Báo cáo khoa học: Effect of magnesium ions on the activity of the cytosolic NADH/cytochrome c electron transport system pptx

Effect of magnesium ions on the activity of the cytosolic
NADH/cytochrome c electron transport system
Gianluigi La Piana
1
, Vincenza Gorgoglione
1
, Daniela Laraspata
1
, Domenico Marzulli
2
and Nicola E. Lofrumento
1
1 Department of Biochemistry and Molecular Biology, University of Bari, Italy
2 Institute of Biomembranes and Bioenergetics (IBBE - CNR), University of Bari, Italy
The oxidative and energetic metabolism of glucose
requires cytosolic NADH to be oxidized by the respi-
ratory chain to gain the maximal production of ATP
and to regenerate the cytosolic NAD
+
necessary for
continuous and efficient glycolytic flux. In mammalian
cells, because of the impermeability of the mitochon-
drial inner membrane (MIM) to pyridine nucleotides,
many translocating pathways are available that may
be involved in the transfer of reducing equivalents
from the cytosol to the mitochondrial matrix, and
vice versa [1]. Among them, two shuttle systems are
well known: the a-glycerophosphate and malate–
aspartate shuttles. All the proposed systems, however,
promote an indirect transfer of reducing equivalents
from cytosolic NADH to either NAD

+
or flavopro-
teins inside the mitochondria. Starting from the obser-
vation that isolated mitochondria oxidize NADH
present outside the mitochondria only if cytochrome c
(cyto-c) is also added to the incubation medium, and
supported by many converging data, we proposed the
existence in liver mitochondria of the cytosolic
NADH/cyto-c electron transport pathway in addition
to and independent of the electron pathway of the
respiratory chain [2–7]. In the presence of a catalytic
Keywords
cytosolic NADH oxidation; magnesium ions
and mitochondrial membrane permeability;
mitochondria, cytochrome c and apoptosis;
mitochondrial contact sites and
respiration; mitochondrial membrane
potential
Correspondence
N. E. Lofrumento, Department of
Biochemistry and Molecular Biology,
University of Bari, via Orabona 4, 70126
Bari, Italy
Fax: +39 80 5443317
Tel: +39 80 5443325
E-mail:
(Received 25 July 2008, revised 5
September 2008, accepted 14 October
2008)
doi:10.1111/j.1742-4658.2008.06741.x

Cytochrome c (cyto-c), added to isolated mitochondria, activates the oxida-
tion of extramitochondrial NADH and the generation of a membrane
potential, both linked to the activity of the cytosolic NADH/cyto-c electron
transport pathway. The data presented in this article show that the protec-
tive effect of magnesium ions on the permeability of the mitochondrial
outer membrane, supported by previously published data, correlates with
the finding that, in hypotonic but not isotonic medium, magnesium pro-
motes a differential effect on both the additional release of endogenous
cyto-c and on the increased rate of NADH oxidation, depending on
whether it is added before or after the mitochondria. At the same time,
magnesium prevents or almost completely removes the binding of exoge-
nously added cyto-c. We suggest that, in physiological low-amplitude swell-
ing, magnesium ions may have the function, together with other factors, of
modulating the amount of cyto-c molecules transferred from the mitochon-
drial intermembrane space into the cytosol, required for the correct execu-
tion of the apoptotic programme and/or the activation of the NADH/
cyto-c electron transport pathway.
Abbreviations
cyto-b
5
, cytochrome b
5
; cyto-c, cytochrome c; FCCP, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone; ferrocyto-c, ferrocytochrome c;
MIM, mitochondrial inner membrane; MIS, mitochondrial intermembrane space; MOM, mitochondrial outer membrane; TMPD,
N,N,N¢,N¢-tetramethyl-p-phenylenediamine; DY
m
, mitochondrial membrane potential change.
6168 FEBS Journal 275 (2008) 6168–6179 ª 2008 The Authors Journal compilation ª 2008 FEBS
amount of cyto-c outside the mitochondria, the
NADH/cyto-c system promotes [2–6,8–10]: (a) the

oxidation of externally added NADH molecules; (b)
the consumption of molecular oxygen by cytochrome
oxidase; and (c) the generation of an electrochemical
membrane potential. We have shown that some com-
ponents of this additional electron transport chain are
sited on the ‘respiratory contact sites’ [5,6], but
endogenous cyto-c, present in the mitochondrial inter-
membrane space (MIS), is not involved in this process
[3]. With no knowledge of our series of publications
and therefore independent of our data, a report in
late 2005 [11], with the support of detailed experimen-
tal approaches, considered a direct interaction
between the voltage-dependent anion channel and
cytochrome oxidase. The existence of ‘a novel type of
contact site’ as such, but with no specific function,
was inferred and outlined in a scheme very similar to
that reported in our paper published at the beginning
of 2005 [6]. The NADH/cyto-c electron transport sys-
tem may have the function, in the physio-pathological
conditions associated with the extra formation of
cytosolic NADH, to promote its oxidation by the
direct transfer of reducing equivalents to cytochrome
oxidase and the generation of an electrochemical pro-
ton gradient [3]. In apoptotic cells, with the impair-
ment of the respiratory chain because of the transfer
of cyto-c from the mitochondria to the cytosol, the
activity of the system may represent an additional,
but necessary, source of energy required for correct
execution of the death programme.
To date, the components identified and involved in

the cytosolic NADH/cyto-c electron transport path-
way are as follows: NAD-dependent dehydrogenases
present in the cytosol; cytosolic NADH; the rotenone-
insensitive NADH/cytochrome b
5
(cyto-b
5
) complex
present on the external leaflet of the mitochondrial
outer membrane (MOM); cyto-c molecules present
outside the mitochondria but not those present in
MIS; the respiratory contact sites between the two
mitochondrial membranes in which the voltage-depen-
dent anion channel or porin of MOM is juxtaposed to
the cytochrome oxidase molecules spanning MIM. All
of these components are required for the correct exe-
cution of the cytosolic NADH/cyto-c system. The
activity of this electron transport pathway was studied
and characterized essentially in liver mitochondria as,
over time, we devised and improved four different
tests for these mitochondria (see Materials and meth-
ods, and [7]) to evaluate the intactness of the two
mitochondrial membranes and to measure their
permeability to both exogenous NADH and cyto-c.
The integrity of isolated mitochondria is a necessary
prerequisite to study the activity of the respiratory
chain, but becomes mandatory for the new, additional
and independent NADH/cyto-c electron transport sys-
tem to counteract the criticism that the results
obtained can be ascribed to damaged or broken mito-

chondria. One test, based on the determination of sul-
fite/cyto-c oxido-reductase activity, is highly specific
for measurement of the permeability of MOM to
exogenous cyto-c, but requires the presence of sulfite
oxidase in MIS. This enzyme is highly expressed in
liver, barely in heart and absent in skeletal muscle
[12]. The rotenone-insensitive NADH/cyto-c oxido-
reductase activity is 10 times lower in the heart than
in rat liver [13]. The NADH/cyto-b
5
complex of
MOM is responsible for the reduction of exogenous
cyto-c, and therefore its activity should not be a limit-
ing factor for the oxidation of cytosolic NADH. With
the support of the four tests in our laboratory, we
routinely utilize mitochondrial preparations containing
more than 98% of mitochondria with MIM not per-
meable to exogenous NADH and with MOM not per-
meable to both endogenous and exogenous cyto-c.In
support of the intactness of MOM, adenylate kinase
and sulfite oxidase, present in MIS, are not released
outside the mitochondria. Recently, unexpected new
findings have shown that exogenous cyto-c does not
permeate into MIS even when mitochondria are incu-
bated in a strongly hypotonic medium [7]. However,
some authors maintain that the NADH/cyto- c system
is catalysed by broken and/or damaged mitochondria,
on the basis of the observation that magnesium ions,
added to mitochondria incubated in a hypotonic med-
ium, promote the oxidation of exogenous NADH even

in the absence of externally added cyto-c [8–10]. The
possibility that the free molecules of endogenous cyto-
c present in the intermembrane space may have the
additional function (proposed in 1969 [14] and
invoked in 1981 [15] and 2002 [16]) to shuttle electrons
between the cyto-b
5
of MOM and the cytochrome oxi-
dase of MIM is in contrast with the already men-
tioned and well-known finding that intact mammalian
mitochondria are unable to oxidize exogenous NADH
unless cyto-c molecules are also present outside the
mitochondria.
With the support of the above-mentioned results
indicating that, even with mitochondria incubated in
hypotonic medium, cyto-c present outside the mito-
chondria is not permeable through MOM [7], we car-
ried out a series of experiments to ascertain the role
and effect of magnesium ions on the activity of the
cytosolic NADH/cyto-c electron transport system of
mitochondria incubated in isotonic 250 mm sucrose
and hypotonic 25 mm sucrose media.
G. La Piana et al. Magnesium ions and cytosolic cytochrome c oxidation
FEBS Journal 275 (2008) 6168–6179 ª 2008 The Authors Journal compilation ª 2008 FEBS 6169
Results
Magnesium stimulates the NADH/cyto-c system
only in mitochondria incubated in hypotonic
medium
Returning to an experiment similar to that carried out
in 1989 and reported in [2], Fig. 1 (trace a) shows the

property of isolated rat liver mitochondria, incubated
in isotonic 250 mm sucrose medium, to promote the
oxidation of exogenously added NADH in the pres-
ence of respiratory chain inhibitors (rotenone and
myxothiazol) and a catalytic amount of exogenous
cyto-c. To test the effect of magnesium ions and avoid
any interference or synergistic activity of other sub-
stances, an incubation medium simply consisting of
sucrose and buffer to stabilize the pH was utilized.
Figure 1 (trace a) also shows that the oxidation rate
was blocked by cyanide, suggesting the involvement of
cytochrome oxidase. The addition of 4 mm MgCl
2
before the cyto-c (Fig. 1, trace b) did not influence the
NADH oxidation rate. Mitochondria incubated in
hypotonic medium (25 mm sucrose; Fig. 1, traces c–e)
oxidized exogenous NADH before the addition of
cyto-c, even if at a very low rate, which was increased
about eight times by magnesium ions (Fig. 1, trace e).
The oxidation rate was further increased by the subse-
quent addition of cyto-c, but reached a value similar
to that obtained in the absence of magnesium (Fig. 1,
trace c). Moreover, when MgCl
2
was already present
in the medium, before the addition of mitochondria
(Fig. 1, trace d), NADH oxidation was higher than in
the control, but, on addition of cyto-c, it was signifi-
cantly lower than that obtained either in the absence
(Fig. 1, trace c) or presence of magnesium added after

the mitochondria (Fig. 1, trace e). Magnesium added
to the isotonic medium before the mitochondria had
no effect on the rate of NADH oxidation (Fig. 1, trace
a). The results illustrated in Fig. 1 are consistent, at
least in part, with those already reported in recent
years by two research groups [8–10], showing that the
exogenous NADH/cyto-c oxidation rate is greatly
increased in hypotonic medium. As a new finding, not
reported previously, we have shown that, in hypotonic
Fig. 1. Effect of magnesium ions on exogenous NADH oxidation by mitochondria incubated in isotonic and hypotonic media. Rat liver mito-
chondria (3 mg protein) were incubated in 3.0 mL of isotonic (traces a, b) or hypotonic (traces c–e) medium containing 250 and 25 m
M
sucrose, respectively, plus 20 mM Hepes (pH 7.4), 6 lM rotenone and 6 lM myxothiazol. After 2 min of incubation, 0.2 mM NADH (N) was
added. Further additions: 10 l
M cytochrome C (C); 4 mM MgCl
2
(Mg); 1 mM potassium cyanide (CN). In trace a, when present, and in trace
d, magnesium ions were added to the medium before mitochondria. The traces reported are representative of 12 obtained with nine differ-
ent mitochondrial preparations, and the values reported are the number of nanomoles of NADH oxidized per minute per milligram of protein.
Statistical significance in hypotonic medium: P = 3.7 · 10
)8
(value 5 of trace d versus value 1.2 of trace e); P = 2.5 · 10
)4
(value 9 of trace
e versus value 5 of trace d); P = 8.8 · 10
)5
(value 24 of trace d versus value 45 of trace e).
Magnesium ions and cytosolic cytochrome c oxidation G. La Piana et al.
6170 FEBS Journal 275 (2008) 6168–6179 ª 2008 The Authors Journal compilation ª 2008 FEBS
medium, magnesium has a differential stimulatory

effect depending on whether it is added to the incuba-
tion medium before or after the mitochondria.
The plurality and differential effects of magnesium
ions on the activity of the respiratory chain, as well as
on many enzymatic reactions and biological processes,
have been studied extensively [17–19]. Experiments,
not reported here, on the effect of Mg
2+
on the oxy-
gen uptake supported by succinate oxidation confirmed
the results reported by Panov and Scarpa [17], and
showed that magnesium, in both isotonic and
hypotonic mitochondria, improves the ratio of state
3/state 4 respiration; moreover, no appreciable
difference was observed when added either before or
after the mitochondria. In the succinate oxidation
experiments, it appears that the prevailing effect of
MgCl
2
consists of the stabilization of ADP and ATP
molecules, the substrate and product of ATP synthase
activity, respectively. We have obtained indications
that the effect of magnesium ions on the oxidation of
substrates present in the matrix space (such as
succinate) and catalysed by the respiratory chain is
completely different from their effect on the oxidation
of exogenous NADH.
Magnesium-dependent binding of exogenous
cyto-c and release of endogenous cyto-c
The effect of Mg

2+
on the distribution of both endog-
enous and exogenous cyto-c of mitochondria incubated
in isotonic and hypotonic media was determined in the
experiments summarized in Fig. 2. The amounts pres-
ent in pellets of 6 mg of mitochondrial protein, incu-
bated in 6 mL of medium and then centrifuged, were
determined to better appreciate the difference between
each sample. As reported in Fig. 2A, we found that
the content of endogenous cyto-c in the pellets of sam-
ples of isotonic mitochondria stopped at zero time (i.e.
immediately after the addition of mitochondria) was
the same as that of samples stopped at the end of a
10-min incubation (samples, m). In addition, in sam-
ples with magnesium present in the medium, the con-
tent of cyto- c was the same when stopped at either 5
A
B
Fig. 2. Effect of magnesium ions on both the content of endoge-
nous cyto-c (A) and the binding of exogenous cyto-c (B) to mito-
chondria incubated in isotonic and hypotonic media. Mitochondria
(6 mg protein) were incubated in 6 mL of 250 m
M (Iso) or 25 mM
(Hypo) sucrose-based medium for a total time of 10 min (A) and
15 min (B), and then centrifuged at 10 000 g for 10 min at 4 °C.
Sequence of additions: (A) at zero time, mitochondria and the reac-
tion stopped by centrifugation either immediately or at 10 min (m);
at 5 min, 4 m
M MgCl
2

and the reaction stopped at 10 min (m-Mg);
at zero time, mitochondria added to the medium already containing
4m
M MgCl
2
and the reaction stopped at either 5 or 10 min
(Mg-m); (B) at zero time, mitochondria, at 5 min addition of 2 l
M
exogenous cyto-c, and reaction stopped at either 10 or 15 min (a);
alternatively, 2 l
M cyto-c added at 10 min and reaction stopped at
15 min (a); at 5 min addition of 4 m
M MgCl
2
, at 10 min addition of
2 l
M cyto-c, and the reaction stopped at 15 min (Mg-c); at 5 min
addition of 2 l
M cyto-c, at 10 min addition of 4 mM MgCl
2
, and the
reaction stopped at 15 min (c-Mg); 4 m
M MgCl
2
present in the
medium, at 5 min addition of 2 l
M cyto-c and reaction stopped at
15 min (Mg-m). In all samples, at 2 min, 6 l
M rotenone plus 6 lM
myxothiazol were added to the incubation medium. The number of

nanomoles of cyto-c present in the pellets of 6 mg protein,
expressed as the mean value (± standard deviation) of duplicate
samples of seven different mitochondrial preparations, are
reported. Statistical significance: (A) **P £ 0.003 (control hypotonic
versus control isotonic); *P £ 0.03 (m-Mg hypotonic versus control
hypotonic); *P £ 0.02 (Mg-m hypotonic versus control hypotonic);
(B) ***P £ 0.0002 (control a hypotonic versus control a isotonic);
**P £ 0.002 (Mg samples in isotonic medium versus control a iso-
tonic).
G. La Piana et al. Magnesium ions and cytosolic cytochrome c oxidation
FEBS Journal 275 (2008) 6168–6179 ª 2008 The Authors Journal compilation ª 2008 FEBS 6171
or 10 min (samples, Mg-m). The same was observed
with hypotonic mitochondria. Therefore, these results
indicate that endogenous cyto-c is released in a rapid
and complete process, and not slowly during the
course of incubation. Moreover, with an isotonic med-
ium, the presence of magnesium, added either before
(Mg-m) or after (m-Mg) the mitochondria, did not
influence the cyto-c content of the pellets. Hypotonic
medium per se promotes the release of no more than
14% of cyto-c from mitochondria (m) and, in this
case, the addition of magnesium after a 5-min incuba-
tion of mitochondria increased to 35% the release of
cyto-c compared with isotonic mitochondria (the
additional release was 21%). The latter result can be
considered in some aspects to be in line with the
concept of the desorption mechanism proposed by
Bodrova et al. [8] and supported by Lemeshko [9,10]
and Scorrano et al. [16]. However, when magnesium
was already present in the hypotonic medium before

the addition of mitochondria (Mg-m), the release of
cyto-c was significantly lower and amounted to 21%
(the additional release was only 7%). The decrease in
the release of cyto-c when magnesium is present in the
medium recalls its protective effect (reported previ-
ously [7]) on the release induced by hypotonic medium
of sulfite oxidase and adenylate kinase, which, similar
to cyto-c, are both present in MIS.
Two experimental protocols have been designed (see
Materials and methods) to analyse the effect of Mg
2+
in preventing or removing the binding of exogenous
cyto-c added to isolated mitochondria. The results of
these experiments are reported in Fig. 2B. In the pres-
ence of exogenously added 2 lm cyto-c, 3.9 nmol was
found in the pellets of 6 mg of mitochondrial protein
incubated in 6 mL of isotonic medium. However, not
all of these molecules are of exogenous cyto-c; accord-
ing to the data reported in Fig. 2A, 1.39 nmol derives
from endogenous cyto-c. More precisely of the total of
12 nmol added, 2.51 nmol remains bound to mito-
chondria, giving a value of 0.42 nmol bound per milli-
gram of protein. The values of the supernatants (not
shown) were complementary to those of the pellets in
both the absence and presence of Mg
2+
. Magnesium
added before cyto-c (Mg-c) greatly limited its binding
to a value of 0.12 nmolÆmg
)1

, corrected for the
1.39 nmol of endogenous cyto-c. From Fig. 1 (traces
a, b), it can be observed that magnesium added before
or after the mitochondria does not influence the activ-
ity of the NADH/cyto-c system. This may suggest that
cyto-c molecules not bound in the presence of
magnesium may not be involved directly in the
oxidation of exogenous NADH. In hypotonic
mitochondria, the binding capability is increased to a
value of 1 nmolÆmg
)1
, calculated after the correction
for the 1.2 nmol of endogenous cyto-c, with an
increase of 2.4 times compared with isotonic mitochon-
dria. It is interesting to note that the extra binding is
almost completely prevented in the presence of magne-
sium as, in both isotonic and hypotonic medium,
2.1 nmol of cyto-c was found in the pellets. More rele-
vant is the finding that the total binding of 2.1 nmol
of cyto-c (endogenous + exogenous) in both isotonic
and hypotonic medium remains the same whether
Mg
2+
is added before (Mg-c; preventing effect) or
after (c-Mg; removal effect) cyto-c. The same value of
2.1 nmol was obtained with magnesium present in the
medium before the addition of mitochondria (Mg-m).
However, in hypotonic medium, the binding of exo-
genous cyto-c in the presence of magnesium, added
either before or after cyto-c, was still higher than that

in isotonic medium, with a value of 0.2 nmolÆmg
)1
obtained by subtracting, from the 2.1 nmol, the value
of 0.91 nmol of endogenous cyto-c reported in Fig. 2A
(and divided by 6 mg of protein). The finding that the
oxidation rate of exogenous NADH after the addition
of cyto-c is essentially the same with the hypotonic
medium, in the absence or presence of magnesium
(Fig. 1, traces c, e), may suggest that only the
nanomoles of cyto-c still bound in the presence of
magnesium are directly involved in the activity of
the NADH/cyto-c system. Notwithstanding that, in
the presence of magnesium and independent of the
sequence of additions, the total binding of cyto-c
remains the same (Fig. 2B, Mg present), the rate of
NADH oxidation is decreased to 24 nmolÆmin
)1
Æmg
)1
(Fig. 1, trace d) with magnesium present in the med-
ium, compared with a value of 45 nmolÆmin
)1
Æmg
)1
with magnesium added after the mitochondria (Fig. 1,
trace e). This may indicate that remodelling of the
mitochondrial structure is involved [16,20].
Magnesium-dependent cyto-c release activates,
in hypotonic medium, the NADH/cyto-c system
with the generation of a membrane potential

As reported for the first time in 1995 [3], the activity of
the cytosolic NADH/cyto-c electron transport pathway
is coupled, similar to the activity of the respiratory
chain, to the generation of an electrochemical proton
gradient determined also as the mitochondrial mem-
brane potential change (DY
m
) [4,21]. The change in
DY
m
generated by the NADH/cyto-c system is similar
to that supported either by succinate oxidation or ATP
hydrolysis [21]. The fluorimetric determination of DY
m
of mitochondria incubated in isotonic medium with
10 lm safranine as probe is reported in Fig. 3. As a
Magnesium ions and cytosolic cytochrome c oxidation G. La Piana et al.
6172 FEBS Journal 275 (2008) 6168–6179 ª 2008 The Authors Journal compilation ª 2008 FEBS
result of the very low electron pressure provided by the
oxidation of endogenous substrates and the activity of
the NADH/cyto-c system, small amounts of both BSA
and EGTA were added to stabilize the membrane
potential. Consistent with the previously reported data
[4,21], Fig. 3 (trace a) shows that DY
m
, supported by
the oxidation of endogenous substrates, is abolished by
the addition of the respiratory chain inhibitors (rote-
none and myxothiazol). The subsequent activation of
the NADH/cyto-c system with the sequential addition

of cyto-c, NAD
+
and alcohol dehydrogenase restores
DY
m
. With this experimental approach, NADH is con-
tinuously produced outside the mitochondria by the
activity of added alcohol dehydrogenase, which cataly-
ses the oxidation of ethanol present in the incubation
medium as the solvent of rotenone and myxothiazol
solutions. It can be observed that the addition of cyto-c
promotes a nonspecific change in fluorescence, which is
proportional to the amount of cyto-c added and is not
abolished by uncouplers, and therefore was electrically
reset down. Figure 3 (trace a) also shows that the
uncoupler carbonyl cyanide p-(trifluoromethoxy)phen-
ylhydrazone (FCCP) dissipates DY
m
, supported by the
oxidation of NADH present outside the mitochondria.
Dissipation of the membrane potential can also be
obtained with the addition of cyanide (Fig. 3, trace b).
These results confirm that the fluorescence signal is the
expression of an electrical charge gradient between the
outside and inside of the mitochondria, and that cyto-
chrome oxidase is involved in this process. Moreover,
the experimental approach in Fig. 3 mimics in vitro the
generation of cytosolic NADH, as well as the activa-
tion of the NADH/cyto-c system when, in physio-path-
ological conditions (e.g. apoptosis), a catalytic amount

of cyto-c is released from the mitochondria. Magne-
sium ions added after the mitochondria, but before the
activation of the NADH/cyto-c system (Fig. 3, trace b),
promote a slight and sometimes not appreciable
decrease in DY
m
linked to the oxidation of exogenous
NADH. Results identical to those illustrated in Fig. 3
(trace b) were obtained when MgCl
2
was added to the
isotonic medium before the mitochondria (not
reported).
Figure 3 (trace c) shows that, also with hypotonic
mitochondria, the activation of the NADH/cyto-c elec-
tron transport system generates a membrane potential.
According to the sequence of additions, it can be seen
that the generation of DY
m
is strictly linked to the
presence of exogenous cyto-c as the electron intermedi-
ate. However, with hypotonic mitochondria, a 1 lm or
lower cyto-c concentration is sufficient to obtain the
full expression of the membrane potential (see Fig. 3,
traces a and c). It should be noted that, in Fig. 3
(traces c–f), mitochondria were preincubated for 5 min
in the presence of respiratory chain inhibitors (rote-
none and myxothiazol) to suppress the membrane
potential generated by the oxidation of endogenous
substrates (Fig. 3, traces a, b). In the presence of mag-

nesium (Fig. 3, trace d), DY
m
was generated even with-
out the addition of exogenous cyto-c, but was still
sensitive to the uncoupler dissipation effect. These
results are substantially consistent with those reported
in [8,9]. Furthermore, as a new and original finding,
Fig. 3 also shows that, if magnesium is present in the
incubation medium before the addition of mitochon-
dria (Fig. 3, trace e), cyto-c is required to generate
DY
m
, similar to the results obtained in the absence of
Mg
2+
(Fig. 3, trace c). The comparison of the results
in Fig. 3 (traces e and f) gives a clear view of the
Fig. 3. Mitochondrial membrane potential generated by the oxida-
tion of either endogenous respiratory substrates or extra mitochon-
drial NADH in isotonic (A) and hypotonic (B) media. (A)
Mitochondria (3 mg protein, ‘mito’) were added to 3 mL of 250 m
M
sucrose isotonic medium containing 0.1 mgÆmL
)1
BSA, 50 lM
EGTA and 10 lM safranine. (B) Mitochondria (3 mg protein) were
incubated for 5 min in 3 mL of 25 m
M sucrose hypotonic medium
containing 6 l
M rotenone, 6 lM myxothiazol, 0.1 mgÆmL

)1
BSA,
50 l
M EGTA and 10 lM safranine. Further additions: 6 lM rotenone
plus 6 l
M myxothiazol (RM); 5 lM and, only in trace c, 1 lM cyto-c
(C); 166 l
M NAD
+
(N); 45 IU alcohol dehydrogenase (A); 4 mM
MgCl
2
(Mg); 1.6 lM FCCP (F); 1 mM potassium cyanide (CN); 30 lM
TMPD (T). In trace e, 4 mM MgCl
2
was already present in the incu-
bation medium before the addition of mitochondria. Experiments
reported are representative of seven performed with five different
mitochondrial preparations.
G. La Piana et al. Magnesium ions and cytosolic cytochrome c oxidation
FEBS Journal 275 (2008) 6168–6179 ª 2008 The Authors Journal compilation ª 2008 FEBS 6173
differential effect of magnesium ions, whether already
present in the hypotonic medium or added after the
mitochondria. It also provides direct evidence that, in
these latter conditions, the addition of magnesium
mimics the results obtained with the addition of
cyto-c, the only difference being that the potential
is lower and N,N,N¢,N¢-tetramethyl-p-phenylene-
diamine (TMPD) must be added to achieve its com-
plete expression.

Effect of magnesium on the two semi-reactions
of the NADH/cyto-c system
The possibility that magnesium, other than having a
protective effect on the permeability of mitochondria
incubated in hypotonic medium (see Fig. 2 and [7]),
may also directly affect the activity of the NADH/
cyto-c system was tested in the experiments reported
in Fig. 4. The activity of the system was split into
two main steps: (a) the reduction of exogenous
cyto-c induced by the oxidation of NADH in
cyanide-inhibited respiration (Fig. 4A); and (b) the
oxidation of exogenous ferrocytochrome c (ferrocyto-c)
(Fig. 4B). It was found (Fig. 4A) that the NADH/
cyto-c reaction rate was similar in both isotonic and
hypotonic mitochondria. This is an expected result if
it is considered that the reaction being catalysed by
the NADH/cyto-b
5
complex sited on the external
side of MOM occurs outside the mitochondria, and
therefore should be independent of the osmolarity of
the medium. In the presence of magnesium (Fig. 4,
trace b), an increased rate of cyto-c reduction, usu-
ally not higher than 15%, was observed in both iso-
tonic and hypotonic media. In Fig. 4 (traces c and d),
it is shown that the oxidation rate of exogenous
cyto-c by isotonic mitochondria is not affected by
the presence of MgCl
2
, added either before or after

the mitochondria. With hypotonic mitochondria, the
oxidation rate is greatly increased (Fig. 4, trace e)
and magnesium decreases this rate only when present
in the medium (Fig. 4, trace f), but has no effect
when added after the mitochondria (Fig. 4, trace e).
The data reported in Fig. 4B are consistent with and
similar to those in Fig. 1 on the oxidation of exo-
genous NADH as the expression of the activity of
the complete system.
Discussion
The data presented provide new insights into the role
of magnesium ions in the permeability of MOM to
both endogenous and exogenous cyto-c, and provide
further support for the functional activity, in liver
mitochondria, of the cytosolic NADH/cyto-c electron
transport system. The oxidation of exogenous NADH
occurs exclusively if cyto-c is also present outside the
mitochondria. It is not relevant if cyto-c is added
externally, as in the case of isotonic mitochondria, or
is released outside from MIS when mitochondria are
incubated in hypotonic medium (Figs 1 and 2). In both
cases, the activity of the system generates DY
m
(Fig. 3), which contradicts the interpretation that the
system could represent the expression of completely
broken mitochondria [8–10] and/or of mitochondria
with MOM broken at leopard’s spots. This interpreta-
tion is also in contrast with the finding that exogenous
cyto-c is unable to react with sulfite oxidase present in
MIS [7], and that dextran sulfate inhibits exogenous

NADH oxidation in intact mitochondria, but not in
mitoplast preparations [6]. Comparing the data
reported in [7] with those in Fig. 2, it is clear that, in
hypotonic mitochondria, endogenous cyto-c is released
outside, although in a limited amount (14%), but
cyto-c present in the medium is unable to move from
outside into MIS [7]. Therefore, MOM of mitochon-
dria incubated in either isotonic sucrose or very low
osmotic medium (25 mm sucrose) is not broken, as
generally believed, but still maintains the function of a
Fig. 4. Reduction and oxidation of exogenous cyto-c by mitochon-
dria incubated in isotonic and hypotonic media. (A) Activity of rote-
none-insensitive NADH-cytochrome c reductase. (B) Oxidation of
ferrocyto-c present outside the mitochondria. Mitochondria (90 lg
protein in A and 1.0 mg protein in B) were incubated for 5 min in
3 mL of 250 m
M (Iso) or 25 mM (Hypo) sucrose-based medium con-
taining 6 l
M rotenone and 6 lM myxothiazol. (A) 15 lM ferricyto-c
plus 1 m
M potassium cyanide were also present, and the reaction
was started with the addition of 0.2 m
M NADH (N). (B) The reaction
was started with 15 l
M ferrocyto-c. Incubations were made in the
absence (traces a and c) or presence of 4 m
M MgCl
2
, added either
before (traces b, d and f) or 2 min after (traces b and d) the addition

of mitochondria; in trace e, magnesium was either absent or added
2 min after the mitochondria. Values on the traces represent the
nanomoles of cyto-c reduced (A) or oxidized (B) per minute per
milligram of protein, and are representative of eight experiments
performed with five different mitochondrial preparations.
Magnesium ions and cytosolic cytochrome c oxidation G. La Piana et al.
6174 FEBS Journal 275 (2008) 6168–6179 ª 2008 The Authors Journal compilation ª 2008 FEBS
protective envelope not permeable to either exogenous
cyto-c or trypsin [7]. The activity of succinate/exoge-
nous cyto-c reductase and the oxidation of exogenous
ferrocyto-c, long proposed to be the expression of bro-
ken and damaged mitochondria [22–24], are both mis-
leading and inappropriate to extrapolate the
percentage of intact mitochondria. Previously pub-
lished results [5–7], and those presented in this report
on the effect of magnesium ions, are consistent with
the view that both the succinate/cyto-c reductase activ-
ity and the oxidation rate of exogenous ferrocyto-c are
correlated with the frequency of specific contact sites.
With regard to the effect of magnesium on the activ-
ity of the cytosolic NADH/cyto-c electron transport
system, our data show a new finding, not elicited pre-
viously, that magnesium presents a dual effect very
clear in hypotonic but not visible in isotonic mitochon-
dria. The first effect involves protection against the
increase in permeability of MOM when magnesium is
present in the hypotonic medium before the addition
of mitochondria. Experimental data have shown that
contact sites [5,6], indicated as respiratory contact
sites, are the mitochondrial structures in which the

main components of the NADH/cyto-c system are
localized. In 1995, we defined the respiratory contact
sites as dynamic but not fixed structures, which could
be visualized as frequently forming and breaking
bridges between different points of the inner and outer
membranes; over time, the two membranes could be
involved in the contact sites, forming these structures
in all their parts [3]. Therefore, as a result of the much
greater area of MIM than MOM, the increase in the
matrix volume by hypotonic medium pushes MIM
against MOM, which becomes stretched; its permeabil-
ity is increased and the contact points between the two
mitochondrial membranes are also expected to
increase. In these conditions, the oxidation rate of
exogenous NADH is greatly increased (Fig. 1). If
Mg
2+
is added before the mitochondria, the release
linked to the hypotonic medium of adenylate kinase,
sulfite oxidase and endogenous cyto-c is, to a large
extent, prevented relative to the findings obtained
when Mg
2+
is added after the mitochondria ([7] and
Fig. 2), and the oxidation rate of NADH is also
decreased from 45–49 to 24 nmolÆmin
)1
Æmg
)1
(Fig. 1).

Tentatively, it could be hypothesized that magnesium,
with its two positive charges, may function as a linker
between the negative charges of phospholipids and/or
proteins. MOM and MIM become more compact,
offering more resistance to the stretching caused by the
pressure induced by the increased volume of the
matrix. The permeability of MOM is decreased signifi-
cantly, together with the frequency of contact sites.
Therefore, the rates of NADH oxidation (Fig. 1) and
of the succinate/exogenous cyto-c reductase [7] are
both decreased greatly.
The second effect is linked to the property of mag-
nesium to prevent and remove the binding of exoge-
nous cyto-c depending on whether it is added before
or after cyto-c (Fig. 2B). In hypotonic medium, cyto-c
binding increases, which tentatively could be the conse-
quence of MOM stretching with the disclosure of addi-
tional nonspecific binding sites. However, this increase
is not responsible for the increased rate of NADH oxi-
dation, as the extra binding is completely removed by
the addition of magnesium, but the oxidation rate is
not affected and still remains high (Figs 1 and 2B).
Inhibition of the rate is observed when magnesium is
already present in the medium to prevent the binding
of cyto-c, which, however, is identical to the binding
observed with magnesium added after the mitochon-
dria or when Mg
2+
is already present in the medium.
Therefore, the decreased rate must be ascribed to the

above-mentioned protective effect of magnesium on
the structural remodelling of the two mitochondrial
membranes, elicited when present in the medium (but
not when added after the mitochondria), rather than
to the binding of cyto-c. All of these considerations
indicate that cyto-c present outside the mitochondria is
essentially in the free form, available to shuttle elec-
trons between the NADH/cyto-b
5
reductase and the
respiratory contact sites (see the scheme in [6]). How-
ever, it can be speculated that, corrected for the
amount of endogenous cyto-c, the 120 pmol of exo-
genous cyto-c bound per milligram of protein of iso-
tonic mitochondria, and insensitive to the presence of
magnesium, may be the expression of the molecules
tightly bound essentially to both the cytosolic side of
the respiratory contact sites and the NADH/cyto-b
5
reductase complex, rather than to nonspecific sites.
Therefore, one possible mechanism may be that, of all
the cyto-c molecules added to isolated mitochondria or
present in the cytosol, only a few, in relation to the
binding sites available, remain firmly bound, some to
the NADH/cyto-b
5
complex and some to contact sites.
The majority of cyto-c molecules are free to move and,
in the oxidized state (with an intermolecular process),
accept electrons from reduced molecules bound to the

NADH/cyto-b
5
system; in the reduced state, they
transfer their electrons to oxidized cyto-c bound to
contact sites. With hypotonic mitochondria, it can be
calculated that the amount of cyto-c bound and insen-
sitive to magnesium added after mitochondria is
200 pmolÆmg
)1
protein. These results correlate with the
differential rate of NADH oxidation by isotonic and
hypotonic mitochondria reported in Fig. 1. As the
G. La Piana et al. Magnesium ions and cytosolic cytochrome c oxidation
FEBS Journal 275 (2008) 6168–6179 ª 2008 The Authors Journal compilation ª 2008 FEBS 6175
number of units per milligram of protein of the
NADH/cyto-b
5
complex should be independent of the
osmolarity of the medium, the increase in the binding
of cyto-c with hypotonic mitochondria and the insensi-
tivity to magnesium ions could essentially be the
expression of the increased number of respiratory con-
tact sites. Additional and direct experimental data are
required to provide further support for these specula-
tions.
The finding that MgCl
2
promotes the release of an
additional amount of endogenous cyto-c with hypo-
tonic but not isotonic mitochondria is consistent with

the view that, in physiological low-amplitude swelling,
similar to the experimental large-amplitude swelling
induced by hypotonic medium, the contact area
between the two mitochondrial membranes is increased
extensively. Cyto-c molecules still bound to the exter-
nal leaflet of MIM turn to face the medium and are
more accessible to displacement by magnesium. This
interpretation correlates with the observation that the
large increase in binding of externally added cyto-c,
observed in hypotonic mitochondria, is completely
removed or prevented by magnesium. However, consis-
tent with the desorption mechanism [8,16], the possibil-
ity that cyto-c bound to MIM is removed by
magnesium, and then released outside because of the
increased permeability of MOM, cannot be excluded.
The data presented here, together with those reported
in [7], show that magnesium may have a dual role in
the permeability of mitochondrial membranes: (a) it
counteracts the remodelling of membrane structures
induced by low-amplitude physiological swelling or, in
general, by cell injury; and (b) it contributes to the
correct execution of the cell death programme by pro-
moting the release of cyto-c from mitochondria. In our
previous publications, we have emphasized that, acti-
vated by the presence of cyto-c outside the mitochon-
dria, the NADH/cyto-c system may have at least two
functions. The first involves the promotion, in healthy
cells, of the oxidation of cytosolic NADH utilizing the
mitochondrial machinery to generate ATP with the
energy preserved in the membrane potential (Fig. 3).

This activity becomes essential for cell survival in the
presence of an impairment of the respiratory chain at
the level of one of the first three respiratory complexes.
The second function concerns its role in the apoptotic
programme. It is well known that, in the early stages
of this process, cyto-c is released into the cytosol where
it participates in the formation of apoptosomes,
responsible for the activation of caspases, leading to
nuclear condensation and the formation of apoptotic
bodies. However, the release of cyto-c from mitochon-
dria promotes an impairment of the respiratory chain,
followed by a relevant decrease in the energy content
of the cell in relation to the amount of cyto-c trans-
ferred into the cytosol. This raises the problem of the
energy source required for the correct execution of the
apoptotic programme. Indeed, in the early stages of
apoptosis, mitochondria continue to generate a mem-
brane potential [25–27] which, according to some
authors, can be ascribed to hydrolysis, inside the mito-
chondria, of ATP generated by glycolytic activity [28].
We maintain that, in these conditions, the cytosolic
cyto-c activates the NADH/cyto- c electron transport
pathway, and more energy is made available for apop-
totic processing before the membrane potential dissipa-
tion step is activated. Indications have been obtained
which support the transient participation of cyto-c in
the formation of apoptosomes, as cyto-c has not been
found in preparations of precipitated native apopto-
somes [29] and a smaller amount of cyto-c has been
found relative to that of Apaf-1 in mature apopto-

somes [30]. The availability of energy, either as ATP
or in the form of a membrane potential, is a prerequi-
site to make apoptotic cell death a programmed and
controlled process distinct from necrosis, which does
not require energy as it is characterized by acute dis-
ruption of cellular metabolism. Therefore, the activa-
tion of the NADH/cyto-c system may represent an
additional source of energy for the correct execution of
the apoptotic programme. Recently, it has been
reported that, in homogenates of apoptotic HeLa cells,
the reduction rate of added cyto-c is lower than that in
homogenates of control cells [31]. In the presence of
azide, the reduction rate is increased and the values
obtained are identical in both types of homogenate.
The decreased rate in the reduction of cyto-c has been
ascribed to an increased involvement of mitochondria
present in homogenates of apoptotic cells, responsible
for the oxidation of reduced cyto-c.
Data have also been reported showing that, in mice
liver mitochondria, magnesium ions are involved in
Bax- and Bid-induced cyto-c release [32,33]. The find-
ing that magnesium ions regulate the permeability of
mitochondria and the binding of cyto-c may have rele-
vant implications in the bioenergetics of the cell, as
well as possible consequences in therapeutic applica-
tions. In tumour cells, an increase in the concentration
of cytosolic cyto-c may contribute to activate the
apoptotic process.
Experiments are in progress in our laboratory to
measure and characterize, in healthy and apoptotic

HeLa cells, the activity of the cytosolic NADH/cyto-c
electron transport system, and the role of magnesium
ions in modulating the transfer of cyto-c from mito-
chondria into the cytosol.
Magnesium ions and cytosolic cytochrome c oxidation G. La Piana et al.
6176 FEBS Journal 275 (2008) 6168–6179 ª 2008 The Authors Journal compilation ª 2008 FEBS
Materials and methods
Incubation of mitochondria
Rat liver mitochondria were isolated by differential centrifu-
gation in 250 mm sucrose medium, as described previously
[3]. Incubations were carried out at 25 °C at pH 7.4 in media
consisting of 20 mm Hepes/Tris and either 250 mm (isotonic
medium) or 25 mm (hypotonic medium) sucrose. The intact-
ness of mitochondrial membranes was routinely determined
by four different but convergent and already described [7]
integrity tests based on the following activities: (a) the oxida-
tion of exogenously added NADH in the absence of both
rotenone and exogenous cyto-c to assess the impermeability
of NADH through MIM; (b) the insensitivity of intermem-
brane adenylate kinase to proteolytic attack by added trypsin
to reveal the increased permeability, if any, of MOM during
the course of incubation; (c) the sulfite/exogenous cyto-c oxi-
do-reductase activity coupled to its sensitivity to trypsin to
assess the impermeability of exogenous cyto-c through
MOM; (d) the succinate/exogenous cyto-c oxido-reductase
activity to reveal the presence of damaged mitochondria with
MIM intact but with MOM permeable to exogenous cyto-c.
Mitochondrial suspensions containing no more than 2% of
damaged mitochondria, according to both the NADH oxida-
tion test (a) and sulfite/exogenous cyto-c test (c), were utilized

(see also [7]). NADH oxidation was determined spectropho-
tometrically at 340–374 nm (e = 4.28 mm
)1
Æcm
)1
) and the
redox state of cyto-c at 548–540 nm (e =21mm
)1
Æcm
)1
)to
minimize the interference of cyto-b
5
sited on MOM, which
has an absorbance peak at 556 nm. The protein content was
determined by the biuret method.
Cyto-c content of mitochondria incubated in the
absence and presence of exogenous cyto-c
The determination of endogenous cyto-c was performed in
pellets of 6 mg protein of mitochondria incubated for
10 min in 6 mL of both isotonic and hypotonic media, and
then centrifuged at 10 000 g for 10 min at 4 °C. Magnesium
ions were added at a concentration of 4 mm according to
the sequence of additions specified in the legend to Fig. 2.
The capability of magnesium ions to both prevent and
remove the binding of exogenously added cyto-c was deter-
mined in isotonic and hypotonic media with two experi-
mental protocols. In the first procedure, 4 mm MgCl
2
was

added 5 min after the mitochondria, but before 2 lm cyto-c
was added at 10 min, and the reaction was stopped at
15 min. In the second procedure, 2 lm of cyto-c was added
at 5 min, 4 mm MgCl
2
at 10 min and the reaction was
stopped at 15 min. Details of the sequence of additions are
reported in the legend to Fig. 2. To increase the reliability
of the results and to better appreciate the changes induced
by magnesium, mitochondria containing 6 mg of protein
were incubated in 6 mL of medium, centrifuged as specified
above and the pellets resuspended in 1.0 mL of 50 mm P
i
(pH 7.4) supplemented with 0.5% Triton X100. The cyto-c
content of the pellets and supernatants was determined
from a reduced minus oxidized differential spectrum in the
wavelength range 500–650 nm, utilizing potassium ferricya-
nide as oxidant and sodium dithionite as reductant. Spectra
and kinetic determinations were carried out with Hitachi-
Perkin Elmer model 557 (Hitachi, Ltd., Tokyo, Japan),
Varian Cary model 50 (Varian Inc., Melbourne, Australia)
and Aminco DW2A double-wavelength [modernized by
OLIS (On Line Instruments Inc., Bogart, GA, USA)] spec-
trophotometers.
Determination of mitochondrial membrane
potential
Time-dependent mitochondrial membrane potential changes
(DY
m
) were followed fluorimetrically with a Perkin-Elmer

LS-5B fluorescence luminometer, with 10 lm safranine O at
wavelengths of 520 nm (excitation) and 580 nm (emission)
[34].
Materials
All reagents were of analytical grade and mainly obtained
from Sigma-Aldrich Chemical Co. (St Louis, MO, USA)
and Roche Spa (Milan, Italy). Ferrocyto-c was prepared
daily as reported in [6].
Acknowledgements
The authors are grateful to Mr Francesco Felice for
his skilled technical assistance. This work was sup-
ported by grants from MIUR (Prin 2005–2007 ‘Bioen-
ergetica: meccanismi molecolari e aspetti fisiopatologici
dei sistemi bioenergetici di membrana’), CNR (Insti-
tute of Biomembranes and Bioenergetics, IBBE, Bari,
Italy) and University of Bari, Bari, Italy.
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