Superoxide generated in photosystem I is scavenged by plastoquinol and
other natural prenyllipids in thylakoid membranes.
J Kruk1, M Jemioła-Rzemińska1, K Burda2, GH Schmid3, K Strzałka1
1Department of Plant Physiology and Biochemistry, Institute of Molecular Biology,
Jagiellonian University, Al. Mickiewicza 3, 31-120 Kraków, Poland
e-mail: firstname.lastname@example.org, fax: (+48 12) 633 69 07
2Institute of Nuclear Physics, ul. Radzikowskiego 152, 32-342 Kraków, Poland
3Fakulät für Biologie, Lehrstuhl Zellphysiologie, Universität Bielefeld, D-33501 Bielefeld,
Keywords: superoxide, photosystem I, plastoquinol, prenyllipids, thylakoid membrane
The main source of oxygen consumption in chloroplasts is chlororespiration and
photoreduction of oxygen by low-potential Fe-S centers in photosystem I (PS I), however,
only the superoxide generation by PS I is directly light dependent. Within PS I, superoxide is
produced in the aprotic, hydrophobic interior of the thylakoid membrane at Fe-S centers: X
and A/B. Because of the aprotic character of the membrane interior, the lifetime of superoxide
is extended. After diffusing to the membrane surface, the superoxide undergoes protonation
and dismutation to hydrogen peroxide. It was suggested (Takahashi et al. 1980) that
superoxide could also reduce oxidized cytochrome f or plastocyanin forming pseudocyclic
electron transport around PS I.
In our study, we examined whether the prenyllipids present in thylakoid membranes, like
plastoquinone (PQ-9), its reduced form (PQH2), tocoquinone (TQ) and tocopherol (Toc) react
with the superoxide generated in PS I. Such a reaction would inhibit and prevent the
formation of the toxic hydrogen peroxide in chloroplasts.
The formation of hydrogen peroxide from oxygen manifests in the oxygen
consumption that can be followed directly using an oxygen electrode. Another indirect
approach used in many previous studies (Asada et al. 1974, Miyake et al. 1998, Takahashi and
Asada 1988) was to observe spectrophotometrically the reduction of the added cytochrome c
that is reduced by superoxide. As a result of this reaction, oxygen is released back from
superoxide and this process manifests in the inhibition of hydrogen peroxide formation and
oxygen consumption. However, our own experiments showed that at low and medium light
intensity cytochrome c is mainly reduced by the PQ-pool in thylakoids and not by superoxide
generated in PS I (Kruk et al., unpublished results). Therefore, the method based on
cytochrome c reduction is not suitable and not specific for the measurements of superoxide
generation in thylakoids and direct methods of oxygen consumption measurements seem to be
only appropriate for this purpose.
Materials and methods
Spinach thylakoids were isolated according to the method described by Robinson and Yocum
(1980). The oxygen consumption measurements were performed in 50 mM Hepes buffer, pH
7.5 containing 10 mM NaCl and 5 mM MgCl2 using a three electrode system (Schmid and
Thibault 1979) in the presence of 50 µM DCMU and 10 mM hydroquinone as an electron
donor. Saturating light flashes of 5 µs (full width at half-maximum) were provided by a xenon
lamp (Stroboscope 1539A from General Radio). The samples were illuminated by 15 flashes
spaced 300 ms apart. Hexane extraction of thylakoids was performed by 3-fold extraction of
lyophylised thylakoids (1 mg Chl) with 1 ml hexane for 20 min, each extraction. After
centrifugation and removing the supernatant, thylakoids were dried in rotatory evaporator and
suspended in the buffer giving final conc. of 1 mg/ml Chl and used as a stock solution. 1 µM
vit. K1 was added to all the extracted samples.
Results and Discussion
We used two approaches in the investigation of the inhibition of superoxide generation by PS
I. In one case, the prenyllipids were added to fresh, dark adapted thylakoids suspension. Since
within thylakoid membranes some of the investigated prenyllipids (PQ-9, Toc, TQ) are
already present, we used lyophylised and hexane-extracted thylakoids to obtain the control
sample devoid of the prenyllipids. This procedure removes selectively prenyllipids from the
membranes preserving their structure and function. Because this extraction removes also
partially vitamin K1 (vit. K1) from PS I, which is the primary electron acceptor in PSI, we
added this vitamin at 1 µM concentration to all extracted samples. First, we measured the
influence of the extracted components added back to the thylakoids (Table 1).
Table 1. Oxygen consumption by flash-illuminated, hexane-extracted spinach thylakoids (150 µg/ml
Chl) in the presence of 10 mM hydroquinone and 50 µM DCMU. The numbers in '( )' denote the
proportion of the added components of the extract in relation to their original concentration in
thylakoids; Y1 and Y2 correspond to oxygen uptake amplitudes at the first and the second flash,
sample oxygen uptake
(% of control)
+ extract (1:1)
+ extract (5:1)
It can be seen from the Table 1 that components of the extract inhibit the oxygen consumption
by PS I. The main prenyllipid components of this extract are PQ-9 and Toc which are
probably responsible for the observed effects. With the increase in the added amount of the
extract, the ratio of the first two signals (Y1/Y2) increases. Similar relationship was observed
in the experiments on extracted thylakoids when the increasing amount of vit. K1 was added
to the extracted thylakoids (data not shown). This indicates that vit. K1 present in the extract
was responsible for the observed changes in the Y1/Y2 ratio.
The results of the experiments on the influence of different native and synthetic (PQ-2)
prenyllipids on the oxygen uptake by PS I measured on hexane-extracted thylakoids are
shown in Table 2.
Table 2. Inhibition of oxygen consumption by flash-illuminated, hexane-extracted spinach thylakoids
in the presence of different prenyllipids at the prenyllipid/chl ratio 1:5 and 1:10 (mol/mol). Other
conditions as in Table 1.
(% of control)
(% of control)
100 - -
The presented data show that all the investigated prenyllipids inhibited the measured
oxygen consumption by PS I. The most effective in this respect was PQH2-9 followed by PQ-
9, PQH2-2 and TQH2. The well-known antioxidant, Toc was relatively poorly active whereas
TQ showed nearly no effect in the investigated reaction. All the three reduced forms of
prenylquinones were more active than the corresponding oxidised forms. Since PQ-2 and PQ-
9 have the same redox potentials, the differences observed for both the reduced and oxidized
forms of these two plastoquinones are probably caused by their different localization in
thylakoid membranes. PQ-2, TQ and Toc are supposed to be located close to the membrane
surface, while the reduced and especially the oxidized form of PQ-9 are supposed to reside
close to the interior of the thylakoid membrane. Since the superoxide is generated in the
hydrophobic, internal part of the membrane, both PQ-9 forms, which are located close or at
the site of superoxide generation, have the highest activity in the reaction with superoxide
among the investigated prenyllipids. The influence of the investigated prenyllipids on the
oxygen consumption for the untreated thylakoids (not extracted) is presented in Table 3.
Table 3. Inhibition of oxygen consumption by flash-illuminated spinach thylakoids in the presence
of different prenyllipids at the prenyllipid/chl ratio 1:2.5 and 1:5 (mol/mol). Other conditions as in
Table 1. The sample with ferredoxin (Fd) was measured additionally in the presence of 30 mM MgCl2.
(% of control)
(% of control)
100 - -
73 - -
73 - -
38 - -
5 µM Fd +
The results show that similar pattern of the inhibition for the reduced prenylquinones and
Toc can be observed as in the case the extracted thylakoids. However, the influence of both
plastoquinones on the oxygen uptake is considerably less pronounced and that of TQ is more
evident than in the case of extracted thylakoids. Generally, higher concentration of the
prenyllipids are required to obtain similar extend of inhibition for the reduced prenylquinones
or Toc. This is probably caused by only partial incorporation of the added compounds into the
lipid bilayer because of the presence of native prenyllipids, as well as peripheral membrane
proteins that are partially removed in the case of extracted thylakoids. The PQH2-9 formed in
thylakoids by enzymatic reduction of PQ-9 with ferredoxin-PQ reductase, after addition of
ferredoxin and NADPH, was even more active in the inhibition of oxygen consumption than
the externally added PQH2-9 (Table 3).
Our results indicate that the PQH2-9/PQ-9 couple, as well as other natural membrane
prenyllipids, such as α-Toc or α-TQH2, play an important role in scavenging superoxide
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radical formed in PS I. This reaction reduces the level of superoxide diffusing towards
membrane surface and inhibits formation of the toxic hydrogen peroxide in chloroplasts.
The model presented below shows the protective function of the investigated prenyllipids
against the superoxide generated by PS I in thylakoid membranes.
Fig. 1. The model shows generation of superoxide radical in PS I and its scavenging by membrane prenyllipids.
The size of the prenyllipid symbols reflects their reactivity with superoxide (based on the results from Table 2).
Supported by the KBN grant No. 6 P04A 031 20.
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