these experiments
with BSA purchased from Sigma (essentially fatty acid-free,
fraction V), as well as extra-purified BSA ([39], a gift from Dr
Paul S. Brookes (Mitochondrial Research and Innovation Group,
University of Rochester Medical Center, Rochester, NY, U.S.A.),
with essentially indistinguishable results (results not shown).
Glutamate consistently stimulates while malate consistently
inhibits succinate-supported superoxide production
To resolve the variability of the rates of H
2
O
2
production with
glutamate + malate + succinate, we dissected the experiment into
its components and measured the rates of H
2
O
2
release with
glutamate + succinate and malate + succinate. The results in
Table 2 show that H
2
O
2
release with glutamate + succinate is
consistently high (∼ 1000 pmol of H
2
O
2
· min
−1
· mg
−1
) and with
malate + succinate is consistently low (∼ 20 pmol of H
2
O
2
·
min
−1
· mg
−1
). This was true whether BSA was present or
c
The Authors Journal compilation
c
2008 Biochemical Society
494 F. L. Muller and others
Table 2 Superoxide production with succinate is stimulated by glutamate
but inhibited b y malate
Superoxide production was measured indirectly as H
2
O
2
release. The substrates glutamate
(10 mM) and malate (10 mM) were used separately rather than together (in the presence or
absence of 10 mM succinate) and the effect of adding 0.05% BSA on the rate of superoxide
production was also determined. The means
+
−
S.E.M. for four experiments are shown. Note the
dramatic increase in H
2
O
2
release when glutamate and succinate are added together, while with
malate, the opposite occurs.
Substrate(s) BSA (%) d[H
2
O
2
]/d
t
(pmol · min
−1
· mg
−1
)
Malate 0 14.89
+
−
2.69
Glutamate 0 47.26
+
−
10.85
Succinate 0 289.45
+
−
80.11
Malate + succinate 0 20.29
+
−
1.65
Glutamate + succinate 0 976.08
+
−
131.09
Malate 0.05 10.24
+
−
1.45
Glutamate 0.05 65.29
+
−
20.37
Succinate 0.05 907.37
+
−
98.6
Malate + succinate 0.05 56.73
+
−
40.58
Glutamate + succinate 0.05 1312.94
+
−
125.52
Figure 2 Superoxide production with succinate + glutamate is observed
even at low substrate concentrations
Superoxide production (indirectly measured as H
2
O
2
release) was determined as described
in the Experimental section, with 0.2 µg/µl of mitochondrial protein. (A) Effect of varying
concentrations of succinate (
x
-axis) on the rate of superoxide production (
y
-axis), in the
absence (open diamonds) or presence of 0.5 mM (grey triangles) or 5 mM (black squares)
glutamate. The experiment shown in (B) was conducted exactly as in (A) except that 0.05 %
BSA was added to all reactions. The graphs show the means
+
−
S.E.M. for five independent
mitochondrial preparations. The range of succinate concentration was 0.25–2 mM.
not, although BSA further increased the rate of H
2
O
2
release
with glutamate + succinate (from ∼ 1000 to ∼ 1300 pmol of
H
2
O
2
· min
−1
· mg
−1
; Table 2). We constructed titration curves
to show the effects of succinate and glutamate or malate on
the rates of H
2
O
2
release. The results in Figure 2 indicate that
contrary to what is observed with succinate alone, the rates of
H
2
O
2
release with glutamate + succinate remain high even at
substrate concentrations below 1 mM. In fact, greater relative
stimulation of H
2
O
2
release by glutamate was observed when
succinate concentrations were low, especially in the absence
of BSA (Figure 2). For example, at 1 mM succinate (in the
Figure 3 Inhibition of superoxide production by malate and oxaloacetate
with varying concentrations of succinate
Superoxide production (H
2
O
2
release) was measured as in Figure 2, with 0.2 µg/µlof
mitochondrial protein. (A) Inhibitory effect of malate or oxaloacetate (5 mM) on superoxide
production with increasing concentrations of succinate. Each data point represents the
mean
+
−
S.E.M. for three independent mitochondrial preparations. (B)Sameasin(A) except
that 0.05% BSA was added to all reactions.
absence of added BSA), the rates of H
2
O
2
release were < 30 pmol
of H
2
O
2
· min
−1
· mg
−1
with succinate alone, but > 400 pmol of
H
2
O
2
· min
−1
· mg
−1
with the further addition of either 0.5 or
5 mM glutamate (> 11-fold increase). At 10 mM succinate,
adding glutamate increased H
2
O
2
release from ∼ 300 to 950 pmol
of H
2
O
2
· min
−1
· mg
−1
(∼ 3-fold increase). The effect of the
interaction of glutamate and succinate on superoxide production
was ‘saturable’: at a succinate concentration of 25 mM, adding
glutamate did not further increase H
2
O
2
release in the presence
of BSA; at the same time, increasing the concentration of
glutamate to 25 mM did not result in any further changes in
superoxide production with succinate (results not shown). Just
as the stimulatory effect of glutamate was more pronounced
at lower concentrations of succinate, so too was the inhibitory
effect of malate. The inhibition of succinate-supported superoxide
production by malate was virtually complete at concentrations
of succinate below 5 mM (Figure 3); there appears to be a
competitive effect between these two substrates. When succinate
concentration was raised to 25 mM, the inhibitory effect of malate
was alleviated (results not shown).
In order to determine why glutamate was stimulatory, while
malate was inhibitory, we asked how other complex I-linked
substrates would affect the rate of H
2
O
2
release with succinate.
We found that pyruvate and α-ketoglutarate, as well as palmitoyl
carnitine, stimulated superoxide production with succinate, to the
same extent as glutamate (results not shown). We hypothesized
that the inhibitory effect of malate on H
2
O
2
release with succinate
was due to the formation of oxaloacetate, a highly potent
competitive inhibitor of complex II. Indeed, as results in Figure 3
c
The Authors Journal compilation
c
2008 Biochemical Society
Superoxide production by complex I 495
Figure 4 Superoxide production with glutamate + succinate is dependent on protonmotive force and is decreased by rotenone
Superoxide production and incubation conditions were the same as described in Figure 2. (A) Effect of rotenone on the rate of superoxide production with 5 mM glutamate, 5 mM succinate and
5 mM glutamate + succinate in the absence (black bars) or presence (white bars) of 150 nM FCCP. Experiments in (B) were conducted exactly as described in (A) except that 0.05% BSA was added
to all reactions. Each bar represents the mean for five independent mitochondrial preparations in (A) and three preparations in (B). Error bars show S.E.M. Note the strong inhibitory effect of FCCP
and rotenone on superoxide production with succinate as well as glutamate + succinate.
show, oxaloacetate was even more effective than malate (on a
concentration basis), at inhibiting H
2
O
2
release with succinate.
When employing oxaloacetate, we took precautions to minimize
the time on ice, because oxaloacetate decays to pyruvate (P.S.
Brookes, personal communication). We noticed that the inhibitory
potency of oxaloacetate became weakened as the aliquots were
kept on ice (results not shown).
Superoxide production with succinate + glutamate i s dependent
on membrane potential and is decreased by rotenone
To identify the site of electron leakage responsible for the high
rates of superoxide production with glutamate + succinate, we
asked how rotenone and FCCP, two agents that dramatically
decrease reverse-electron transfer [7–9,12,13], would affect the
rate of superoxide production with glutamate + succinate. Results
in Figure 4 (compare white and black bars) show that just as
for succinate alone, superoxide production with glutamate +
succinate is completely eliminated by the addition of the
uncoupler FCCP, indicating complete dependence on proton-
motive force (this was the case whether BSA was present or not).
Results in Figure 4 also show that superoxide production with
glutamate + succinate is decreased by ∼ 20 % by the addition of
rotenone in the absence and by ∼ 60 % decrease in the presence
of BSA. The reason for this apparently stronger rotenone in-
hibition in the presence of BSA is that the rates of superoxide
production are higher with glutamate + succinate in the presence
of BSA (as compared with glutamate + succinate without BSA).
The fact that superoxide production with glutamate + succinate
is not further decreased can be partly explained by the fact that
superoxide production in the forward-electron transfer direction
is greatly stimulated by rotenone [8,9,14], a process further
stimulated by imposition of a protonmotive force (as would
happen when succinate is added to mitochondria respiring on
glutamate but inhibited with rotenone [14]). Indeed, the rates of
superoxide production with glutamate + succinate + rotenone are
noticeably decreased by the addition of FCCP.
DISCUSSION
In the present study, we address a contentious and unresolved
question in mitochondrial ROS metabolism: can reverse-
electron transfer-mediated superoxide production, the strongest
source of ROS in isolated mitochondria, actually occur under
substrate conditions that are physiologically plausible [8,14,40–
42]? The major arguments against are that supraphysiological
concentrations of succinate are required [8] and that it putatively
cannot occur when NAD
+
, the terminal electron acceptor
during reverse-transfer, is not available [18], i.e. the in vivo
concentrations of succinate are low and NAD is predominantly in
the reduced state).
To address these outstanding issues, we have measured the
rates of reverse-electron transfer-mediated superoxide production
as a function of succinate concentration and in the presence or
absence of NADH-linked substrates, in isolated skeletal-muscle
mitochondria. We find that high rates of superoxide production
can occur at low substrate concentrations and when the NAD
+
pool is reduced. While the present study was in progress, a
recent article by Zoccarato et al. [26] in brain mitochondria
reached the same conclusion. The main advance, as compared
with Zoccarato et al.’s [26] report, is that the present study
shows that certain NAD-linked substrates actually stimulate
succinate-supported superoxide production, while others are
c
The Authors Journal compilation
c
2008 Biochemical Society
496 F. L. Muller and others
inhibitory and that this can be explained by oxaloacetate
inhibition of succinate dehydrogenase. Further, our study provides
evidence that superoxide production with NAD-linked substrates
in combination with succinate is due to reverse-electron transfer
through complex I.
High rates of superoxide production with glutamate + succinate
are observed even at low substrate concentration and are due to
reverse-electron transfer
Succinate-supported reverse-electron transfer is, according to
most studies, the strongest source of superoxide in isolated
mitochondria; in our hands, it is highly variable, and on average,
is in the low range of previously published estimates [43]. During
the course of the present study, we discovered that the source
of this variability and lower values was due to omission of BSA
in the Amplex
TM
Red assay medium (Figure 1). A very recent
report in brain mitochondria came to the same conclusion [41].
BSA is typically added to respiratory experiments because it
increases the respiratory control ratio, which is generally felt
to be due to the removal of non-esterified (‘free’) fatty acids
(which can act as uncouplers [44,45]). BSA can also act as a
calcium buffer [46]. It is known that succinate-reverse-electron
transfer-mediated superoxide production is extremely sensitive to
even small decreases in protonmotive force, much more so than
other substrate/inhibitor conditions [7–9,47,48]. Tretter et al. [41]
concluded that BSA increases superoxide production with
succinate by hyperpolarizing protonmotive force; we also find
that adding BSA increases the strength of the membrane potential
as measured by Safranin O with succinate, although it has no
dramatic effects with other substrates (results not shown). It is
debatable whether the addition of BSA is warranted: one might
argue that it restores respiratory control ratios to what they would
be in vivo (presumably very tightly coupled mitochondria), or that
it introduces an experimental artefact. Because non-esterified fatty
acids are released from mitochondria as the preparation ‘ages’ on
ice [45], we are inclined to accept the former proposition. Because
we cannot resolve this issue and to make our results easier to
compare with those in the literature, we performed all subsequent
experiments both in the presence and in the absence of 0.05 %
fatty acid-free BSA.
Even with BSA present (although more evident in its absence),
we find that, in agreement with the previous study by Hansford
et al. [8], superoxide production with succinate exhibited a
strong substrate concentration dependence, becoming essentially
negligible below 1 mM succinate (Figure 2). While most previous
studies have used concentrations of succinate between 10 and
5 mM succinate [8,9,49], the in vivo concentration of succinate
has been reported to b e < 0.5 mM in heart [50] and < 0.3 mM
in brain [51]. Studies in cell culture suggest levels as low as
120 µM intracellular succinate concentration [52]. These results
would indicate that succinate concentrations are far too low under
normal in vivo conditions to allow for high rates of succinate-
driven reverse-electron transfer-mediated superoxide production
(but see below).
Because NAD
+
is the terminal electron acceptor during reverse-
electron transfer from succinate [18], it is thought that the presence
of NADH would be inhibitory towards reverse-electron transfer-
mediated superoxide production. We therefore measured the rates
of superoxide production with both succinate and NADH-linked
substrates. Unexpectedly, we discovered that the combination of
both NADH-linked substrates (glutamate/malate) and succinate
resulted in higher rates of superoxide production than with suc-
cinate alone. The rates were highly variable, raging from as little
as 30 to over 2000 pmol of H
2
O
2
/min per mg of mitochondrial pro-
tein (Table 1). This extreme variability could be eliminated when
the glutamate + malate + succinate combination was broken
down to glutamate + succinate and malate + succinate, the former
combination resulting in consistently high rates of superoxide
production and the latter in consistently low rates (this held
true whether BSA was present or not; Table 2). We thus suggest
that the apparent variability of the succinate + glutamate + malate
measurement (Table 1) is the result of competing actions of
glutamate (stimulatory) and malate (inhibitory).
We performed concentration dependence measures of the rate
of superoxide production with glutamate + succinate and suc-
cinate + malate. While the rate of superoxide production with
succinate alone decreases dramatically at lower concentrations
of substrate, that with a combination of glutamate (either 5 or
0.5 mM) + succinate remains considerable even at concentrations
as low as 0.25 mM succinate (Figure 2). At 0.5 mM glutamate +
1 mM succinate, the rates of superoxide production were as high
or higher (depending on whether BSA was present or not) as
those observed with 10 mM succinate alone (compare Table 2 and
Figure 2). Just as the stimulatory effect of glutamate was more
evident at a low concentration of succinate, the inhibitory effect
of malate on the superoxide production with succinate + malate
was also more pronounced at low concentrations of succinate
(Figure 3 and see the Results section).
We are aware of only two other publications comparing the
rates of superoxide production with succinate versus succinate +
glutamate + malate in mitochondria. In Hansford et al.’s
[8] report, the succinate + glutamate + malate experiment was
performed only once (Table 1 in [8]), resulting in considerably
lower superoxide production than with succinate alone. In a
very recent publication, Zoccarato et al. [26] reported that
moderate concentrations (< 3 mM) of glutamate + malate only
partially inhibited the rate of superoxide production with suc-
cinate in brain mitochondria (in experiments performed with
BSA [26]), thus leading them to conclude that high rates of
superoxide production could indeed occur under physiologically
realistic substrate conditions. This inhibition was modest unless
the concentration of succinate was < 1 mM, very similar to our
results with malate in Figure 3, showing that malate inhibition
of succinate supporter superoxide production is much more
pronounced at low concentrations of succinate. Because both
Hansford et al. [8] and Zoccarato et al. [26] used a combination
of glutamate + malate + succinate, rather than separating the
experiment into the glutamate + succinate and malate + succinate
components, they were unable to tease apart the competing effects
of glutamate (stimulatory) and malate (inhibitory). As far as
we are aware, the present study is the first to do so. Our data
could explain the contrasting results of Hansford et al. [8] and
Zoccarato et al. [26]. While one could invoke organ differences
(rat heart compared with rat brain mitochondria), Zoccarato et al.
[26] indicated that heart mitochondria behaved similar to brain
mitochondria. Zoccarato et al. [26] used BSA in their assay
medium, while Hansford et al. [8] did not; whereas Zoccarato et al.
[26] used a maximum of 3 mM glutamate/malate, Hansford
et al. [8] used 5 mM. Our results indicate that the inhibitory
effect of malate is dose-dependent and is more evident in the
absence of BSA than in its presence. Regarding the question
of whether high rates of superoxide production can occur under
physiologically relevant substrate conditions, we must point out
that contrary to Zoccarato et al.’s [26] results, who showed that
glutamate + malate exerted stronger inhibitory effects on
superoxide production at lower (i.e. more physiological) succinate
concentrations, our results demonstrate that glutamate added in
combination with succinate provided stronger relative stimulation
at lower succinate concentrations.
c
The Authors Journal compilation
c
2008 Biochemical Society
Superoxide production by complex I 497
We next queried the origin of superoxide produced in the
presence of both NADH-linked and complex II-linked substrates
(i.e. with glutamate + succinate). In Zoccarato et al.’s [26] study,
it was demonstrated that glutamate oxidation was still proceeding
(albeit at a depressed rate) when glutamate + malate + succinate
were added together, indicating that forward-electron transfer
was taking place, although the authors concluded that in all
likelihood, forward- and reverse-electron transfers were occurring
concomitantly. Zoccarato et al. [26] also demonstrated that
addition of nigericin (which collapses pH) partially decreases
H
2
O
2
release with glutamate + malate + succinate, which is con-
sistent with reverse-electron transfer being responsible for
superoxide production (although not conclusive, because forward-
electron transfer-mediated superoxide production can also be
partially decreased by nigericin [7,14]). No further attempt was
made to identify the site of superoxide production.
We found that as with succinate alone, glutamate + succinate
supported superoxide production could be essentially eliminated
by the addition of FCCP, indicating complete dependence on
protonmotive force (compare glutamate + succinate in black and
white bars of Figure 4). In this regard, superoxide production
with glutamate + succinate behaves very similarly to succinate-
mediated reverse-electron transfer ([8,9,47] and see Figure 4).
Reverse-electron transfer can be inhibited to a large extent by
the addition of rotenone, which prevents ubiquinol reduction of
complex I [9,10]. We found that rotenone can indeed decrease
the rate of superoxide production with glutamate + succinate
(Figure 4). This effect was modest in the absence of BSA (∼ 20 %
decrease), but could inhibit the rate of superoxide production with
glutamate + succinate by over 60 % in the presence of BSA.
This was due to the fact that BSA yielded higher rates of
superoxide production with glutamate + succinate (although this
stimulatory effect was less pronounced as compared with what
we found with succinate alone, Figure 2) and led to lower rates
of superoxide production with glutamate + succinate + rotenone,
as compared with experiments performed in the absence of
BSA. Why BSA would decrease the rate of superoxide pro-
duction with glutamate + succinate + rotenone is at present
unclear; we consistently observed this effect, more so with
higher concentrations of BSA (results not shown). Regardless, the
decrease in superoxide production with glutamate + succinate +
rotenone is all the more noteworthy considering that rotenone
stimulates superoxide production when electrons are added
via NADH [8,9,12–15,53–55]. If one adds to that the rate
of superoxide production with succinate + rotenone alone, the
additive effects of the rates of superoxide production with
glutamate + rotenone and rotenone + succinate can account for
∼ 60 % of the rate of superoxide production with glutamate +
succinate + rotenone (Figure 4). There appears to be a synergistic
effect between superoxide production with succinate + rotenone
and glutamate + rotenone (i.e. the actual measurement is higher
than the sum of its individual components): this is probably the
result of imposing a protonmotive force on rotenone-inhibited
complex I, probably caused by the same underlying phenomenon
as the observation by Lambert and Brand [14] that adding
ATP to mitochondria treated with glutamate/malate + rotenone
results in a further increase in superoxide production (by
imposing a protonmotive force by hydrolysis of ATP [14]).
Consistent with this idea, while the rate of superoxide formation
with glutamate + rotenone was unaffected by the addition of
FCCP, a large fraction of the rate of superoxide production
with glutamate + succinate + rotenone was in fact dependent on
protonmotive force (Figure 4). Based on these considerations,
we argue that the 20–60 % inhibition by rotenone of the rate of
superoxide production with glutamate + succinate is a minimum
estimate of the fraction that is due to reverse-electron transfer, and
we suggest that the majority probably originates from this source.
Taken together, these results indicate that high rates of reverse-
electron transfer-mediated superoxide production can occur in
situations that cause even a moderate increase in succinate
concentrations (even if NAD is in the reduced state). One might
expect elevations in succinate concentrations during ischaemia,
which could then result in high rates of reverse-electron transfer-
mediated superoxide production during reperfusion. In support
of this idea, administration of rotenone, which prevents reverse-
electron transfer, is actually protective in mouse models of
ischaemia/reperfusion injury [56,57].
The divergent effects of glutamate and malate on
succinate-supported superoxide production can be explained
by oxaloacetate inhibition of complex II
In addition to glutamate, other NADH-linked substrates, pyruvate,
α-ketoglutarate and palmitoyl carnitine, stimulated superoxide
production in combination with succinate (to a similar extent as
glutamate), while citrate and isocitrate left the rate of superoxide
production in combination with succinate unchanged (results
not shown). Thus only malate was inhibitory towards succinate-
supported superoxide production.
We propose that these contrasting effects on superoxide pro-
duction can be explained by ‘relieving’ the well-known inhibition
of complex II by oxaloacetate [27,28]. Succinate alone as a
substrate only supports weak state 3 respiration rates [31] because
of oxaloacetate formation, and inhibition, during continuous
succinate oxidation. The addition of rotenone is necessary not
only to prevent reverse-electron transfer (which would reduce P/O
ratios), but also to block oxaloacetate formation: rotenone blocks
oxaloacetate formation indirectly by locking the NAD pool in the
reduced state, thereby inhibiting the NAD
+
-dependent oxidation
of malate to oxaloacetate [27].
Why malate inhibits succinate-supported superoxide produc-
tion becomes evident when considering that malate is converted
into oxaloacetate by malate dehydrogenase, which accumulates
unless acetyl-CoA is available. In direct support of the hypo-
thesis that malate conversion into oxaloacetate is responsible
for inhibiting succinate reverse-electron transfer superoxide
production, we found that direct addition of oxaloacetate
was indeed profoundly inhibitory to superoxide production
with succinate (Figure 3). That oxaloacetate is a potent
competitive (competitive with succinate) inhibitor of complex
II/succinate dehydrogenase has been known since the 1970s,
although the physiological significance of this inhibition has
so far remained unclear [27]. Based on the above results,
we speculate that oxaloacetate inhibition of complex II is
a deliberate mechanism evolved to minimize reverse-electron
transfer-mediated superoxide production.
In this hypothesis, the stimulatory effect of pyruvate, glutamate,
α-ketoglutarate and palmitoyl carnitine can be explained by the
fact that, one way o r another, their immediate metabolism leads
to the consumption of oxaloacetate, and hence to a relief of the
inhibition of succinate dehydrogenase (allowing succinate-driven
reverse-electron transfer). In the case of pyruvate, oxaloacetate
is consumed by citrate synthase concomitantly with acetyl-CoA
generated by pyruvate dehydrogenase [44,58]. Glutamate leads to
the removal of oxaloacetate because glutamate and oxaloacetate
are co-substrates for transaminases yielding aspartate and α-keto-
glutarate [44,58]. The α-ketoglutarate carrier exchanges malate
for α-ketoglutarate [44], and palmitoyl carnitine generates acetyl-
CoA, which is again used by citrate synthase to consume
c
The Authors Journal compilation
c
2008 Biochemical Society
498 F. L. Muller and others
oxaloacetate [44,58]. A detailed summary of the metabolism of
these classical mitochondrial substrates has been compiled by
E. Gnaiger (Oroboros protocols at http://www.oroboros.at).
In summary, we have found that skeletal-muscle mitochondria
respiring on both complex I- and complex II-linked substrates
show high rates of reverse-electron transfer-mediated superoxide
production even when substrate concentrations are low. These
results counter some of the major arguments against the possibility
that superoxide production by this pathway can occur in vivo.
The results also suggest a physiological role for the long known
phenomenon of oxaloacetate inhibition of complex II, namely the
minimization of reverse-electron transfer-mediated superoxide
production. The finding that reverse-electron transfer-mediated
superoxide production can still occur in the presence of NAD-
linked substrates has important implications for the mechanism of
superoxide production by complex I as well as the mechanism
of its normal catalytic turnover. The minimal interpretation of
these results, we feel, is that the pathways of electron transfer in
the forward and reverse direction are different and branched at
one point or another.
We acknowledge the many useful discussions with Dr Paul S. Brookes and Dr Adrian
Lambert (Medical Research Council Dunn Human Nutrition Unit, Cambridge, U.K.). We
also thank Dr Sergey I. Dikalov (Free Radicals in Medicine Core, Division of Cardiology,
Emory University School of Medicine, Atlanta, GA, U.S.A.) for directing our attention
to the phenomenon of oxaloacetate inhibition. Financial support was provided by NIA
(National Institute of Aging) training grant no. 5T3-AG021890-02 (F. L. M.), NIH (National
Institutes of Health) grants P01AG19316 and P01AG020591 and the San Antonio Nathan
Shock Aging Center (1P30-AG13319). We also thank Dr Arlan Richardson (Director,
Barshop Institute for Longevity and Aging Studies) for constant encouragement and
support.
REFERENCES
1 Muller, F. L., Lustgarten, M. S., Jang, Y., Richardson, A. and van Remmen, H. (2007)
Trends in oxidative aging theories. Free Radical Biol. Med. 43, 477–503
2 Beal, M. F. (1996) Mitochondria, free radicals, and neurodegeneration.
Curr. Opin. Neurobiol. 6, 661–666
3 Beckman, K. B. and Ames, B. N. (1998) The free radical theory of aging matures.
Physiol. Rev. 78, 547–581
4 Halliwell, B. and Gutteridge, J. (1999) Free Radicals in Biology and Medicine, Oxford
University Press, New York
5 Muller, F. (2000) The nature and mechanism of superoxide production by the electron
transport chain: its relevance to aging. J. Am. Aging Assoc. 23, 227–253
6 Loschen, G., Azzi, A., Richter, C. and Flohe, L. (1974) Superoxide radicals as precursors
of mitochondrial hydrogen peroxide. FEBS Lett. 42, 68–72
7 Lambert, A. J. and Brand, M. D. (2004) Superoxide production by NADH:ubiquinone
oxidoreductase (complex I) depends on the pH gradient across the mitochondrial inner
membrane. Biochem. J. 382, 511–517
8 Hansford, R. G., Hogue, B. A. and Mildaziene, V. (1997) Dependence of H
2
O
2
formation
by rat heart mitochondria on substrate availability and donor age. J. Bioenerg. Biomembr.
29, 89–95
9 Votyakova, T. V. and Reynolds, I. J. (2001) DeltaPsi(m)-dependent and -independent
production of reactive oxygen species by rat brain mitochondria. J. Neurochem. 79,
266–277
10 Cino, M. and Del Maestro, R. F. (1989) Generation of hydrogen peroxide by brain
mitochondria: the effect of reoxygenation following postdecapitative ischemia. Arch.
Biochem. Biophys. 269, 623–638
11 Zoccarato, F., Cavallini, L., Deana, R. and Alexandre, A. (1988) Pathways of hydrogen
peroxide generation in guinea pig cerebral cortex mitochondria. Biochem. Biophys.
Res. Commun. 154, 727–734
12 Starkov, A. A., Polster, B. M. and Fiskum, G. (2002) Regulation of hydrogen peroxide
production by brain mitochondria by calcium and Bax. J. Neurochem. 83, 220–228
13 Starkov, A. A. and Fiskum, G. (2003) Regulation of brain mitochondrial H
2
O
2
production
by membrane potential and NAD(P)H redox state. J. Neurochem. 86, 1101–1107
14 Lambert, A. J. and Brand, M. D. (2004) Inhibitors of the quinone-binding site allow rapid
superoxide production from mitochondrial NADH:ubiquinone oxidoreductase (complex I).
J. Biol. Chem. 279, 39414–39420
15 Gyulkhandanyan, A. V. and Pennefather, P. S. (2004) Shift in the localization of sites of
hydrogen peroxide production in brain mitochondria by mitochondrial stress.
J. Neurochem. 90, 405–421
16 Boveris, A., Oshino, N. and Chance, B. (1972) The cellular production of hydrogen
peroxide. Biochem. J. 128, 617–630
17 Loschen, G. and Azzi, A. (1976) On the formation of hydrogen peroxide and oxygen
radicals in heart mitochondria. Recent Adv. Stud. Cardiac Struct. Metab. 7,3–12
18 Hinkle, P. C., Butow, R. A., Racker, E. and Chance, B. (1967) Partial resolution of the
enzymes catalyzing oxidative phosphorylation. XV. Reverse electron transfer in the
flavin-cytochrome beta region of the respiratory chain of beef heart submitochondrial
particles. J. Biol. Chem. 242, 5169–5173
19 Staniek, K. and Nohl, H. (2000) Are mitochondria a permanent source of reactive oxygen
species? Biochim. Biophys. Acta 1460, 268–275
20 Forman, H. J. and Azzi, A. (1997) On the virtual existence of superoxide anions in
mitochondria: thoughts regarding its role in pathophysiology. FASEB J. 11, 374–375
21 Adam-Vizi, V. and Chinopoulos, C. (2006) Bioenergetics and the formation of
mitochondrial reactive oxygen species. Trends Pharmacol. Sci. 27, 639–645
22 Krishnamoorthy, G. and Hinkle, P. C. (1988) Studies on the electron transfer pathway,
topography of iron-sulfur centers, and site of coupling in NADH-Q oxidoreductase.
J. Biol. Chem. 263, 17566–17575
23 Chen, Q., Vazquez, E. J., Moghaddas, S., Hoppel, C. L. and Lesnefsky, E. J. (2003)
Production of reactive oxygen species by mitochondria: central role of complex III.
J. Biol. Chem. 278, 36027–36031
24 St-Pierre, J., Buckingham, J. A., Roebuck, S. J. and Brand, M. D. (2002) Topology of
superoxide production from different sites in the mitochondrial electron transport chain.
J. Biol. Chem. 277, 44784–44790
25 Muller, F. L., Liu, Y. H., Richardson, A. and Van Remmen, H. (2006) Two distinct sites of
superoxide production within complex I. Free Radical Biol. Med. 41, S122
26 Zoccarato, F., Cavallini, L., Bortolami, S. and Alexandre, A. (2007) Succinate modulation
of H
2
O
2
release at NADH:ubiquinone oxidoreductase (complex I) in brain mitochondria.
Biochem. J. 406, 125–129
27 Huang, L. S., Shen, J. T., Wang, A. C. and Berry, E. A. (2006) Crystallographic studies of
the binding of ligands to the dicarboxylate site of complex II, and the identity of the ligand
in the ‘oxaloacetate-inhibited’ state. Biochim. Biophys. Acta 1757, 1073–1083
28 Kearney, E. B., Ackrell, B. A. and Mayr, M. (1972) Tightly bound oxalacetate and the
activation of succinate dehydrogenase. Biochem. Biophys. Res. Commun. 49, 1115–1121
29 Ackrell, B. A., Kearney, E. B. and Mayr, M. (1974) Role of oxalacetate in the regulation of
mammalian succinate dehydrogenase. J. Biol. Chem. 249, 2021–2027
30 Chappell, J. B. and Perry, S. V. (1954) Biochemical and osmotic properties of skeletal
muscle mitochondria. Nature 173, 1094–1095
31 Ernster, L. and Nordenbrand, K. (1967) Skeletal muscle mitochondria. Methods Enzymol.
10, 86–94
32 Zhou, M., Diwu, Z., Panchuk-Voloshina, N. and Haugland, R. P. (1997) A stable
nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen
peroxide: applications in detecting the activity of phagocyte NADPH oxidase and other
oxidases. Anal. Biochem. 253, 162–168
33 Muller, F. L., Liu, Y. and Van Remmen, H. (2004) Complex III releases superoxide to both
sides of the inner mitochondrial membrane. J. Biol. Chem. 279, 49064–49073
34 Muller, F., Crofts, A. R. and Kramer, D. M. (2002) Multiple Q-cycle bypass reactions at the
Q
o
site of the cytochrome
bc
1
complex. Biochemistry 41, 7866–7874
35 Muller, F. L., Roberts, A. G., Bowman, M. K. and Kramer, D. M. (2003) Architecture of the
Q(o) site of the cytochrome
bc
(1) complex probed by superoxide production.
Biochemistry 42, 6493–6499
36 Akerman, K. E. and Wikstrom, M. K. (1976) Safranine as a probe of the mitochondrial
membrane potential. FEBS Lett. 68, 191–197
37 Genova, M. L., Ventura, B., Giuliano, G., Bovina, C., Formiggini, G., Parenti Castelli, G.
and Lenaz, G. (2001) The site of production of superoxide radical in mitochondrial
complex I is not a bound ubisemiquinone but presumably iron-sulfur cluster N2.
FEBS Lett. 505, 364–368
38 Starkov, A. A., Fiskum, G., Chinopoulos, C., Lorenzo, B. J., Browne, S. E., Patel, M. S.
and Beal, M. F. (2004) Mitochondrial alpha-ketoglutarate dehydrogenase complex
generates reactive oxygen species. J. Neurosci. 24, 7779–7788
39 Chen, R. F. (1967) Removal of fatty acids from serum albumin by charcoal treatment.
J. Biol. Chem. 242, 173–181
40 Liu, Y., Fiskum, G. and Schubert, D. (2002) Generation of reactive oxygen species by the
mitochondrial electron transport chain. J. Neurochem. 80, 780–787
41 Tretter, L., Mayer-Takacs, D. and Adam-Vizi, V. (2007) The effect of bovine serum albumin
on the membrane potential and reactive oxygen species generation in
succinate-supported isolated brain mitochondria. Neurochem. Int. 50, 139–147
42 Jezek, P. and Hlavata, L. (2005) Mitochondria in homeostasis of reactive oxygen species
in cell, tissues, and organism. Int. J. Biochem. Cell Biol. 37, 2478–2503
c
The Authors Journal compilation
c
2008 Biochemical Society
Superoxide production by complex I 499
43 Muller, F. L., Song, W., Jang, Y., Liu, Y., Sabia, M., Richardson, A. and Van Remmen, H.
(2007) Denervation-induced skeletal muscle atrophy is associated with increased
mitochondrial ROS production. Am. J. Physiol. Regulatory Integrative Comp. Physiol.
293, R1159–R1168
44 Nicholls, D. G. and Ferguson, S. J. (2002) Bioenergetics 3, Academic Press, London
45 Chan, S. H. and Higgins, Jr, E. (1978) Uncoupling activity of endogenous free fatty acids
in rat liver mitochondria. Can. J. Biochem. 56, 111–116
46 Pedersen, K. O. (1971) Binding of calcium to serum albumin. I. Stoichiometry and
intrinsic association constant at physiological pH, ionic strength, and temperature.
Scand. J. Clin. Lab. Invest. 28, 459–469
47 Miwa, S., St-Pierre, J., Partridge, L. and Brand, M. D. (2003) Superoxide and hydrogen
peroxide production by Drosophila mitochondria. Free Radical Biol. Med. 35, 938–948
48 Starkov, A. A. (1997) ‘Mild’ uncoupling of mitochondria. Biosci. Rep. 17, 273–279
49 Herrero, A. and Barja, G. (1998) H
2
O
2
production of heart mitochondria and aging rate are
slower in canaries and parakeets than in mice: sites of free radical generation and
mechanisms involved. Mech. Ageing Dev. 103, 133–146
50 Lewandowski, E. D., Doumen, C., White, L. T., LaNoue, K. F., Damico, L. A. and Yu, X.
(1996) Multiplet structure of
13
C NMR signal from glutamate and direct detection of
tricarboxylic acid (TCA) cycle intermediates. Magn. Reson. Med. 35, 149–154
51 Brockmann, K., Bjornstad, A., Dechent, P., Korenke, C. G., Smeitink, J., Trijbels, J. M.,
Athanassopoulos, S., Villagran, R., Skjeldal, O. H., Wilichowski, E. et al. (2002) Succinate
in dystrophic white matter: a proton magnetic resonance spectroscopy finding
characteristic for complex II deficiency. Ann. Neurol. 52, 38–46
52 Selak, M. A., Armour, S. M., MacKenzie, E. D., Boulahbel, H., Watson, D. G., Mansfield,
K. D., Pan, Y., Simon, M. C., Thompson, C. B. and Gottlieb, E. (2005) Succinate links TCA
cycle dysfunction to oncogenesis by inhibiting HIF-alpha prolyl hydroxylase. Cancer Cell
7, 77–85
53 Herrero, A. and Barja, G. (1997) Sites and mechanisms responsible for the low rate of free
radical production of heart mitochondria in the long-lived pigeon. Mech. Ageing Dev. 98,
95–111
54 Cadenas, E., Boveris, A., Ragan, C. I. and Stoppani, A. O. (1977) Production of
superoxide radicals and hydrogen peroxide by NADH-ubiquinone reductase and
ubiquinol-cytochrome
c
reductase from beef-heart mitochondria.
Arch. Biochem. Biophys. 180, 248–257
55 Takeshige, K. and Minakami, S. (1979) NADH- and NADPH-dependent formation of
superoxide anions by bovine heart submitochondrial particles and NADH-ubiquinone
reductase preparation. Biochem. J. 180, 129–135
56 Chen, Q., Moghaddas, S., Hoppel, C. L. and Lesnefsky, E. J. (2006) Reversible
blockade of electron transport during ischemia protects mitochondria and decreases
myocardial injury following reperfusion. J. Pharmacol. Exp. Ther. 319,
1405–1412
57 Ichikawa, H., Takagi, T., Uchiyama, K., Higashihara, H., Katada, K., Isozaki, Y., Naito, Y.,
Yoshida, N. and Yoshikawa, T. (2004) Rotenone, a mitochondrial electron transport
inhibitor, ameliorates ischemia-reperfusion-induced intestinal mucosal damage in rats.
Redox Rep. 9, 313–316
58 Voet, D. and Voet, J. G. (1990) Biochemistry, John Wiley and Sons, New York
Received 23 August 2007/4 October 2007; accepted 5 October 2007
Published as BJ Immediate Publication 5 October 2007, doi:10.1042/BJ20071162
c
The Authors Journal compilation
c
2008 Biochemical Society
