Báo cáo khoa học: Modular kinetic analysis reveals differences in Cd2+ and Cu2+ ion-induced impairment of oxidative phosphorylation in liver pot

Modular kinetic analysis reveals differences in Cd2+ and
Cu2+ ion-induced impairment of oxidative phosphorylation
in liver
Jolita Ciapaite1, Zita Nauciene1,2, Rasa Baniene2, Marijke J. Wagner3, Klaas Krab3 and
Vida Mildaziene1
1 Centre of Environmental Research, Faculty of Natural Sciences, Vytautas Magnus University, Kaunas, Lithuania
2 Institute for Biomedical Research, Kaunas Medical University, Lithuania
3 Department of Molecular Cell Physiology, Institute for Molecular Cell Biology, VU University, Amsterdam, The Netherlands

Keywords
cadmium and copper; lipid peroxidation;
metabolic control analysis; modular kinetic
analysis; oxidative phosphorylation
Correspondence
J. Ciapaite, Centre of Environmental
Research, Faculty of Natural Sciences,
Vytautas Magnus University, Vileikos 8,
LT-44404 Kaunas, Lithuania
Fax: +370 37 327916
Tel: +370 37 455193
E-mail:
(Received 29 January 2009, revised 18 April
2009, accepted 5 May 2009)
doi:10.1111/j.1742-4658.2009.07084.x

Impaired mitochondrial function contributes to copper- and cadmiuminduced cellular dysfunction. In this study, we used modular kinetic analysis and metabolic control analysis to assess how Cd2+ and Cu2+ ions
affect the kinetics and control of oxidative phosphorylation in isolated rat
liver mitochondria. For the analysis, the system was modularized in two
ways: (a) respiratory chain, phosphorylation and proton leak; and (b) coenzyme Q reduction and oxidation, with the membrane potential (Dw) and
fraction of reduced coenzyme Q as the connecting intermediate, respectively. Modular kinetic analysis results indicate that both Cd2+ and Cu2+
ions inhibited the respiratory chain downstream of coenzyme Q. Moreover,

Cu2+, but not Cd2+ ions stimulated proton leak kinetics at high Dw values. Further analysis showed that this difference can be explained by Cu2+
ion-induced production of reactive oxygen species and membrane lipid
peroxidation. In agreement with modular kinetic analysis data, metabolic
control analysis showed that Cd2+ and Cu2+ ions increased control of the
respiratory and phosphorylation flux by the respiratory chain module
(mainly because of an increase in the control exerted by cytochrome bc1
and cytochrome c oxidase), decreased control by the phosphorylation
module and increased negative control of the phosphorylation flux by the
proton leak module. In summary, we showed that there is a subtle difference in the mode of action of Cd2+ and Cu2+ ions on the mitochondrial
function, which is related to the ability of Cu2+ ions to induce reactive
oxygen species production and lipid peroxidation.

Many pollutants, even at low effective concentrations,
can harm living organisms by weakening their ability
to cope with long-term environmental challenges. At
excess amounts, the heavy metals cadmium and copper
are toxic and carcinogenic [1]. The ability of cadmium

and copper to accumulate in the bones, liver and kidneys determines their toxicity. Their deleterious effects
can be ameliorated to some extent by binding to
metallothionein [2]. Cellular dysfunction induced by
cadmium and copper is thought to involve alterations

Abbreviations
CiJP , flux control coefficient, quantifying the control of phosphorylation flux JP by module i; CiJR , flux control coefficient, quantifying the
control of respiratory flux JR by module i; CoQ, coenzyme Q; COX, cytochrome c oxidase; DCPIP, 2,6-dichlorophenolindophenol; JL, proton
leak flux; JP, phosphorylation flux; JR, respiratory flux; MCA, metabolic control analysis; ROS, reactive oxygen species; SDH, succinate
dehydrogenase; TBA, 2-thiobarbituric acid; TBARS, thiobarbituric acid reactive substances; XCoQ, fraction of reduced coenzyme Q;
Dp, proton-motive force; Dw, mitochondrial transmembrane electric potential.

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J. Ciapaite et al.

in mitochondrial metabolism. Initially, the accumulation of metal ions in mitochondria may protect the cell
against metal overload, however, later, their incorporation may cause complex disturbances in mitochondrial
function [3–5], resulting in severe defects in cellular
metabolism.
Cd2+ ions are transported into the mitochondria via
a Ca2+ -uniporter [6], whereas the accumulation of
Cu2+ in mitochondria proceeds via a different, energyindependent mechanism [7]. Both metal ions interact
with important functional groups (in particular, thiol
groups) in a variety of enzymes in the matrix and inner
mitochondrial
membrane
[8].
At
micromolar
concentrations, Cd2+ ions uncouple oxidative phosphorylation and inhibit respiration in actively
ADP-phosphorylating (state 3) isolated rat liver mitochondria [4]. At higher concentrations, Cd2+ ions inhibit succinate dehydrogenase (SDH) and H+-ATPase
[5,9]. Increasing the amount of Cu2+ ions added per mg
of mitochondrial protein has been shown to successively
cause inhibition of phosphate transport, accumulation
of K+ ions, membrane aggregation, stimulation of respiration in the absence of active ADP phosphorylation
(state 4), an increase in passive membrane permeability
to cations and anions, uncoupling and swelling, and
inhibition of respiration in state 3 [3,4]. Furthermore, it

has been suggested that Cu2+ ions inhibit SDH [10].
Cd2+ and Cu2+ ions induce cell death by necrosis
and apoptosis via mechanisms involving opening of
the mitochondrial permeability transition pore and
increased generation of reactive oxygen species (ROS)
[11–13]. In turn, metal-induced stimulation of ROS
production has been suggested to stem from both
increased ROS production by the mitochondrial respiratory chain and decreased activity of the antioxidant
enzymes [9,11,14–16].
Although the effects of Cd2+ and Cu2+ ions on
some individual mitochondrial enzymes and processes
have been studied extensively, few attempts have been
made to elucidate the mode of action of Cd2+ and
Cu2+ ions at the system level, which, in turn, would
allow us to understand the complex metabolic effects
of these substances.
Metabolic control analysis (MCA) is a useful tool
for studying complex biological systems because it
allows the quantification of the contribution made by
each system component to system behavior (e.g. fluxes,
metabolite concentrations) in terms of control coefficients [17,18]. In turn, knowledge of the system’s control structure is valuable in that it allows identification
of system components that are potentially most important in mediating the effects of external effectors on the
system (i.e. the component with the highest control

Metal-induced impairment of mitochondrial function

coefficient) [19]. A ‘top-down’ elasticity analysis (or
modular kinetic analysis) was developed to simplify
experimental assessment of the control structure of the
complex system via MCA [20], and was initially used to

study the control of fluxes and intermediates in the oxidative phosphorylation system [21]. The method is also
valuable in determining the sites of action of external
effectors within a system [22–27]. In this type of analysis, the system of interest is conceptually subdivided
into functional modules (reaction blocks) in such a way
that the selected modules interact via a single connecting intermediate. In the further analysis, each module is
treated as a single enzyme. Figure 1A shows how the
oxidative phosphorylation system can be subdivided
into three functional modules (respiratory chain, phosphorylating and proton leak module) with membrane

Fig. 1. Modularization of the system. Division of the oxidative phosphorylation system into (A) the respiratory chain, phosphorylation
and proton leak modules, with Dw as the connecting intermediate;
(B) the CoQ-reducing and CoQ-oxidizing modules with fraction of
reduced CoQ (XCoQ) as the connecting intermediate; and (C) the
CoQ-reducing, cytochrome bc1 + COX, phosphorylation and proton
leak modules with Dw and XCoQ as the connecting intermediates.
Succ, succinate; SDH, dicarboxylate carrier and succinate dehydrogenase; cyt bc1, cytochrome bc1, COX, cytochrome c oxidase, XCoQ,
fraction of reduced CoQ. Arrows marked e and h indicate electron
and trans-membrane proton flux, respectively (in this study all fluxes
were analyzed in terms of oxygen consumption flux).

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J. Ciapaite et al.

potential (Dw) as the connecting intermediate [21]. To

obtain module kinetics, titrations with module-specific
inhibitors are performed and the flux and level of the
connecting intermediate measured. Elasticity coefficients, quantifying sensitivity of the flux through the
module to a change in the level of the connecting intermediate, can be calculated from the slope of the inhibitor titration curves at a steady state. In turn, elasticity
coefficients and steady-state flux values can be used to
calculate the flux control coefficients of the modules
[21]. Repeating the procedure in the presence of a fixed
concentration of external effector reveals how that
effector affects the kinetics of each module and the
magnitude of the control that each module exerts
over system fluxes. The drawback of the ‘top-down’
approach to MCA is that it yields a coarse picture of
the control structure of the system. Different ways of
modularizing the system of interest may allow a more
resolved picture. However, this is often limited by the
feasibility of assigning modules that interact via a single
connecting intermediate [28].
In this study, we used modular kinetic analysis and
MCA to determine the effects of low concentrations
(5 lm) of CdCl2 and CuCl2 on the kinetics and control
of oxidative phosphorylation in isolated rat liver mitochondria respiring on succinate. To obtain a more
resolved picture of the effects of Cd2+ and Cu2+ ions
on the system, we subdivided the oxidative phosphorylation system into modules in different ways (Fig. 1).
We showed that at the concentration tested, both
metal ions inhibited respiratory chain module components downstream of coenzyme Q (CoQ). In addition,
Cu2+ ions increased the permeability of the inner
membrane to ions at high Dw levels. We tested a
hypothesis that the latter effect resulted from Cu2+
ion-induced formation of ROS and lipid peroxidation.

Results

ence of Cd2+ ions than in their absence, when compared at the same Dw value (Fig. 2B). The kinetics of
the proton leak and phosphorylation modules were not
significantly affected by Cd2+ ions (Fig. 2A,C), as indicated by similar values for the proton leak (JL) and
phosphorylation (JP) flux in the presence and absence
of CdCl2, when the fluxes are compared at the same
Dw value. Inhibition of the respiratory chain module
by Cd2+ ions resulted in a decrease in Dw by 6 mV in
state 3 (i.e. the state of maximal ADP phosphorylation)
(Table 1). Furthermore, Cd2+ ions decreased JR and
JP by 23 and 25%, respectively. However, Cd2+ ions
had no significant effect on JL in state 3.
Three-modular kinetic analysis of the effects of Cu2+
ions is shown in Fig. 3. Similar to Cd2+, Cu2+ ions
inhibited the respiratory chain module (Fig. 3B),
although to a lesser extent. Cu2+ ions had no significant
effect on the kinetics of the phosphorylation module
(Fig. 3C), but clearly stimulated proton leak kinetics
(Fig. 3A). The increase in JL was more prominent at
higher Dw values corresponding to state 4 (i.e. the state
with no ADP phosphorylation) (Fig. 3A). In state 4,
Cu2+ ions stimulated JL by 42% and caused a decrease
of 11 mV in Dw. In state 3, Cu2+ ions inhibited JR by
16% and JP by 17%, respectively, but had no significant
effect on JL (Table 1). Despite moderate effects on the
fluxes, in state 3 Cu2+ ions had a strong effect on Dw,
which decreased by 12 mV (Table 1).
It should be noted that the effects of Cd2+ and Cu2+
ions were determined in two separate series of experiments (performed in spring and autumn, respectively)

resulting in two sets of flux and Dw values under control
conditions (K1 and K2; Table 1). The difference
between the two data sets may have been caused by
hormone-related seasonal variations in mitochondrial
properties (e.g. the expression levels of the enzymes
involved in the process of oxidative phosphorylation),
as observed in different tissues in rodents [29,30].

Three-modular kinetic analysis of effects of Cd2+
and Cu2+ ions on oxidative phosphorylation

Bimodulular kinetic analysis of the effects of Cd2+
and Cu2+ ions on oxidative phosphorylation

To determine which oxidative phosphorylation components were affected by Cd2+ and Cu2+ ions in liver
mitochondria oxidizing succinate, we first used threemodular kinetic analysis with Dw as the connecting
intermediate (Fig. 1A). We assessed the effects of a
low metal ion concentration, which did not induce
mitochondrial swelling (results not shown).
Figure 2 shows the effect of 5 lm CdCl2 on the
kinetics of the three modules. The plots indicate that
Cd2+ ions inhibited the respiratory chain module
because the respiratory flux (JR) is lower in the pres-

The data in Figs 2 and 3 indicate that Cd2+ and Cu2+
ions affected the respiratory chain module. Therefore,
as a next step, we set out to pinpoint the components
of the respiratory chain module affected by Cd2+ and
Cu2+ ions. To achieve this, we conceptually subdivided the oxidative phosphorylation system into two
modules: (a) CoQ reducing, comprising dicarboxylate

carrier, fumarase and succinate dehydrogenase; and (b)
CoQ oxidizing, comprising cytochrome bc1, cytochrome c oxidase (COX) and the rest of the oxidative
phosphorylation system, including proton leak and

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J. Ciapaite et al.

Metal-induced impairment of mitochondrial function

JR (nmol O·min–1·mg protein–1)

JL (nmol O·min–1·mg protein–1)

A

B
State 3

150

150

State 4

50

120

140
160
Δψ (mV)

State 3

100

50
State 4

0
100

180

C

150

100

100

0
100

200

JP (nmol O·min–1·mg protein–1)

200

200

120

140
160
Δψ (mV)

180

50

0
100

120

140
160
Δψ (mV)

180

Fig. 2. Effect of Cd2+ ions on the kinetics of the proton leak module (A), the respiratory chain module (B) and the phosphorylation module
(C). The kinetics of the proton leak module were obtained by titrating with a specific inhibitor of the respiratory chain module, malonate
(0–12.5 lM), when phosphorylation module activity is fully blocked with oligomycin (0.7 lgỈmL)1). The kinetics of the respiratory chain

module were obtained by titrating with a specific inhibitor of the phosphorylation module, carboxyatractyloside (0–0.5 lM). The kinetics of
the phosphorylation module were obtained by titrating with a specific inhibitor of the respiratory chain module, malonate (0–3.125 lM), and
subsequently calculating JP by subtracting JL from JR at the same value of Dw [21]. JR, respiratory flux; JP, phosphorylation flux; JL, proton
leak flux. Open symbols, no CdCl2 added; closed symbols, plus 5 lM CdCl2. Average of n = 6 independent experiments ± SEM.

Table 1. Effect of Cd2+ and Cu2+ ions on system properties in
state 3. Average of n = 4 independent experiments ± SEM. K1 and
K2, control experiments with no CdCl2 or CuCl2 added; JR, respiratory flux; JP, phosphorylation flux; JL, proton leak flux.
K1
JR (nmol min)1
Ỉmg protein)1)
JP (nmol min)1
Ỉmg protein)1)
JL (nmol min)1
Ỉmg protein)1)
Dw (mV)

5 lM CdCl2

K2

5 lM CuCl2

171 ± 14

131 ± 16*

140 ± 7

118 ± 1*

162 ± 14

122 ± 15*

131 ± 6

109 ± 2*

8±1

9±2

143 ± 2

137 ± 2*

9±1
140 ± 3

10 ± 2
128 ± 1*

*P < 0.05 versus the condition with no CdCl2 or CuCl2 added.

enzymes involved in ATP synthesis. We used the fraction of CoQ (XCoQ) as the connecting intermediate
(Fig. 1B).
The results of bimodular kinetic analysis with XCoQ
as the connecting intermediate are presented in Fig. 4.
A similar JR value at any given XCoQ indicates that

the kinetics of the CoQ-reducing module was not significantly affected by either Cd2+ (Fig. 4B) or Cu2+
ions (Fig. 4D). Both metal ions inhibited the CoQ-oxidazing module (Fig. 4A,C) because lower JR values
were observed when comparison was made at the same
XCoQ level. Because three-modular analysis showed
that neither of the ions had any effect on the enzymes
involved in ATP synthesis (Figs 2C and 3C) or on the
proton leak kinetics close to state 3 (Figs 2A and 3A),
we can conclude that the site of action of Cd2+ and
Cu2+ ions must be cytochrome bc1 and ⁄ or COX.

Effects of Cd2+ and Cu2+ ions on the activity of
succinate dehydrogenase
Bimodular kinetic analysis showed that SDH (a component of the CoQ-reducing module) was not significantly affected by either Cd2+ or Cu2+ ions. However,
literature reports suggest that SDH is the target of
both metal ions [5,9,10]. To check whether data
obtained using modular kinetic analysis were correct,
we determined the effect of Cd2+ and Cu2+ ions on
SDH activity in isolated rat liver mitochondria. The
dependence of SDH activity on the concentration of
CdCl2 and CuCl2 is shown in Fig. 5A and B, respectively. At 5 lm neither CdCl2 nor CuCl2 had any
significant effect on SDH activity, which was
51 ± 8 nmol 2,6-dichlorophenolindophenol (DCPIP)Ỉ
min)1Ỉmg protein)1 under control conditions and
52 ± 7 and 48 ± 9 nmol DCPIPỈmin)1Ỉmg protein)1
in the presence of 5 lm CdCl2 and 5 lm CuCl2, respectively. A significant effect on SDH activity was
observed only at CdCl2 and CuCl2 concentrations
exceeding 10 lm (Fig. 5).
Effects of Cd2+ and Cu2+ ions on H2O2 production
and lipid peroxidation
We hypothesized that stronger stimulation of proton

leak kinetics by Cu2+ ions (Fig. 3A) compared with
Cd2+ ions (Fig. 2A) may be explained by the ability of
Cu2+ ions to stimulate ROS production and induce
peroxidation of the membrane lipids. Therefore, we

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Metal-induced impairment of mitochondrial function

A

150

B
State 3

150

100

50

120

140
160
Δψ (mV)

State 3

100

50
State 4

0
100

180

C

150

100

State 4

0
100

200
JP (nmol O·min–1·mg protein–1)

200
JR (nmol O·min–1·mg protein–1)

JL (nmol O·min–1·mg protein–1)

200

J. Ciapaite et al.

120

140
160
Δψ (mV)

180

50

0
100

120

140
160
Δψ (mV)

180

Fig. 3. Effect of Cu2+ ions on the kinetics of the proton leak module (A), the respiratory chain module (B) and the phosphorylation module
(C). The kinetics of the modules were obtained as described in the legend for Fig. 2. JR, respiratory flux; JP, phosphorylation flux; JL, proton
leak flux. Open symbols, no CuCl2 added; closed symbols, plus 5 lM CuCl2. Average of n = 5 independent experiments ± SEM.

200

State 3

150

200

State 3

100

50
0
20 30 40 50 60 70 80
Fraction of reduced CoQ (%)

250

JR (nmol O·min–1·mg protein–1)

B

150

100

50
0

20 30 40 50 60 70 80
Fraction of reduced CoQ (%)

250

C

200

JR (nmol O·min–1·mg protein–1)

250

A

State 3

150
100
50
0
0
20
40 60
80 100
Fraction of reduced CoQ (%)

JR (nmol O·min–1·mg protein–1)

JR (nmol O·min–1·mg protein–1)

250

D

200

State 3

150
100
50
0

0
20
40 60
80 100
Fraction of reduced CoQ (%)

assessed how Cd2+ and Cu2+ ions affect overall ROS
production in isolated mitochondria oxidizing succinate
in state 2 (i.e. the resting state with no ADP phosphorylation). Figure 6A shows that 5 lm CdCl2 (i.e. the
concentration used for modular kinetic analysis) had no
significant effect on overall H2O2 production, as indicated by the unchanged oxidation rate of 2¢,7¢-dichlorofluorescin (DCF). In turn, 5 lm CuCl2 stimulated the
3660

Fig. 4. Effect of Cd2+ and Cu2+ ions on the
kinetics of the CoQ-reducing and CoQoxidizing modules. Effect of Cd2+ on the
kinetics of the CoQ-oxidizing module (A) and

CoQ-reducing module (B). Effect of Cu2+ on
the kinetics of the CoQ-oxidizing module (C)
and CoQ-reducing module (D). The kinetics
of the CoQ-oxidizing module were obtained
by titrating with a specific inhibitor of
the CoQ-reducing module, malonate
(0–3.125 lM). The kinetics of the CoQreducing module were obtained by titrating
with a specific inhibitor of the CoQ-oxidizing
module, myxothiazol (0–80 nM). JR, respiratory flux. Open symbols, no CdCl2 or CuCl2
added; closed symbols, plus 5 lM CdCl2
(A,B) or 5 lM CuCl2 (C,D). Average of n = 4
(A,B) and n = 2 (C,D) independent
experiments ± SEM.

rate of DCF oxidation by 43% (Fig. 6A). Increasing the
concentration of CdCl2 and CuCl2 to 10 lm resulted in
DCF oxidation rates that were 1.7 (P < 0.01) and 2.1
(P < 0.01) times higher, respectively.
Next, we assessed the ability of both metal ions to
induce lipid peroxidation in isolated rat liver mitochondria respiring on succinate in state 2. Figure 6B shows
the effects of Cd2+ and Cu2+ ions on the formation of

FEBS Journal 276 (2009) 3656–3668 ª 2009 The Authors Journal compilation ª 2009 FEBS


Metal-induced impairment of mitochondrial function

A

A

100
80
60
40
20
0

0

20

40 60 80
CdCl2 (µM)

100 120

SDH activity (% of control)

120

B

B

100
80
60
40
20

0

2+

20 40 60 80 100 120 140
CuCl2 (µM)
2+

Fig. 5. Effect of Cd (A) and Cu (B) ions on succinate dehydrogenase (SDH) activity. SDH activity under control conditions (i.e.
without CdCl2 or CuCl2) was 50.8 ± 7.8 and 50.8 ± 8.3 nmol
DCPIPỈ min)1Ỉmg protein)1 (set to 100%) in the experiments
shown in (A) and (B), respectively. Average of n = 2 independent
experiments ± SEM.

thiobarbituric acid reactive substances (TBARS), which
indicate levels of the lipid peroxidation product malondialdehyde. At the amounts tested (5 and 10 nmol
Cd2+ỈmgỈmitochondrial protein)1), Cd2+ ions had no
significant effect on TBARS formation. However, for
Cu2+ ions, addition of 5 nmolỈmg protein)1 significantly
increased (by 26%) the amount of TBARS per mg of
mitochondrial protein. Increasing the amount of added
Cu2+ ions to 10 nmolỈmg protein)1 did not further
increase the amount of TBARS formed (Fig. 6B).
Effects of Cd2+ and Cu2+ ions on the control of
fluxes in oxidative phosphorylation
Using MCA, we assessed the contribution made by
each oxidative phosphorylation module to the control
of JR and JP (Table 2 and Fig. 7). Three-modular
analysis (Fig. 1A) revealed that control of JR and JP
was shared between the respiratory chain and phosphorylation modules, with the former exerting somewhat more control. As expected for state 3 conditions,

the proton leak module exerted low levels of positive

**

1000

**

*

500

0

CuCl2

0 nmol·mg protein–1
5 nmol·mg protein–1
10 nmol·mg protein–1

0.15

* *

0.1

0.05

0

0

0 μM
5 μM
10 μM

1500

CdCl2

TBARS (nmol·mg protein–1)

SDH activity (% of control)

120

2',7'-dichlorofluorescin oxidation
rate (RFU·min–1·mg protein–1)

J. Ciapaite et al.

CdCl2

CuCl2

Fig. 6. Effect of Cd2+ and Cu2+ ions on H2O2 production (A) and
lipid peroxidation (B). Average of n = 3 (A) and n = 4 (B) independent experiments ± SEM. *P < 0.05 and **P < 0.01 versus the
condition with no CdCl2 or CuCl2 added, respectively. RFU, relative
fluorescence units; TBARS, thiobarbituric acid reactive substances.

control over JR and low levels of negative control over
JP (the latter is because stimulation of this module
decreases flux through the phosphorylation branch of
oxidative phosphorylation). A somewhat different fluxcontrol pattern was obtained from two separate sets of
experiments performed in the absence of CdCl2 and
CuCl2 (K1 and K2; Table 2). It has previously been
shown that the flux-control structure of the oxidative
phosphorylation system is influenced by hormones
[31]. Therefore, the observed difference may have been
caused by seasonal variations in the hormonal state of
the animals. Bimodular analysis (Fig. 1B) showed that
the CoQ-reducing module (comprising enzymes
involved in substrate transport and SDH) exerted relatively low levels of control over JR and JP compared
with the control exerted by the remaining system
components. Combining the results of three- and
bimodular MCA made it possible to deduce the control of fluxes exerted by the respiratory chain complexes downstream of the CoQ (i.e. cytochrome bc1
and COX) (Fig. 1C). The data obtained showed that
cytochrome bc1 and COX together exerted stronger
control over fluxes than the components of the respiratory chain upstream of CoQ (Table 2).

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J. Ciapaite et al.

Table 2. Effect of Cd2+ and Cu2+ ions on the metabolic control of fluxes. Average of n = 4 independent experiments ± SEM. K1 and K2,

control experiments with no CdCl2 or CuCl2 added; C, flux control coefficient; JR, respiratory flux; JP, phosphorylation flux.
CiJR
K1

Module, i
Respiratory
Phosphorylation
Proton leak
CoQ-oxidizing
CoQ-reducing
Cytochrome bc1 + COX

0.66
0.33
0.02
0.84
0.16
0.50

5 lM CdCl2
±
±
±
±
±
±

0.04
0.04
0.00

0.03
0.03
0.07

K2

0.78
0.20
0.02
0.78
0.22
0.56

±
±
±
±
±
±

0.02*
0.02*
0.00
0.04
0.04
0.05

0.53
0.45
0.03

0.84
0.16
0.37

5 lM CuCl2
±
±
±
±
±
±

0.06
0.06
0.00
0.01
0.01
0.05

0.94
0.06
0.01
0.89
0.11
0.83

±
±
±
±

±
±

0.02*
0.02*
0.00*
0.00*
0.00*
0.02*

±
±
±
±
±
±

0.02*
0.02*
0.01*
0.00
0.00
0.01*

CiJP
Module, i

K1

Respiratory

Phosphorylation
Proton leak
CoQ-oxidizing
CoQ-reducing
Cytochrome bc1 + COX

0.68
0.36
)0.03
0.84
0.16
0.51

5 lM CdCl2
±
±
±
±
±
±

0.05
0.04
0.01
0.03
0.03
0.08

K2

0.82
0.24
)0.06
0.82
0.18
0.64

±
±
±
±
±
±

0.02*
0.02*
0.01
0.01
0.01
0.02

0.54
0.49
)0.03
0.88
0.12
0.43

5 lM CuCl2
±

±
±
±
±
±

0.07
0.06
0.00
0.01
0.01
0.08

1.00
0.08
)0.08
0.89
0.11
0.89

*P < 0.05 versus condition with no CdCl2 or CuCl2 added.

Control coefficient of JR

A 1
0.8
0.6
0.4
0.2
0

Control coefficient of JP

CdCl2

K2

CuCl2

K1

B

K1

CdCl2

K2

CoQ reduction
bc1 + COX
Phosphorylation
Proton leak

CuCl2

1

0.8
0.6

0.4
0.2
0

Fig. 7. Control distribution of the respiratory (A) and phosphorylation
flux (B) among the CoQ-reducing, cytochrome bc1 + COX, phosphorylation and proton leak modules. Division of the system of oxidative
phosphorylation into modules is depicted schematically in Fig. 1C.
Average of n = 4 data sets. JR, respiratory flux; JP, phosphorylation
flux; K1 and K2, control experiments with no CdCl2 or CuCl2 added.

Cd2+ ions induced a redistribution in the control over
fluxes through the system (Table 2 and Fig. 7). Cd2+
ions tended to increase the control over JR exerted by
3662

the respiratory chain module and significantly decreased
the control over JR exerted by the phosphorylation
module. A similar trend was observed for control over
JP; control by the respiratory chain module increased
significantly, whereas control by the phosphorylating
module decreased significantly. In addition, negative
control of JP by the proton leak module increased
slightly, but significantly. Analysis of the control exerted
by the respiratory chain components revealed that control of JR and JP exerted by the CoQ-reducing module
was not affected by Cd2+ ions. Meanwhile, control of
JR and JP by cytochrome bc1 and COX tended to
increase in the presence of Cd2+ ions.
The effects of Cu2+ ions on the control pattern followed the same trend as the effects of Cd2+ ions but
were more pronounced (Table 2 and Fig. 7). Threemodular MCA showed that in the presence of Cu2+
ions the respiratory chain module acquired almost

complete control of JR and JP. Combination of threeand bimodular analysis revealed that this resulted from
a dramatic increase in the control of fluxes by cytochrome bc1 and COX. Moreover, in agreement with
the observation that Cu2+ ions are more potent stimulators of proton leak kinetics, we showed that
the effect of Cu2+ ions on the control of fluxes by the
proton leak module was stronger than the effect of
Cd2+ ions.
Taken together, the changes in flux control distribution were consistent with the results of modular kinetic
analysis, which revealed that both Cd2+ and Cd2+
ions interfere with oxidative phosphorylation function-

FEBS Journal 276 (2009) 3656–3668 ª 2009 The Authors Journal compilation ª 2009 FEBS


J. Ciapaite et al.

ing by inhibiting the respiratory chain downstream of
CoQ (i.e. cytochrome bc1 and ⁄ or COX).

Discussion
Living systems are continuously exposed to low levels
of multi-component pollution, which may affect many
cellular processes simultaneously. Although no cellular
process is hampered severely, there may be a cumulative effect on the functioning of various metabolic
pathways and this may ultimately challenge cellular
metabolism as a whole. Many pollutants, including the
heavy metal ions Cd2+ and Cu2+ are expected to
interfere with several enzymes at the same time because
of a rather nonspecific interaction with their functional
groups. In this study, we used modular kinetic analysis
and MCA to elucidate the molecular mechanisms

underlying Cd2+ and Cu2+ ion-induced impairment of
the main aerobic energy production pathway in the
cell, i.e. oxidative phosphorylation. By subdividing oxidative phosphorylation into modules in different ways
we were able to obtain a detailed picture of the effects
of Cd2+ and Cu2+ ions. We showed that Cd2+ ions
interfere with oxidative phosphorylation solely through
their inhibitory effect on respiratory chain complexes
downstream of CoQ. This resulted in lower respiratory
and phosphorylation fluxes and lower Dw values, as
well as an increase in flux control by the respiratory
chain module. The overall effect of Cu2+ ions on oxidative phosphorylation functioning was similar to that
seen with Cd2+ ions, however, it was caused not only
by inhibition of respiratory chain, but also by stimulation of proton leak module activity at high Dw. The
latter effect was caused, at least in part, by stimulation
of ROS production and the subsequent peroxidation
of membrane lipids.
Modular kinetic analysis uses natural properties of
metabolism, i.e. its organization into recognizable functional units, simplifying the analysis and making cellular
complexity manageable [20,32,33]. We demonstrated
how different modularization of the system of interest
and subsequent application of modular kinetic analysis
allows identification of molecular targets of Cd2+ and
Cu2+ ions. In the first application of the analysis, we
conceptually divided oxidative phosphorylation into
three functional modules based on the production and
consumption of Dw (Fig. 1A). We showed that Cd2+
and Cu2+ ions inhibited the respiratory chain module
(Figs 2B and 3B) but had no significant effect on the
phosphorylation module (Figs 2C and 3C). Interestingly, Cu2+ ions had a stronger ability to uncouple oxidative phosphorylation than did Cd2+ ions, as indicated
by stimulation of proton leak module kinetics at high

Metal-induced impairment of mitochondrial function

Dw values (Figs 2A and 3A). Our data are in agreement
with earlier findings that Cu2+ ions can effectively
increase membrane permeability, whereas Cd2+ ions are
much less effective; comparable swelling of isolated rat
liver mitochondria was obtained at 5 lm Cu2+ and
40 lm Cd2+ [34]. It has been shown that the ability of
Cd2+ ions to increase membrane permeability is dependent on inorganic phosphate transport and increases
with increasing phosphate concentration in the medium
[35]. Therefore, under certain experimental conditions
characterized by a high inorganic phosphate concentration, low Cd2+ concentrations can induce uncoupling
[35,36]. However, at a low inorganic phosphate concentration (as in this study) a much higher Cd2+ concentration is needed to stimulate membrane ion permeability
and induce uncoupling [37].
Three-modular kinetic analysis has been used previously to localize the sites of action of Cd2+ ions in isolated potato tuber mitochondria respiring on succinate,
and has shown that different concentrations of Cd2+
ions inhibited the respiratory chain module, had no
significant effect on the phosphorylation module and
stimulated proton leak kinetics at 3.5 lm free Cd2+
[22]. The apparent contradiction between the latter
finding and our observation that 5 lm CdCl2 has no
significant effect on the proton leak kinetics is
explained by the fact that we used a lower amount
of Cd2+ ions per mg of mitochondrial protein, and
therefore uncoupling did not occur.
After establishing that the respiratory chain module is
the target of both metal ions, we conceptually divided
the system into CoQ-reducing and CoQ-oxidizing modules with XCoQ as the connecting intermediate (Fig. 1B).
This approach revealed that 5 lm CdCl2 or CuCl2 interfered with the respiratory chain complexes downstream

of CoQ and had no significant effect on the dicarboxylate carrier and SDH (Fig 4). By contrast, earlier studies
identified SDH as the target of both metal ions [5,9,10].
We investigated this and found that 5 lm CdCl2 or
CuCl2 did not affect SDH activity under our experimental conditions (Fig. 5), confirming that modular kinetic
analysis yielded the correct results.
In this study, we showed that at low concentrations,
Cd2+ and Cu2+ ions affected a rather limited number of
components in the oxidative phosphorylation system,
i.e. the respiratory chain downstream of CoQ (i.e. cytochrome bc1 and COX) and the mitochondrial inner membrane. This suggests that because these components are
affected by a low concentration of Cd2+ and Cu2+ ions,
they are the most sensitive and are therefore responsible
for the early response of the system when it is chronically
exposed to low levels of pollutants. Support for this
hypothesis comes from the observation that mitochon-

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J. Ciapaite et al.

dria from liver, kidney and muscle of cadmium-treated
rats display a rapid early decrease in COX activity, followed by partial restoration after 6 months of treatment,
and a progressive decrease in SDH activity [9].
The variation in the number of molecular targets of
Cd2+ or Cu2+ ions reported in the literature may be
explained by their differing sensitivity to these ions,

resulting in a greater number of affected system components at higher ion concentrations. For example, the
effects on mitochondrial ATPase were examined at
Cd2+ concentrations in the mm range (up to 10 mm)
[5]. It is questionable whether such high concentrations
of Cd2+ ions can be achieved even in a heavily intoxicated cell. Furthermore, it has been shown that
ATPase activity was induced by very low concentrations of Cu2+ (4–6 nmolỈmg protein)1). However, the
same ion concentration induced maximal uncoupling
under the experimental conditions used by Hwang
et al. [34], and the effect might therefore be explained
by uncoupling. In contrast to these findings, our data
(Figs 2C and 3C) did not reveal any noticeable effects
of 5 lm CdCl2 and 5 lm CuCl2 on the components of
the ADP phosphorylation machinery.
We hypothesized that the different effect of Cd2+ and
Cu2+ ions on the proton leak kinetics observed in our
study may be determined by the ability of Cu2+ ions to
stimulate ROS production, which in turn may lead to
lipid peroxidation and membrane damage. We showed
that 5 lm CuCl2, but not 5 lm CdCl2, stimulated the
oxidation of DCF significantly, indicating increased
H2O2 formation in the mitochondrial matrix. Because
oxygen favors reduction by one electron at a time, the
primary ROS that is formed by the action of Cu2+ ions
must be a superoxide anion radical. The latter may
reduce Cu2+ to Cu+, leading to formation of the hydroxyl radical via the Haber–Weiss cycle [38]. In turn, the
hydroxyl radical is a potent inductor of lipid peroxidation. In support of this, we showed that Cu2+ ions, but
not Cd2+, cause accumulation of TBARS, suggesting
that Cu2+ may increase membrane permeability by stimulating lipid peroxidation. It has also been shown that,
in intact hepatocytes, Cu2+ ions are much more potent
inductors of lipid peroxidation than Cd2+ ions [14].

In addition to the evaluation of Cd2+ and Cu2+ ioninduced changes in the kinetics of the individual
oxidative phosphorylation components, we were also
interested in how these changes affect the control structure of the system. MCA was designed to deal with the
effects of a small disturbance in enzyme activity on system fluxes and is a useful method with which to analyze
and diagnose cell sickliness caused by agents that simultaneously affect many enzymes. In this study, we
assessed metabolic control of the respiratory (JR) and
3664

phosphorylation (JP) fluxes by the respiratory chain,
phosphorylation and proton leak modules, as well as
control of the respiratory flux by CoQ-reducing and
CoQ-oxidizing modules, and determined how Cd2+ and
Cu2+ ions affected this control. In agreement with previously published data [21,24], we found that control of
both fluxes is mainly shared between the respiratory
chain and phosphorylation modules, with only slight
control being exerted by the proton leak module (positive in the case of JR and negative in the case of JP)
(Table 2 and Fig. 7). Five micromolar CdCl2 and CuCl2
caused an increase in the control of JR and JP by the
respiratory chain module, with cytochrome bc1 and
COX being the main contributors. This is in agreement
with our observation that both metal ions interfere with
the respiratory chain components downstream of CoQ.
The effect of Cu2+ ions was stronger than that of
Cd2+. Furthermore, Cu2+ ions increased negative control of JP by the proton leak module more than Cd2+
ions, because the former had a stronger effect on the
permeability of the inner membrane to ions. This observation illustrates how interfering with a system component that makes a relatively low contribution to the
control of system fluxes (i.e. proton leak) may compromise the system via control of other vital system
properties (e.g. stronger membrane damage by Cu2+
ions may contribute to inhibition of cytochrome bc1
and COX, which are membrane proteins, leading to

increased flux control by these enzymes).
Flux control is a property of the whole system
rather than of individual components of that system
(i.e. enzymes, modules), as illustrated by the summation theorem for flux control coefficients [17,18]. As a
result, individual flux control coefficients for each system component cannot change independently. Consequently, we see that an increase in flux control by the
respiratory chain and proton leak modules results in
decreased control of both fluxes by the phosphorylation module, although this module is not affected
directly by Cd2+ and Cu2+ ions.
Taken together, we have demonstrated how modular
kinetic analysis could be gradually applied to identify
the sites of action of external effectors such as Cd2+
and Cu2+ ions in a multienzyme system like oxidative
phosphorylation. Although we found that Cd2+ ions
affect system behavior by acting on a single target,
whereas Cu2+ interferes with two, there is no doubt
that this method is valuable, especially when assessing
multisite effects of toxic substances on complex metabolic systems. The next step in this type of analysis
may be to elucidate the combined effects of several
effectors (e.g. a mixture of Cd2+ and Cu2+ ions) with
individual known sites of action. Repeated modular

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J. Ciapaite et al.

kinetic analysis using different modularization
approaches might reveal the causes and consequences
of their competition for binding to the same molecular
component or synergism of their action.

Materials and methods
Materials
Rotenone, myxothiazol, oligomycin, creatine phosphokinase, CoQ1, 2¢,7¢-DCF, 2¢,7¢-dichlorofluorescin diacetate
and 2-thiobarbituric acid (TBA) were from Sigma (SigmaAldrich, St Louis, MO, USA).

Isolation of mitochondria
The handling of the animals conformed to the rules defined
by the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific
Purposes (License No. 0006 of State Veterinary Service for
working with laboratory animals). Mitochondria were isolated from the livers of male Wistar rats using a standard
differential centrifugation procedure as described previously
[24], using 250 mm sucrose, 10 mm Tris, 3 mm EGTA and
2 mgỈmL)1 BSA (pH 7.7) as the isolation medium. Mitochondria were suspended in the medium containing 250 mm
sucrose and 5 mm Tris (pH 7.3). Protein was estimated
according to Bradford [39] using BSA as the standard.

Measurement of respiration and membrane
potential
Prior to each measurement, mitochondria (1 mgỈmL)1 mitochondrial protein) were incubated for 3 min with or without
5 lm CdCl2 or 5 lm CuCl2 in the assay medium containing
110 mm KCl, 20 mm Tris, 5 mm KH2PO4, 50 mm creatine,
an excess of creatine kinase and 1 mm MgCl2, pH 7.2. Mitochondrial respiration rate and Dw were measured simultaneously at 37 °C in a closed, stirred and thermostated glass
vessel equipped with a Clark-type oxygen electrode and
TPP+-sensitive electrode, as described previously [40]. We
used 5 mm succinate (+ 2 lm rotenone) as the respiratory
substrate. ATP (1 mm) was added to initiate state 3 respiration. Data were processed using the chart program supplied
with MacLab (AD Instruments, Chalgrove, UK).

Determination of CoQ reduction level

CoQ reduction level was determined in mitochondria
(1 mgỈmL)1 mitochondrial protein) incubated under the conditions used in Dw measurements in a thermostated (37 °C)
vessel equipped with platinum and oxygen electrodes, by
polarographically measuring the redox state of exogenous
CoQ1 (2 lm) [41]. To calibrate the platinum electrode traces,

Metal-induced impairment of mitochondrial function

samples were taken from incubations of mitochondria in
standard assay medium without further additions (state 1)
and mitochondria were incubated with substrate (5 mm succinate + 2 lm rotenone). One milliliter of sample was
quenched with 1 mL of 0.2 m HClO4 in methanol (0 °C).
CoQ was extracted with 3 mL of petroleum ether (40–60 °C)
and determined by HPLC, as described previously [42]. Data
were processed using the chart and powerchrom programs
supplied with MacLab.

Determination of SDH activity
SDH activity was determined spectrophotometrically at
600 nm from the rate of reduction of DCPIP in the presence
of CoQ1, as described in Ragan et al. [43]. Briefly, intact
mitochondria (1 mgỈmL)1 mitochondrial protein) were incubated for 3 min with 5 lm CdCl2 or 5 lm CuCl2 under the
experimental conditions used in the respiration and Dw measurements. Medium was then collected and mitochondrial
membranes were ruptured by four freeze–thaw cycles. Measurement of SDH activity was carried out in the presence of
1 mgỈmL)1 CoQ1 and 100 lm DCPIP. SDH activity was calculated using a molar extinction coefficient of 21 mm)1Ỉcm)1.
The dependence of SDH activity on concentrations exceeding
5 lm Cd2+ or Cu2+ ions was determined using a slightly
modified protocol: varying amounts of CdCl2 or CuCl2 were
added directly to four-times freeze–thawed mitochondrial
suspension (i.e. without 3 min preincubation with intact

mitochondria) in an assay medium containing 110 mm KCl,
20 mm Tris, 5 mm KH2PO4, 2.24 mm MgCl2, pH 7.2, and
SDH activity was determined as described above.

Measurement of H2O2 production
Oxidation of DCF was used as an indicator of H2O2 production in the mitochondrial matrix. Isolated mitochondria were
incubated with 5 lm 2¢,7¢-dichlorofluorescin diacetate for
30 min. Next, mitochondria were resuspended in medium
containing 250 mm sucrose, 5 mm Tris (pH 7.3), centrifuged
at 7300 g for 10 min at 4 °C, and the supernatant discarded.
To assess H2O2 production, mitochondria (1 mgỈmL)1 mitochondrial protein) were incubated at 37 °C in assay medium
(as in SDH activity determination) supplemented with 5 mm
succinate and different concentrations of CdCl2 and CuCl2
(0, 5 and 10 lm). The oxidation of DCF was measured
spectrofluorimetrically (kex = 485 nm, kem = 535 nm) for
3 min using GENios Pro reader (Tecan, Mannedorf, Swită
zerland) and the DCF oxidation rate was expressed as relative fluorescence unitsỈ min)1Ỉmg protein)1. To correct for
changes in mitochondria-derived background fluorescence,
the same measurement was carried out with mitochondria,
which were not loaded with 2¢,7¢-dichlorofluorescin diacetate
and the rate obtained was subtracted from the DCF oxidation rate.

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J. Ciapaite et al.

Assessment of lipid peroxidation
Malondialdehyde content was determined from the formation of TBARS using a TBA assay [44]. Isolated mitochondria (5 mgỈmL)1 mitochondrial protein) were incubated in a
thermostated (37 °C) vessel in 1 mL of assay medium (as in
the SDH activity determination) supplemented with 5 mm
succinate and different amounts of CdCl2 and CuCl2 (0, 5
and 10 nmolỈmg protein)1). After 3 min of incubation,
medium with mitochondria was collected, centrifuged at
10 000 g for 1 min at 4 °C and resuspended in 1 mL of
cold fresh incubation medium. The washing step was
repeated twice to remove any sucrose present in the mitochondrial suspension because sucrose interferes with the
TBA assay [44]. Then, 1 mL of TBA (0.5% in 20% of
trichloroacetic acid) was added and the mixture was incubated in a boiling water bath for 30 min. Precipitated protein was removed by centrifugation at 10 000 g for 10 min
at 4 °C. The absorbance of the sample was measured at
532 and 600 nm (to correct for turbidity) and the amount
of TBARS formed (expressed as nmol TBARSỈmg protein)1) was calculated using a molar extinction coefficient
of 1.56 · 105 m)1Ỉcm)1.

Modular kinetic analysis
In the first application of the analysis, the mitochondrial
oxidative phosphorylation system was conceptually divided
into three functional modules based on the production and
consumption of Dw: (a) respiratory chain module, (b) phosphorylation module, and (c) proton leak module [21]. In
this study, we measured mitochondrial Dw instead of proton-motive force (Dp) because we have previously shown
that measurement of Dw (one component of Dp) instead of
Dp does not introduce significant error. This is because the
proton concentration gradient (DpH) (the second component of Dp) is small and does not change under our experimental conditions [25]. In the second application of the
analysis, respiratory chain enzymes were grouped into two
functional modules with the fraction of CoQ (XCoQ) as the
connecting intermediate: (a) a CoQ-reducing module, and

(b) a CoQ-oxidizing module [23]. The kinetics of the modules were determined by titration with specific inhibitors
and simultaneous measurement of levels of connecting
intermediates (Dw or XCoQ) and oxygen consumption rate
as a measure of flux, as described previously [21,23]. To
determine the effects of Cd2+ and Cu2+ ions on the
module kinetics, the procedure was repeated in the presence
of these substances.

Metabolic control analysis
The control of a pathway flux by a specific enzyme (or
group of enzymes, i.e. module) can be assessed quantitatively using MCA [17–19]. The flux control coefficient (C)

3666

quantifies the importance of each enzyme (module) in the
pathway in controlling the flux through that pathway at a
steady state. It represents the fractional change in the
system property (flux, J) in response to an infinitesimal
change in the activity of the enzyme (or module) of the
system [17–19]. In this study, the flux control coefficients of
the oxidative phosphorylation modules over respiration (JR)
and phosphorylation (JP) flux were calculated from the
steady-state fluxes and elasticity coefficients (obtained from
the corresponding inhibitor titration curves depicted in
Figs 2–4) using summation and connectivity theorems, as
described previously [21]. The flux control coefficient values
obtained from the three- and bimodular analysis (Fig. 1A,B,
respectively) were used to deduce the control coefficients of
JR and JP by cytochrome bc1 and COX (Fig. 1C). The
calculations were made assuming that the sum of all flux

control coefficients in the system equals 1 [17,18].

Data presentation and statistical analysis
Data were obtained from n = 2–7 experiments, carried out
using independent mitochondrial preparations and expressed
as average ± SEM. The effects of Cd2+ and Cu2+ ions
were determined in separate series of experiments, which
resulted in two sets of data under conditions without Cd2+
or Cu2+ ions added (referred to as K1 and K2 in the text).
The statistical significance of Cd2+ and Cu2+ ion effects
was evaluated using a paired Student’s t-test. Differences
were assumed to be statistically significant when P < 0.05.

Acknowledgements
This research was supported by grant IC15 CT960307
from the European Community.

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