Protective effect of active oxygen scavengers on protein
degradation and photochemical function in photosystem I
submembrane fractions during light stress
Subramanyam Rajagopal*, David Joly, Alain Gauthier, Marc Beauregard and Robert Carpentier
Groupe de Recherche en Biologie Ve
´
ge
´
tale, Universite
´
du Que
´
bec a
`
Trois-Rivie
`
res, Que
´
bec, Canada
Excessive light causes the inactivation of photosyn-
thesis. The detailed mechanism of PSII inactivation
has been extensively characterized both in vivo and
in vitro [1–5] and it has been suggested that the inac-
tivation develops at either acceptor or donor side of
the photosystem [2,6]. PSI was first believed to be
tolerant to strong light. However, several in vitro
studies suggested that PSI photochemical activity
could be inhibited under strong illumination [7,8]. In
these early experiments, photoinactivation of PSI was
not observed in the absence of oxygen. Later, Satoh
& Fork [9] demonstrated the inactivation of PSI in

res, C.P. 500, Trois-Rivie
`
res,
Que
´
bec, Canada, G9A 5H7
Fax: 1 819 376 5057
E-mail:
*Present address
School of Life Sciences and Center for the
Study of Early Events in Photosynthesis,
Arizona State University, Tempe, Arizona
85287, USA
(Received 17 August 2004, revised 23
November 2004, accepted 2 December
2004)
doi:10.1111/j.1742-4658.2004.04512.x
The protective role of reactive oxygen scavengers against photodamage
was studied in isolated photosystem (PS) I submembrane fractions illumin-
ated (2000 lEÆm
)2
Æs
)1
) for various periods at 4 °C. The photochemical
activity of the submembrane fractions measured as P700 photooxidation
was significantly protected in the presence of histidine or n-propyl gallate.
Chlorophyll photobleaching resulting in a decrease of absorbance and
fluorescence, and a blue-shift of both absorbance and fluorescence maxi-
mum in the red region, was also greatly delayed in the presence of these
scavengers. Western blot analysis revealed the light harvesting antenna

PSI core particles such as spinach PSI-180 and PSI-
100, as well as cyanobacterial PSI membranes exposed
to strong light [14]. In PSI core particles illuminated
with strong light, damage to the light-harvesting com-
plex (LHC) and degradation of reaction-center pro-
teins as well as acceptor side proteins were observed
[15]. We have recently shown that exposure of PSI
submembrane fractions to strong light under low tem-
perature altered the structure of chlorophyll (Chl)–pro-
tein (CP) complexes and decreased the photochemical
activity and the efficiency of excitation energy migra-
tion [16–18]. The damages were associated with the
formation of reactive oxygen species. It was also found
that the above photoinactivation of PSI was retarded
by glycinebetaine and sucrose [18].
In intact leaves, PSI was also found to be inacti-
vated by light. In Cucumis sativus leaves, weak light
induced the photoinactivation of PSI at chilling tem-
peratures, while practically no damage to PSII was
reported [19,20]. Sonoike et al. [20] demonstrated that
electron carriers located on the acceptor side of PSI
(A
1
, F
X
, F
A
, and F
B
) were damaged during the photo-

addition of glucose oxidase, glucose, and catalase to
scavenge dissolved oxygen suppressed the photodamage
of PSI submembrane fractions [17].
However, the protective role of specific active oxy-
gen scavengers against photo-induced changes in the
photosystems was discussed only in a limited number
of reports. LHCII protein degradation was analyzed
during strong light illumination of isolated LHCII or
BBY PSII subcomplexes [28]. Random cleavage, start-
ing in the NH
2
terminal region resulted in the
complete degradation of the antenna proteins. The
addition of scavengers such as histidine, DABCO, and
n-propyl gallate, retarded the above damages to the
antenna proteins indicating mainly
1
O
2
was involved
[28]. In spinach thylakoids, illumination at low light
intensity resulted in the degradation of PsaB gene
product into two fragments of 51 and 45 kDa [30].
These fragments were absent with added n-propyl gal-
late, which removes hydroxyl radicals [30]. There are
no reports regarding the protective role of oxygen
scavengers against reactive oxygen species under strong
light in PSI submembrane fractions.
We used PSI submembrane particles as model sys-
tem to obtain a better insight into the photo-induced

830
Exposure of isolated PSI submembrane fractions to
strong white light at 4 °C resulted in the loss of ability
of P700 to undergo reversible redox changes. Similar
S. Rajagopal et al. Photoprotective effect of active oxygen scavengers
FEBS Journal 272 (2005) 892–902 ª 2005 FEBS 893
results were obtained at room temperature (data not
shown). Figure 1 shows the magnitude of DA
830
signal
remained stable during the first 4 h of strong light
treatment of PSI submembrane fractions in the
absence of active oxygen scavengers. It indicated an
unchanged amount of photooxidizable P700 during
this period. However, further irradiation caused a
rapid decline in DA
830
. The addition of histidine and
n-propyl gallate to the submembrane fractions signifi-
cantly prevented the decline of absorbance changes
(DA
830
). The magnitude of DA
830
was almost constant
until 10 h of light exposure with the above reactive
oxygen scavengers. In contrast, in presence of cata-
lase or superoxide dismutase, or both, the amplitude
of DA
830

Photoprotective effect of active oxygen scavengers S. Rajagopal et al.
894 FEBS Journal 272 (2005) 892–902 ª 2005 FEBS
ation. This becomes particularly clear from the changes
in first derivative spectra that further demonstrate the
gradual time-dependent alteration in the position of the
major peak in the red region (Fig. 2, insert). On the
other hand, in the presence of the scavengers histidine
or n-propyl gallate, the absorption maximum at 680 nm
was less affected by the strong illumination and even
after a 10-h exposure this absorption was higher than in
control (6 h). Consequently, the blue shift of the maxi-
mum absorption peak at 680 nm was also less pro-
nounced (Fig. 2B,C). In the presence of histidine the
peak shift was about 5 nm after 6 h, while in n-propyl
gallate this peak shift was only 3 nm after 10 h.
Fluorescence emission changes
The 77 K fluorescence emission spectra and their first
derivatives measured in PSI submembrane fractions
illuminated for various times at 4 °C are shown in
Fig. 3. The intensity of the peak at 736 nm associated
to the PSI complex decreased by about 24% after
1 h of strong light illumination. It declined with
further light exposure, with only 30% of its initial
magnitude remaining after a 3-h illumination. Similar
to the absorbance spectra, a blue shift of the position
of the major fluorescence peak was observed. The
extent of that shift was larger in the fluorescence
emission spectra and reached 15–18 nm after 6 h
(Fig. 3A,C). The shift in fluorescence maximum was
evident from both absolute (Fig. 3A) and first deriv-

ured at the maximum in the presence or absence of scavengers.
Results are means ± SD. n ¼ 3. (C) Changes in the position of the
fluorescence maximum obtained from the first derivative of the
fluorescence spectra of PSI complexes. Control (s), histidine (h),
and n-propyl gallate (,). For further details see Experimental proce-
dures. These experiments were repeated three times and yielded
identical spectra; a typical spectrum is presented.
S. Rajagopal et al. Photoprotective effect of active oxygen scavengers
FEBS Journal 272 (2005) 892–902 ª 2005 FEBS 895
particles during strong illumination. The reaction-
center protein PsaA was stable until 4 h and then
decreased very slightly with further illumination. How-
ever, PsaB protein degradation started after a 1-h
exposure and a degradation product appeared, whereas
after a 5-h exposure this protein mostly disappeared
(Fig. 4). These reaction-center proteins were protected
in the presence of histidine and n-propyl gallate (data
not shown). The stromal ridge constituted of PsaC,
PsaD, and PsaE polypetides provides the docking site
for the soluble electron acceptors ferredoxin and flavo-
doxin [33,34]. Among these polypeptides, PsaC was
relatively sensitive to strong light and this protein star-
ted to degrade even after 1 h of illumination and fur-
ther illumination accelerated the degradation (Fig. 4,
Table 1). PsaD was less sensitive (Fig. 4) and after 6 h
of exposure this polypeptide degraded only by 30%
(Table 1). PsaE was the most sensitive, after a 3-h
exposure more than 60% of this protein content was
decreased (Table 1). In comparison, the integral mem-
brane protein PsaF was more stable (Fig. 4). Addition

this subunit was altered by 25 and by 50% after 6 h of
Table 1. Quantification of photosystem I polypeptides from immuno-
blots obtained from Fig. 5.
Control (%)
Histidine
(%)
n-Propyl
gallate (%)
0h 3h 6h 3h 6h 3h 6h
PsaC 100 68 0 105 109 55 45
PsaD 100 106 72 103 103 106 109
PsaE 100 36 0 73 55 95 86
Lhca1 100 71 0 91 77 97 106
Lhca2 100 45 0 54 38 63 58
Lhca3 100 62 9 83 37 93 77
Lhca4 100 74 52 83 65 91 76
Fig. 4. Quantitative immunoblot analysis of PSI submembrane frac-
tions illuminated with strong light at 4 °C for different time dur-
ation. All experimental conditions are given in Experimental
procedures.
Fig. 5. Quantitative immunoblot analysis of PSI submembrane frac-
tions illuminated with strong light at 4 °C for different time duration
in the presence of histidine or n-propyl gallate. All experimental
conditions are given in Experimental procedures.
Photoprotective effect of active oxygen scavengers S. Rajagopal et al.
896 FEBS Journal 272 (2005) 892–902 ª 2005 FEBS
illumination. Histidine and n-propyl gallate retarded
the photodegradation. The sensitivity of LCHI pro-
teins to strong illumination was Lhca2 > Lhca1 >
Lhca3 > Lhca4 (Fig. 4).

protective role of reactive oxygen scavengers against
strong light in PSI submembrane fractions. Our data
clearly demonstrated that some specific scavengers sig-
nificantly protected both the structure and function
of PSI.
Several authors postulated that oxidative mecha-
nisms are the basis of PSI photodamage in intact
leaves [19,20,22], isolated chloroplasts [7,8,11], or iso-
lated PSI fragments [15–18]. The selective action of
reactive oxygen scavengers used here indicated that the
species involved in the photodamage of proteins and
photochemical functions in the PSI submembrane frac-
tions were
1
O
2
, OH, and alkoxyl radicals. However,
H
2
O
2
and
•
O
2
–
were apparently not implicated in the
damaging processes. Addition of n-propyl gallate pro-
vided more protection than histidine (Figs 1–3). It is
known that n-propyl gallate protects against OH and

O
2
. It is well known that
•
OH and alkoxyl radi-
cals can be produced from reactions of
1
O
2
or
•
OH
with organic molecules [36,37].
Generation of singlet oxygen by Chl is expected dur-
ing strong illumination as it depends on the population
of excited Chls molecules and is formed by energy
transfer from Chl molecules in triplet state to oxygen
[27 and references therein, 38]. The involvement of
P700 triplet states appearing as a consequence of
charge recombination between P700
+
and reduced A
1
or A
0
[39] could not represent a significant source for
1
O
2
production in isolated submembrane fractions

noblot analysis showed that among the Lhca proteins,
Lhca2 is more sensitive and Lhca4 is stable to strong
light. The sequence of the changes observed in Lhca
proteins was: Lhc2 > Lhca1 > Lhca3 > Lhca4
(Fig. 4).
As in the PSI core antenna, excitonically coupled
dimers or trimers of Chl a or b in the Lhca were also
suggested to form a pool of red pigments of low-
energy [45–47], more specifically in the Lhca4 subunit
of the LHCI-730 complex [45,48,49]. Recent findings
confirmed that Lhca2 and Lhca3 are also having low-
energy red pigments [44] but this is more pronounced
in Lhca3 [35]. If the absorbed energy migrates towards
PSI holochromes with higher-absorption wavelengths
[27], then, the Chl molecules with an absorption max-
ima in the red located at a relatively long wavelength
in Lhca3 and Lhca4 should be bleached first. A blue
shift in absorption and fluorescence maximum in the
red was clearly observed in the PSI submembrane frac-
tions (Figs 2 and 3, see also [16–18]). Thus, the pig-
ment aggregates absorbing at these long-wavelengths
could be involved in photoprotection [16,17,27]. This
phenomenon is in agreement with our results showing
that Lhca4 and Lhca3, which have red pigments with
higher absorption wavelength, are more stable. More-
over, Lhca1 and Lhca2 have a higher content in bulk
Chl. Thus, the fast degradation of Lhca1 and Lhca2 is
due to generation of reactive oxygen species during
strong light illumination which leads to some alter-
ation in the Chl–protein interaction. Hui et al. [15]

These authors proposed that the degradation of PsaB
was mainly due to formation of superoxide and
hydroxyl radicals and this was protected by n-propyl
gallate. In another report using barley leaves illumin-
ated with weak light at low temperatures, damage to
both reaction-center proteins of PsaA and PsaB was
reported [23]. The involvement of reactive oxygen
Photoprotective effect of active oxygen scavengers S. Rajagopal et al.
898 FEBS Journal 272 (2005) 892–902 ª 2005 FEBS
species such as superoxide, hydrogen peroxide, and
hydroxyl radicals was suggested. They also showed
that
1
O
2
was involved in the damage of reaction-
center proteins. In vivo, the photooxidative damages
in PSI may occur with a similar pattern as in PSII
where reactive oxygen species are thought to initiate
protein degradation that is completed by chloroplast
proteases [21,30]. It was recently shown that the deg-
radation of PsaA ⁄ B proteins and alteration of photo-
chemical activity in spinach thylakoids are more
intensive at room temperature than at low tempera-
ture under strong light [13], which suggested that the
photoinactivation may involve an enzymatic contribu-
tion to the phenomenon.
As mentioned above, in PSI submembrane fractions,
PsaA was more resistant to strong light compare to
PsaB. PsaA was stable until 4 h of illumination and

Apart from PsaA and PsaB, other smaller polypep-
tides of PSI were degraded during strong light illumin-
ation. PsaC together with PsaD and E comprises the
stromal side of PSI. PsaC anchors the two Fe
4
S
4
clus-
ters F
A
and F
B
, which are needed to carry out the elec-
tron transfer from F
x
. The order of sensitivity to
strong light illumination was PsaE > PsaC > PsaD
(Fig. 5, Table 1). Similar results were observed in PSI
core particles [15]. This order closely corresponds with
the degradation profile observed during disassembly
studies using urea treatment [50]. The crystal structure
of plant and cyanobacterium PSI revealed that loops
at the stromal surface of PsaA ⁄ B partly contribute the
binding interface to the PsaC, D and E subunits
[51,52]. These observations are in agreement with our
data showing that the degradation of PsaC subunit
ensue almost simultaneously the degradation of Lhca2
and PsaB subunits (Figs 4 and 5). PsaD is stable even
after 5 h of illumination, whereas, PsaE is more
susceptible to strong light. The structural model of

Isolation of photosystem I submembrane
fractions
PSI submembrane particles were isolated from fresh spin-
ach leaves obtained from the local market, according to the
procedure of Peters et al. [31] with some modifications [32].
The isolated preparations with Chl content of 1–2 mg
ChlÆmL
)1
were suspended in a medium containing 20 mm
Tricine ⁄ KOH buffer (pH 7.8), 10 mm NaCl, 10 mm KCl,
and 5 mm MgCl
2
, and stored at )80 °C until its use. The
Chl a ⁄ b ratio was found to be higher than 6.0 in isolated
PSI submembrane fractions. Chl was determined in 80%
acetone according to Porra et al. [54].
Light treatment
The PSI preparations (500 lg ChlÆmL
)1
) were illuminated
for 5 h with continuous stirring at 4 °C using strong white
light (WL) (2000 lEÆm
)2
Æs
)1
) from a 150 W quartz ⁄ halogen
projector lamp. WL was passed through a 5-cm layer of
S. Rajagopal et al. Photoprotective effect of active oxygen scavengers
FEBS Journal 272 (2005) 892–902 ª 2005 FEBS 899
water containing CuSO

All measurements were carried out at an identical sensitiv-
ity of the PAM fluorometer. The absorbance changes at
830 nm represented only the oxidation and reduction of
P700 as no contribution to the absorbance changes due to
plastocyanin redox transformations can be detected with
the ED-P700DW unit. White actinic light was obtained
from the KL 1500 projector (Walz, Effeltrich, Germany).
The aliquots of 60 lL taken from the suspension of PSI
submembrane particles during strong light treatment were
added to 140 lL of suspension buffer containing 200 lm
ascorbate as an artificial donor. Chl concentration during
the measurements of DA
830
was 30 lgÆmL
)1
in the control
experiments (prior to the illumination).
Measurement of absorption spectra
The room temperature absorption spectra and their first
derivatives were recorded using a PerkinElmer Lambda 40
spectrophotometer (Wellesley, MA, USA). Ten microlitres
of the suspension of submembrane particles were repeatedly
taken during the illumination. Such aliquots taken from the
suspension of untreated particles contained 5 lg of Chl,
whereas the Chl content decreased gradually in the samples
taken during strong illumination.
Fluorescence spectroscopy
Low temperature (77 K) spectra of fluorescence emission
excited at 436 nm were measured as reported previously
[16] using a PerkinElmer LS55 spectrofluorometer. The Chl

To identify and quantify the PSI polypeptides, immuno-
blotting was carried out essentially as described by Towbin
et al. [57]. Western blotting was performed by electropho-
retic transfer of proteins to nitrocellulose membranes
(0.45 lm, Millipore, Billerica, MA, USA). The membrane
was incubated with polyclonal antibodies raised in rabbits
against PSI complex. Subsequently, secondary antibodies
ligated to alkaline phosphate were applied. Bromo-chloro-
indolyl-phosphate and tetrazolium blue were used for the
coloring reaction. The developed membranes were analyzed
by using Bio-Rad Gel-Doc 2000 system (Hercules, CA,
USA).
Acknowledgements
The authors wish to thank Drs J.H. Golbeck, K.
Sonoike and P. Chitnis for antibodies and J. Harnois
for helpful professional assistance. This research was
supported by the Natural Sciences and Engineering
Research Council of Canada and by Fonds Que
´
be
´
cois
de la Recherche sur la Nature et les Technologies.
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