Full text of DOI:10.1007/BF02901162

the secondary electron donor in D1 protein TyrZ+), which
promote the D1 degradation[4]. Using various free radical
scavengers, many scholars[5,6] demonstrated that active
oxygen species are involved in the process of photoinhibi-
Effect of Mn cluster on the
formation of superoxide
radicals in photoinhibition of
photosystem Ċ
tion. However, the role of
In this work, we used 5,5-dimethyl-1-pyrroline-N-
is still controversial[7—13]
.
Oꢀ2x
oxide (DMPO) as the spin trap, associated with EPR spec-
Oꢀ2x
troscopy, to carry out an comparative study on
ZHANG Suping1, TU Tiecheng2, WENG Jun1,
PAN Jingxi2, XU Chunhe1 & YAO Side2
formation during photoinhibition of control Spinach PSĊ
membranes and Mn-depleted PSĊ membranes. Mn clus-
ter is composed of four differnent valent Mn atoms. It can
act as a catalyst with the help of other cofactors to split
H2O into O2, and afford electron to P680[14]. NH2OH
treatment can remove Mn while producing little effect on
extrinsic polypeptides[15]. When the electron transport on
the donor-side is blocked, illumination of Mn-depleted PS
1. Laboratory of Photosynthesis, Institute of Plant Physiology, Chinese
Academy of Sciences, Shanghai 200032, China;
2. Laboratory of Radiation Chemistry, Institute of Nuclear Research,
Chinese Academy of Sciences, Shanghai 201800, China
Correspondence should be addressed to Xu Chunhe (e-mail: xch@iris.
sipp.ac.cn)
Abstract
To further realize the action of superoxide
Ċ
membranes can result in the donor-side-induced
radicals (O2ꢀꢁ.) in photoinhibition of photosystem Ċ (PSĊ),
we employed 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a
spin trap, associated with EPR spectroscopy, to study the
photoinhibition, which is quite different from the accep-
tor-side-induced photoinhibition when normal PSĊ par-
ticles are as experimental materials. The possible mecha-
nism was also discussed in this manuscript.
Oꢀ2x
effect of illumination time on
formation during high
light photoinhibition in PSĊ membranes and Mn-depleted
PSĊ membranes. Results indicated that the removal of Mn
cluster from PSĊ membranes has a strong influence on the
dynamics of superoxide formation. The relative mechanism
was also discussed. These novel findings may further pro-
mote the studies of the structure and function of PSĊ and
the mechanism of photoinhibition.
1
Materials and methods
Oxygen-evolving PSĊ membranes were prepared
from market Spinach leaves according to ref. [16] with
minor modification. Freshly isolated PSĊ membranes
were suspended in SMN solution (0.4 mol/L sucrose, 5
mmol/L MgCl2 and 10 mmol/L NaCl, 50 mmol/L
MES-NaOH, pH 6.5) and stored at 77 K until use. Chlo-
rophyll (Chl) content of the samples was determined ac-
cording to Arnon[17]. The oxygen-evolution activity of
PS Ċ membranes was about 280 Pmol O2 (mg of
Keywords: Mn cluster, photosystem Ċ, photoinhibition, superoxide
radicals, EPR.
In green plants, light energy harvested by antenna
chlorophyll-protein complexes was transferred to the
chlorophyll of PSĊ reaction center (RC) to initiate pho-
tochemical reaction in PSĊ and result in the plas-
toquinone reduction and the water oxidation to molecular
oxygen. But prolonged illumination with high intensities
leads to the functional impairment of PSĊ electron
transport and the structural damage of the D1 protein in
PSĊ RC. This is described as the so-called photoinhibi-
Chl)ꢀ1ghꢀ1.
Mn-depleted PSĊ membranes were prepared from
PSĊ membranes as described in ref. [18] with slight
modifications as follows. 2 mL PSĊ membranes (1 mg
Chl/mL) were diluted with an equal volume of 10 mmol/L
NH2OH, and kept at 4ć in darkness for 30 min. Then the
membranes were collected after centrifugation at 40000×g
for 20 min and washed once with SMN (~5 mL). The re-
sultant pellet of PSĊ particles was suspended in SCN
solution (0.4 mol/L sucrose, 10 mmol/L CaCl2 and 180
mmol/L NaCl, 50 mmol/L Mes-NaOH, pH 6.5). The
Mn-depleted PSĊ membranes (5 mg Chl/mL) were fro-
zen in liquid nitrogen and kept at 77 K until use. The oxy-
gen-evolution activity was about 15 Pmol O2 (mg of
Chl)ꢀ1ghꢀ1 (only about 5% of that in control PSĊ mem-
branes).
ton[1,2]
.
In recent years, great progress has been made in the
research on the molecular mechanism of photoinhibition,
but there still exist many problems to be resolved. Gener-
ally, photoinhibition is considered to proceed by two dif-
ferent molecular mechanisms: One is the so-called accep-
tor-side-induced photoinhibition, it is mainly due to the
over-reduction of QA, which results in the charge recom-
bination to form the triplet states of P680, and subse-
quently to generate singlet oxygen, which promotes the
D1 degradation[3]. The other is the donor-side-induced
photoinhibition, it is mainly caused by the light-induced
accumulation of highly oxidizing species (P680+ and/or
Photosynthetic oxygen evolution was measured with
a Clark-type oxygen electrode in SMN solution at 25ć.
0.8 mmol/L 2,6-dichloro-p-benzoquinone (DCBQ) was
used as the artificial electron acceptor.
Chinese Science Bulletin Vol. 46 No. 22 November 2001
1877

NOTES
Illumination was performed directly in the EPR cav-
At present, DMPO is the most extensively used spin trap
for O2ꢀ.. It can react with O2ꢀ. to form DMPO-OOH adducts,
showing the characteristic EPR signal. In this work, we
also used this spin trap to study the formation of O2ꢀ. in
photoinhibition process of PSĊ.
ity using a 500-W Xenon lamp. Light wavelength less
than 400 nm was cut off by a long-pass filter. The light
intensity on the surface of the cavity was 4.8×102
Jgmꢀ2gsꢀ1. A water filter was used for protection from
far-red irradiation. Photoinhibition was performed in 10
mmol/L sodium phosphate (pH 6.5) containing 100
mmol/L sucrose, 5 mmol/L MgCl2 and 50 mmol/L NaCl.
X-band EPR spectra were recorded at room tem-
perature on a Varian E-112 spectrometer at 9.17 GHz. PS
Acceptor-side-induced photoinhibition was often
achieved when the PSĊ functional donor side was intact.
However, donor-side-induced photoinhibition was com-
monly studied with impairment of oxidizing side of PSĊ.
In the absence of external electron acceptors, no EPR sig-
nal appeared when PSĊ membranes (150 Pg Chl/mL)
were mixed with DMPO (50 mmol/L) in darkness (curve
1 in fig. 2). But after 1 min illumination, the EPR spec-
trum was immediately detected (as shown in curve 2 of
fig. 2). The spectrum was well consistent with the stan-
dard DMPO-OOH signal (DN = 14.2×10ꢀ4 T, DEH = 11.2×
Ċ samples and DMPO were quantitatively injected into a
flat quartz cell. Typical spectrometer parameters for spin
trapping experiments were set as follows: microwave
power, 50 mW; modulation amplitude, 1.25×10ꢀ5 T; time
constant, 0.125 s; receiver gain, 5u104.
The EPR spectrum of Mn was directly recorded in
10ꢀ4 T and DJH = 1.3×10ꢀ4 T)[20]
.
cavity after mixing PSĊ samples with 0.5 mol/L HCl.
EPR parameters were kept as the following: microwave
power, 30 mW; modulation amplitude, 1×10ꢀ3 T; time
constant, 0.25 s; receiver gain, 2u104.
Mn-depleted PS Ċ membranes were used as a
model system for study of donor-side-induced photoin-
hibition. When the samples were mixed with DMPO, no
EPR signals were detected in darkness (spectrum not
shown). But upon illumination, the EPR signals of
DMPO-OOH adducts also appeared (curve 3 in fig. 2). It
is suggested that superoxide radicals should have been
involved in both acceptor-side- and donor-side-induced
photoinhibition of PSĊ. After 1 min illumination (fig. 2),
the relative signal intensity of DMPO-OOH adduct (also
calculated from the ralative amplitude of the third line) in
donor-side-induced photoinhibition was stronger than that
in acceptor-side-induced photoinhibition.
2
Results
(ν) Manganese (Mn) content of PSĊ and Mn-de-
pleted PSĊ membranes. In order to compare the con-
tents of Mn in control PSĊ membranes and Mn-depleted
PSĊ membranes, both membranes in the presence of 0.5
mol/L HCl were quantitatively injected into a flat quartz
cell. The EPR spectra of Mn2+ are shown in fig. 1. The
characteristic EPR spectra of Mn2+ (curve 1) were well
consistent with literature report[19]. Curve 2 in fig. 1 shows
that a great part of Mn was released after NH2OH (5
mmol/L) treatment of PSĊ membranes. According to
relative amplitude of the third-line in EPR spectrum, Mn
content in the NH2OH-treated PSĊ samples was calcu-
lated to be ~20% of control PSĊ samples. This indicated
that the Mn-depleted PSĊ membranes were suitable for
the further study.
Fig. 1. EPR spectra of aqueous Mn2+ released from: (1) PSĊ mem-
branes (0.5 mg/mL) in the presence of 0.5 mol/L HCl; (2) NH2OH-
treated PSĊ membranes, other conditions as (1). For EPR parameters,
see “Materials and methods”.
Fig. 2. EPR spectra for PSĊ samples with different treatments in the
presence of DMPO (50 mmol/L) at room temperature: (1) control, (2)
after 1-min illumination, (3) NH2OH-treated after 1-min illumination.
Chl concentrations were 150 Pg Chl/mL. For EPR parameters, see Sec-
tion 1 “Materials and methods”.
(ξ) Relationship of acceptor-side- and donor-side-
induced photoinhibition of PSĊwith superoxide radicals.
1878
Chinese Science Bulletin Vol. 46 No. 22 November 2001

branes[21], since SOD can inhibit the formation of
;
Oꢀ2x
(ο) Effect of illumination time on EPR intensities of
DMPO-OOH adducts. To further understand the action
Second, there was probably no external electron donor to
maintain the PSĊ electron transfer after the removal of
Mn by NH2OH washing. Our additional work demon-
strated that superoxide radicals were generated probably
from the reaction of QBꢀ and O2 (to be published). The ter-
mination of temporary electron transport may greatly in-
fluence the superoxide formation. And last, the large
Oꢀ2x
mechanism of
in acceptor-side- and donor-side-
induced photoinhibition of PSĊ, the effect of illumination
time on EPR intensities of DEPMPO-OOH adducts was
studied with PSĊmembranes and Mn-depleted PSĊ
membranes (as shown in fig. 3). It can be seen clearly that
Oꢀ2x
amount of
formed rapidly after illumination of Mn-
depleted PSĊmembranes with high intensity can lead to
the fast oxygen exhaustion, which in turn results in the
appearance of the maximal EPR signal of DMPO-OOH.
In fact, we obtained the similar results when using 5-di-
ethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEP-
MPO) as spin trap. However, the life-time of DEPMPO-
OOH (~14 min) is longer than that of DMPO-OOH
(~1 min), so the maximal EPR signals of DEPMPO-OOH
appeared relatively late than that of DMPO-OOH (to be
published).
In conclusion, superoxide radicals are shown to be
involved in both acceptor-side- and donor-side-induced
Fig. 3. Effect of illumination time on EPR signal intensities of DEP-
MPO-OOH adducts in different PS Ċ membranes. 1, Control; 2,
NH2OH-treated. Other experimental conditions were the same as in fig.
1.
photoinhibition of PSĊ. Removal of Mn cluster from PS
Ċ membranes has a strong influence on the dynamics of
superoxide formation. These novel findings may promote
the maximal EPR signal intensities of DMPO-OOH ad-
ducts of Mn-depleted PSĊ membranes (for about 1-min
the studies of the structure and function of PSĊ and the
mechanism of photoinhibition.
illumination) appeared earlier than those of PSĊ mem-
branes (for about 5-min illumination).
Acknowledgements We are grateful to Yu Xinjian for his help in
preparation of biological samples. This work was supported by the Na-
tional Natural Science Foundation of China (Grant No. 39870161) and
the National Key Basic Research and Development Plan (Grant No.
G1998010100).
3
Discussion
Considering the above results, it is proposed that
the production of superoxide radicals could be initiated by
both acceptor-side- and donor-side-induced photoinhibi-
tion of PSĊ (fig. 2), which is in contrast to the results of
Hedig et al.[9,10]. They used DMPO as the spin trap to
study the radicals generated in photoinhibition of PSĊ,
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