Biologia 64/5: 877—880, 2009
Section Cellular and Molecular Biology
DOI: 10.2478/s11756-009-0150-3
Evaluation of Fe(III) reduction by mitochondria induced
with a respiratory substrate NADH or succinate,
using a Fe(II)-specific chelator bathophenanthroline disulfonate
in Saccharomyces cerevisiae
Daisuke Tsugama1, Shenkui Liu2 & Tetsuo Takano1*
1
Asian Natural Environmental Science Center (ANESC), The University of Tokyo, 1-1-1, Midori-cho, Nishitokyo-shi,
Tokyo, 188-0002, Japan; e-mail: takano@anesc.u-tokyo.ac.jp
2
Alkali Soil Natural Environmental Science Center (ASNESC), Northeast Forestry University, Harbin 150040, People’s
Republic of China
Abstract: Mitochondria are the centers of the cellular iron metabolism. Iron utilization by mitochondria is deeply related
to their respiratory chain activity. We isolated mitochondria from Saccharomyces cerevisiae and examined Fe(III) reduction
induced by a respiratory substrate (NADH or succinate), using a Fe(II)-specific chelator (bathophenanthroline disulfonate).
In the presence of either 50 µM NADH or 5 mM succinate, the amount of reduced Fe(III) was linearly correlated with the
amount of mitochondria. As the concentration of the substrate increased, the rate of the mitochondrial Fe(III) reduction
reached a plateau. In the presence of 1 mM ADP or 1 mM ATP, the extramitochondrial Fe(III) reduction was repressed
when succinate was used as the substrate, but not when NADH was used. ADP had an inhibitory effect even under low
concentration of succinate, suggesting that ADP and ATP acted in a manner of both competitive and uncompetitive
inhibition.
Key words: Fe(III) reduction; iron; mitochondria; respiratory chain; Saccharomyces cerevisiae; superoxide.
Abbreviations: ALA, 5-aminolevulinate; BPS, bathophenanthroline disulfonate; INT, 2-(4-iodophenyl)-3-(4-nitrophenyl)5-phenyl-2H-tetrazolium chloride; ISC, iron-sulfur cluster; SCS, succinyl-CoA synthase.
Introduction
Iron is essential for living organisms. It is present in
many proteins in the form of a heme or iron-sulfur cluster (ISC), and often plays a crucial role in the function
of the protein. In eukaryotic cells, mitochondria have
machineries for ISC biogenesis and heme synthesis. In
yeast mutants that are defective in mitochondrial ISC
biogenesis or transport, mitochondria accumulate more
iron, and activities of ISC-containing proteins in the cytosol decrease (Kispal et al. 1997, 1999). On the other
hand, in yeast mutants that are defective in mitochondrial heme synthesis, transcription of iron uptake genes
is repressed (Crisp et al. 2003). Mitochondrial ISC biogenesis, which regulates the activity of ferrochelatase
and the resultant heme synthesis (Lange et al. 2004), is
also thought to underlie many human diseases (Rouault
& Tong 2008). Thus mitochondria can play a key role
in iron homeostasis and protein functions of the whole
cell.
It has been suggested that iron is transported into
mitochondria as Fe(II), and that mitochondrial iron ac-
cumulation is deeply related to respiratory chain activity (Flatmark & Romslo 1975). The mitochondrial electron transport chain generates superoxide, and other
kinds of reactive oxygen species derived from superoxide. Because Fe(III) is spontaneously reduced by reacting with superoxide, which is known as the HarberWeiss reaction, mitochondrial superoxide production
may contribute to the reduction of external iron and
the resultant iron accumulation in mitochondria. Complex I and complex III are reported to be the major sites of mitochondrial superoxide production (Orrenius 2007), and thus Fe(III) reduction possibly occurs
around them. Mitochondrial carrier proteins MRS3 and
MRS4 are thought to be involved in iron import into
mitochondria (Foury & Roganti 2002). After being imported into mitochondria, iron is used for ISC biogenesis by the iron donor frataxin (for a review, see Lill
& Mühlenhoff 2005), and for heme synthesis by ferrochelatase (for a review, see Ajioka et al. 2006). Subsequently ISC and heme are incorporated into mitochondrial apoproteins, and also into cytosolic apoproteins
after being exported from mitochondria. Because free
* Corresponding author
c 2009 Institute of Molecular Biology, Slovak Academy of Sciences
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D. Tsugama et al.
878
iron is highly reactive and toxic, the mitochondrial iron
reduction might be coupled with the import, ISC/heme
synthesis, and export, to minimize damage to DNA,
proteins and lipids caused by free iron. This idea is further supported by the fact that more amount of free
iron accumulates in mitochondria and leads to oxidative stress if mitochondrial ISC biogenesis or export is
aborted (Senbongi et al. 1999; Pandolfo 2002).
Though there has been such suggestions for mitochondrial iron metabolism, external Fe(III) reduction by whole mitochondria has not been studied well.
In this study, we isolated mitochondria from baker’s
yeast Saccharomyces cerevisiae, and evaluated Fe(III)
reduction by mitochondria induced with a respiratory
substrate (NADH or succinate), using a Fe(II)-specific
chelator, bathophenanthroline disulfonate (BPS). Because ADP and ATP are deeply related to the mitochondrial energy states, their effects on Fe(III) reduction were also examined.
Material and methods
Yeast growth and mitochondrial isolation
The S. cerevisiae strain BY4741 was grown aerobically at
30 ◦C in media containing 2% (v/v) lactate, 3 g/L yeast extract, 0.5 g/L glucose, 0.5 g/L CaCl2 , 0.5 g/L NaCl, 0.6 g/L
MgCl2 , 1.0 g/L KPi, and 1.0 g/L NH4 Cl at pH 5.6. Yeast
cells were harvested during logarithmic growth phase (A600
= 1.8–2.2). Mitochondria were isolated from spheroplasts
and purified using Percoll density gradient centrifugation as
previously described (Daum et al. 1982; Murakami et al.
1988). The isolated mitochondria were suspended in 0.6 M
mannitol, 20 mM HEPES (K+ ), and 1 mM phenylmethylsulfonyl fluoride, pH 7.2. Protein concentration was determined using Bradford method using BSA as a standard.
Fe(III) reduction assay
To evaluate effects of mitochondria concentration on Fe(III)
reduction, the isolated mitochondria were suspended to 18,
27, 36, or 53 µg protein/mL in the assay buffer containing 0.51 M mannitol, 17 mM HEPES (K+ ), 0.5 mM FeCl3 ,
1mM BPS, and 0.05 mM NADH as a respiratory substrate,
or they were suspended to 22, 43, 67, or 91 µg protein/mL
in the assay buffer above, which was containing 5 mM succinate instead of 0.05 mM NADH as a substrate. Because
Fe(III) was slowly but clearly reduced in the assay buffer
without mitochondria, the assay buffers were freshly prepared in each experiment. The mixture was incubated at
room temperature for 10 min and centrifuged at 13,000
rpm for 2 min, and the A520 of the supernatant was measured. A blank was treated in the same way except without
mitochondria. The amount of reduced Fe(III) was calculated from a standard curve obtained using known amount
of FeSO4 . We also tested the specificity of the reaction to
Fe(III), substituting 0.5 mM FeCl3 in the assay buffer above
with 0.5 mM 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl2H-tetrazolium chloride (INT). In this case, 70% ethanol
was added after an incubation to dissolve formazan resulting from INT, then the solution was centrifuged, and the
A490 of the supernatant was measured. The amount of reduced INT was calculated from the extinction coefficient
for INT-formazan of 15,000 M−1 at 490 nm according to
the manufacturer of INT (Dojindo Molecular Technologies,
Inc.; http://www.dojindo.com/). For kinetics study, mitochondria were suspended to 25 µg protein/mL in the assay
Fig. 1. Effect of mitochondria concentration on the amount of
reduced Fe(III). The reaction was allowed to go for 10 min at
room temperature. Values are means ± SEM (n = 3). Lines are
regression lines.
buffer containing 0.51 M mannitol, 17 mM HEPES (K+ ), 0.5
mM FeCl3 , 1 mM BPS, and 0.01, 0.025, 0.05, 0.075, 0.125,
0.25, 0.5, 1, 2.5, or 5 mM NADH, and the mixture was incubated at room temperature for 15 min. When succinate was
used as a substrate, mitochondria were suspended to 67 µg
protein/mL in the assay buffer containing 0.01, 0.025, 0.05,
0.25, 0.5, 1, or 5 mM succinate instead of NADH, and incubated at room temperature for 20 min. The amount of
reduced Fe(III) was measured as described above. In some
experiments, ADP or ATP was added to 0.5 mM or 1 mM
into the assay buffer.
Results and discussion
NADH is the substrate for complex I in the respiratory chain, while succinate is the substrate for complex II. With both substrates (at concentrations of 50
µM and 5 mM, respectively), the amount of reduced
Fe(III) was linearly correlated with the amount of mitochondria (Fig. 1), confirming that Fe(III) reduction
in the assay buffer was dependent on the concentration of mitochondria. A greater amount of Fe(III) was
reduced when NADH was used as the substrate than
when succinate was used as the substrate, even though
the concentration of NADH was lower than that of succinate. This might be due to the inhibitory effect of
BPS on the oxidation of succinate by mitochondrial
complex II as reported previously (Rich et al. 1977).
Succinate is a substrate for not only complex II but
also succinyl-CoA synthase (SCS) in mitochondria. 5Aminolevulinate (ALA) is a heme precursor whose rate
of synthesis is largely controlled by the activity of ALA
synthase. A subunit of SCS physically interacts with
ALA synthase, and this interaction is thought to promote efficient synthesis of ALA and resultant heme (Furuyama & Sassa 2000). Because heme synthesis is coordinated with mitochondrial iron reduction and transport as described above, the exogenous succinate may
have more directly affected mitochondrial iron reduction than NADH.
Next, we tested the kinetics of extramitochondrial
Fe(III) reduction. The kinetics followed the MichaelisMenten equation as shown in Figure 2. This suggests
that the substrate-induced Fe(III) reduction by mitochondria was dependent on the activity of mitochonUnauthenticated
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Study of Fe(III) reduction with respiratory substrates by yeast mitochondria
879
Fig. 2. Kinetics of extramitochondrial Fe(III) reduction using NADH (A) and succinate (B) as substrate. (A) Assay buffer contained
mitochondria at concentration of 25 µg protein/mL (designated as “mt protein” in the y-axis). Incubation was for 15 min at room
temperature. Values are means ± SEM (n = 3). The curve was obtained by fitting the concentrations of NADH to the Michaelis-Menten
equation of Vmax = 135 nmol/min/mg mt protein and Km = 0.337 mM. (B) Assay buffer contained mitochondria at concentration of
67 µg protein/mL, with or without 1 mM ADP. Incubation was for 20 min at room temperature. The curve of a solid line (for the assay
without ADP) was obtained by fitting the concentrations of succinate to the Michaelis-Menten equation of Vmax = 5 nmol/min/mg
mt protein and Km = 42.7 µM. The curve of a broken line (for the assay with 1 mM ADP) was obtained by that of Vmax = 2.67
nmol/min/mg mt protein and Km = 118 µM.
Table 1. Effects of ADP and ATP on extramitochondrial Fe(III) reduction induced by NADH and succinate.
Reduced Fe(III) (nmol/min/mg mt protein)a
Nucleotide concentration (mM)
ADP
ATP
0.05 mM NADH
5 mM succinate
0
1
0
0
0
1
10.10 ± 0.49
10.49 ± 2.10
9.16 ± 1.43
4.74 ± 0.84
2.32 ± 1.53
2.56 ± 1.32
a
Mitochondria concentrations were 36 µg/mL for NADH and 67 µg/mL for succinate. Incubation was for 10 min at room temperature.
The amount of reduced Fe(III) was measured as described in Figure 2. The values are means ± SEM (n = 3). The values for succinate
are those shown in Figure 2.
drial enzymes such as complex I and complex II to
promote electron flow from NADH and succinate, respectively, into the respiratory chain. The maximum
rate of extramitochondrial Fe(III) reduction was higher
with NADH as the substrate than with succinate. This
might be explained by the inhibitory effect of BPS on
mitochondrial complex II, which limits the electron flow
into the respiratory chain, or by the direct effect of
succinate on mitochondrial iron metabolism as mentioned above. When the NADH concentration was high,
Fe(III) was easily reduced even without mitochondria,
so it was necessary to use the assay buffer without mitochondria as a blank. The substrate-induced Fe(III)
reduction could be caused by superoxide production,
probably from mitochondrial complex I and complex
III, the main sites of mitochondrial superoxide production, because the amount of reduced INT was more than
the amount of reduced Fe(III) in the same assay solution (data not shown).
In the presence of 1 mM ADP or 1 mM ATP, extramitochondrial Fe(III) reduction was repressed when
succinate was used as the substrate, but not when
NADH was used (Fig. 3, Table 1). The kinetics of extramitochondrial Fe(III) reduction in the presence of
ADP suggests that both ADP and ATP acted in a
manner of competitive and uncompetitive inhibition
(Fig. 3). According to Mitchell et al. (1975), ATP has
a promoting effect on succinate-driven reverse electron
flow, namely, by driving the reduction of NAD+ by
Fig. 3. Double-reciprocal plots for extramitochondrial Fe(III) reduction induced by succinate in the presence or absence of ADP.
Extramitochondrial Fe(III) reduction was also measured in the
presence of 0.5 mM ADP in the same way as described in Figure 2.
mitochondrial complex I, while ADP and a structural
analogue of ADP have an inhibitory effect on the reverse electron flow. In our study, however, ADP and
ATP inhibited the succinate-induced Fe(III) reduction
by mitochondria in a similar manner, but did not inhibit the NADH-induced Fe(III) reduction, suggesting
that ADP and ATP did not directly affect the sites
of superoxide production. One possibility is that ADP
and ATP inhibited the electron flow from succinate to
those sites and thus decreased superoxide production
from mitochondria. Because SCS can be activated by
ATP-derived phosphorylation in vitro (Schwartz et al.
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880
1983), it is also possible that the exogenous ADP was
converted into ATP and the ATP affected mitochondrial iron metabolism by promoting the SCS activity.
Because ATM1, which is essential for maturation of
cytosolic ISC-containing proteins, is a mitochondrial
ATP-binding cassette (ABC) transporter (Kispal et al.
1999), whose activity is coupled with ATP hydrolysis
(Kuhnke et al. 2006), the additional ADP and ATP possibly affected ATM1 activity and resultant iron export
from the mitochondria. Further studies are required to
elucidate the relation between mitochondrial electron
chain activity and iron metabolism of whole mitochondria.
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Received February 4, 2009
Accepted June 1, 2009
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