Journal of Photochemistry and Photobiology B: Biology 78 (2005) 115–123

www.elsevier.com/locate/jphotobiol

Role of LHCII organization in the interaction of substituted1,4-anthraquinones with thylakoid membranes

Emilia Apostolova a,*, Sashka Krumova a, Tzvetelina Markova a, Tzvetanka Filipova b,Maria T. Molina c, Ivana Petkanchin d, Stefka G. Taneva a

a Institute of Biophysics, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., B1.21, Sofia 1113, Bulgariab Department of Organic Chemistry, University of Chemical Technology and Metallurgy, Blv. Kl. Ohridski 8, Sofia, Bulgaria

c Insituto de Quımica Medica, C.S.I.C., Calle Juan de la Cierva 3, 28006 Madrid, Spaind Institute of Physical Chemistry, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria

Received 18 June 2004; received in revised form 4 October 2004; accepted 6 October 2004

Available online 2 December 2004

Abstract

The chlorophyll fluorescence, photochemical activity and surface electric properties of thylakoid membranes with different stoi-

chiometry of pigment–protein complexes and organization of the light-harvesting chlorophyll a/b protein complex of photosystem II

(LHCII) were studied in the presence of substituted 1,4-anthraquinones. Data show strong dependence of the quenching of the chlo-

rophyll fluorescence on the structural organization of LHCII. The increase of the LHCII oligomerization, which is associated with

significant reduction of the transmembrane electric charge asymmetry and electric polarizability of the membrane, correlates with

enhanced quenching effect of substituted 1,4-athraquinones. Crucial for the large quinone-induced changes in the membrane electric

dipole moments is the structure of the quinone molecule. The strongest reduction in the values of the dipole moments is observed

after interaction of thylakoids with 3-chloro-9-hydroxy-1,4-anthraquinone (TF33) which has the highest quenching efficiency. The

quinone induced changes in the photochemical activity of photosystem II (PSII) correlate with the total amount of the supramo-

lecular LHCII–PSII complex and depend on the number of substituents in the 1,4-anthraquinone molecule.

� 2004 Elsevier B.V. All rights reserved.

Keywords: Chlorophyll fluorescence; Thylakoid membranes; Light-harvesting chlorophyll a/b protein complex; 1,4-Anthraquinones; Photochemical

activity; Surface electric properties

1. Introduction

The light reactions of photosynthesis are driven by

the excitation energy absorbed by the antenna pigments

and transferred to the reaction centers. High degree oforganization of the pigment–protein complexes is re-

quired for efficient utilization of the solar energy by pho-

tosynthetic organisms. Quinones play an important role

as electron and proton carriers in the electron-transport

1011-1344/$ - see front matter � 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.jphotobiol.2004.10.002

* Corresponding author. Tel.: +359 2 979 26 21; fax: +359 2 971 24

93.

E-mail address: emya@obzor.bio21.bas.bg (E. Apostolova).

system [1,2]. Exogenous quinones are able to compete

with plastoquinone for electrons and quench the fluores-

cence from antenna chlorophyll in chloroplasts, algae

and green bacteria [3–11]. They can be partially or com-

pletely reduced under illumination with loss of theirability to act as quenchers [4]. As an analog of the pho-

tosystem II (PSII) plastoquinone electron acceptor, QB,

substituted quinones compete with QB for a common

binding domain and thereby inhibit the QB function

[12].

In our previous study we found that the quenching of

the chlorophyll fluorescence by exogenous 1,4-anthra-

quinones (1,4-AQ) in thylakoid membranes is due to

116 E. Apostolova et al. / Journal of Photochemistry and Photobiology B: Biology 78 (2005) 115–123

interaction of quinones with chlorophyll molecules and

that the quenching efficiency of halogen-substituted

1,4-anthraquinones strongly depends on the position

of the Cl atom [13]. The present investigation is focussed

on the contribution of light-harvesting chlorophyll a/b

protein complex of PSII (LHCII) in the interaction ofthylakoid membranes with 1,4-AQ. The quenching effect

of 1,4-AQ was determined in thylakoid membranes with

different stoichiometry of the chlorophyll–protein com-

plex and structural organization of LHCII. Combining

the fluorescence with the electro-optic data demon-

strates a relationship between the quinone-induced

changes in the chlorophyll fluorescence, the electric

properties and the LHCII amount and its oligomeriza-tion state.

2. Materials and methods

2.1. Substituted 1,4-anthraquinones

In the present study were used 1,4-anthraquinone(QA6) and substituted 1,4-anthraquinones: 9-acetoxy-

1,4-anthraquinone (QA4); 9-hydroxy-1,4-anthraquinone

(QA5); 9-methoxy-1,4-anthraquinone (QA7); 10-chloro-

9-hydroxy-1,4-anthraquinone (TF11); 9-acetoxy-3-bro-

mo-1,4-anthraquinone (TF23); 9-acetoxy-3-chloro-1,4-

anthraquinone (TF29); 3-chloro-9-hydroxy-1,4-anthra-

quinone (TF33); 3-bromo-9-hydroxy-1,4-anthraquinone

(TF34). The substituted 1,4-anthraquinones were syn-thesized as in [13]. The type of the substituent groups,

R3, R9 and R10, for the different substituted 1,4-anthra-

quinones (1,4-AQ) is given in Table 2.

2.2. Thylakoid membranes

Thylakoid membranes were isolated from pea (Pisum

sativum L. cv. Borec and its mutants Coeruleovireus 2/16and Costata 2/133 and P. sativum L. cv. Auralia and its

mutant Chlorotica XV/1422) and barley (Hordeum vulg-

are L. and the chlorophyll b-deficient barley mutant

Chlorina f2) as described in [14] and [15], respectively.

The total chlorophyll concentration was determined by

the method of Lichtentaler [16].

2.3. Chlorophyll fluorescence

Chlorophyll fluorescence at room temperature in the

presence of 1,4-anthraquinone and substituted 1,4-

anthraquinones was measured with Jobin–Yvon JY3

spectrofluorimeter as in [13]. The quinone concentration

was 200 lM. The chlorophyll fluorescence was excited at

480 nm. The thylakoid membranes were suspended in 50

mM HEPES–NaOH (pH 7.5), 0.1 M sucrose, 5 mMNaCl and 20 lM DCMU. The chlorophyll concentra-

tion was 10 lg chl/ml. Before the measurements, thyla-

koid membranes were incubated with 1,4-AQ for 15

min.

The Stern–Volmer quenching constant (KSV) was

used as a characteristic of the quenching effect of the

quinones on the chlorophyll fluorescence [7]:

F m=F 0m � 1 ¼ Ksv½CQ�, ð1Þ

where Fm and F 0m are the fluorescence intensities in ab-

sence and presence of quinone, respectively; CQ is the

quinone concentration.

2.4. Photochemical activity of PS II

The photochemical activity of PSII was measured

polarographically with a Clark-type electrode at 20 �C,with 1,4-benzoquinone as electron acceptor as in [13].

The concentration of 1,4-anthraquinones was 200 lM.

2.5. Electric light scattering

The electric polarizability (ci) and the permanent di-

pole moment (p^) were estimated from the dependence

of the electro-optic effect on the frequency (m) of the ap-plied a.c. field for ellipsoid particles [17]. The electro-op-

tic effect a = (IE�I0)/I0 was defined as a relative change

in the intensity of the scattered light. IE and I0 are the

intensities of the light scattered by the membrane sus-pension when an external electric field of strength E is

applied and without field, respectively. At a low degree

of orientation (i.e., U/kT�1, U is the energy of orienta-

tion, kT is the thermal energy) is expressed by the

equation:

a ¼ AðKl,KdÞI0ðKl,KdÞ

ðb2 þ cÞE2, ð2Þ

where A(Kl,Kd) and I0(Kl,Kd) are optical functions

dependent on the dimensions of the particles and the

geometry of the experimental setup; K = (2p/k 0)sin2(h/2); k 0 is the wavelength of the light in the suspension,

h is the angle of observation (90� in this case); l and d

are the long and short axis of the ellipsoid particles.

b = p^/kT and c = (ci�c^)/kT, where p^ is the permanent

dipole moment arising from the asymmetric charge dis-tribution on the membrane surfaces, i.e., along the par-

ticle symmetry axis; ci and c^ are the electric

polarizabilities along and perpendicular to the mem-

brane surface, respectively; k is the Boltzmann constant

and T is the absolute temperature.

The dimensions of the membrane particles were esti-

mated from the relaxation time of particle disorientation

(at high degree of orientation) after switching off theelectric field as described in [18]. The surface area, S,

of the thylakoid membranes was calculated assuming

that they are prolate ellipsoids with axial ratio 2 [18].

The measuring set-up is described in detail elsewhere

[19]. Electric field pulses were applied to the suspension

E. Apostolova et al. / Journal of Photochemistry and Photobiology B: Biology 78 (2005) 115–123 117

in an electro-optical cell (optical horn) with horizontal

platinum electrodes at a fixed distance of 2.6 mm. Elec-

tric pulse generator (Waveteck 185) and power-ampli-

filer (Model PA25, Optimatium, JRL, New York) were

used for low and high intensity electric field pulses,

respectively. The light source was a halogen lamp. Thetransient signal was detected by a photomultiplier

(FQS 35, Zeiss, Yena), the output signal recorded on a

digital storage oscilloscope (Philips PM 3335) and ana-

lyzed with the aid of a personal computer.

The electro-optic measurements were carried out in a

medium containing 0.25 mM sorbitol (pH 5.3–5.8 and

conductivity 4.56 lS/cm). The chlorophyll concentration

was 5 lg chl/ml.The data of the electric dipole moments are mean val-

ues from 4 to 5 independent measurements.

3. Results

3.1. Characteristics of the thylakoid membranes

In this work thylakoid membranes from pea wild

types Borec and Auralia, two mutants of the variety

Borec (Coeruleovireus 2/16 and Costata /133), one mu-

tant of the variety Auralia (Chlorotica XV/1422), barley

wild type and the chlb-deficient mutant of barley Chlo-

rina f2 were studied. The composition and electric prop-

erties of thylakoid membranes from these higher plants

were characterized in our previous investigation [18,20].It was determined that the total amount of both the

LHCII and the supramolecular complex of LHCII–PSII

in thylakoid membranes from Auralia wt and its mutant

Chlorotica XV/1422 is smaller than those from Borec wt

and its mutants Costata 2/133 and Coeruleovireus 2/16

(Table 1; [18,20]). Besides, the organization of LHCII

(i.e., the ratio of oligomeric to monomeric form of

LHCII (LHCIIo/LHCIIm)) varies with the plant type(Table 1; [24,25]). It was also shown that the increase

of the ratio LHCIIo/LHCIIm strictly correlates with

the decrease in the values of membrane electric dipole

moments (electric polarizability (ci) and permanent di-

Table 1

Relative amount of the chlorophyll-protein complexes of pea thylakoid memb

XV/1422; Costata 2/133 and Coeruleovireus 2/16) determined by nondenatura

form; LHCIIm – monomeric form) and PSII (protein containing P680 and a

Plant type LHCII

Chloroticaa XV/1422 33.90 ± 0.29

Auraliaa wt 34.12 ± 0.49

Costatab 2/133 47.03 ± 0.39

Borecb wt 45.86 ± 0.79

Coeruleovireusb 2/16 45.25 ± 1.86

The data are taken from our previous papers [18,20].a Data from [20].b Data from [18].

pole moment (p^)) (data for the electric moments with-

out 1,4-AQ are given in Table 4 and [18,20]).

Chlorina f2 barley mutant is characterized by strongly

reduced pigment content, reduced antenna size of the

PSII and lack of the oligomeric structure of LHCII as

compared to the wild type [15,21].

3.2. Chlorophyll fluorescence of thylakoid membranes

from different plant species in presence of

1,4-anthraqinones

The interaction of 1,4-AQ with thylakoid membranes

from the different plant species results in a strong de-

crease in the intensity of the chlorophyll emission at686 nm without change in the position of the emission

maximum. Stern–Volmer quenching constants (Ksv) for

the 686 nm emission band are shown on Table 2. The

Ksv values of the 1,4-AQ depend on the organization

of thylakoid membranes. KSV for thylakoid membranes

from Auralia wt and its mutant (Chlorotica XV/1422)

are smaller than those from Borec wt and its mutants

(Costata 2/133 and Coeruleovireus 2/16) (Table 2). Theresults clearly show that the quenching effect of 1,4-

AQ is bigger in the membranes containing higher

amount of LHCII (Tables 1 and 2). Similar tendency

is observed for the quenching efficiency of 1,4-AQ in

thylakoids from barley and Chlorina f2 mutant. The

constants (Ksv) of 1,4-AQ for the mutant thylakoids

are smaller than those for barley wild type (Fig. 1).

The increase of the quenching constant with the in-crease of the amount of LHCII is more pronounced

for mono-substituted 1,4-anthraquinone and 3-halo-

gen-9-acetoxy-1,4-anthraquinones than for halogen

substituted 9-hydroxy-1,4-anthraquinones (Table 2).

Comparison of the chlore substituted 9-hydroxy-1,4-

anthraquinones (TF33 and TF11) shows that the posi-

tion of the chlore atom strongly affects the quenching

efficiency of the quinone in all studied thylakoid mem-branes. Independent of the plant type the biggest

quenching effect is found in the presence of 3-chloro-9-

hydroxy-1,4-anthraquinone (TF33). The same is true

for thylakoids from barley wild type and its mutant

ranes from wild types (Auralia and Borec) and the mutants(Chlorotica

ting SDS–PAGE: LHCII (LHCII – total content; LHCIIo – oligomeric

ntenna complex) thylakoid membranes

LHCII + PSII LHCPIIo/LHCPIIm

51.10 ± 0.56 2.45

47.24 ± 0.69 2.82

59.73 ± 0.42 3.34

59.46 ± 0.86 4.57

59.35 ± 1.54 6.62

Table 2

Stern–Volmer quenching constants (KSV) for 686 nm room-temperature chlorophyll fluorescence of thylakoid membranes from Auralia wt, Borec wt

and the mutants Chlorotica XV/1422, Costata 2/133 and Coeruleovireus 2/16 incubated with 1,4-anthraquinone and substituted 1,4-anthraquinones

1,4-AQ Substituent group Chlorotica XV/1422

KSV [M�1]

Auralia wt

KSV [M�1]

Costata 2/133

KSV [M�1]

Borec wt

KSV [M�1]

Coerul. 2/16

KSV [M�1]R3 R9 R10

TF23 Br OCOCH3 H 5.30 · 103 5.15 · 103 6.50 · 103 11.60 · 103 15.90 · 103

TF11 H OH Cl 10.70 · 103 13.90. · 103 15.40 · 103 14.10 · 103 18.00 · 103

TF29 Cl OCOCH3 H 21.70 · 103 23.50 · 103 34.23 · 103 34.20 · 103 32.50 · 103

TF34 Br OH H 42.90 · 103 44.20 · 103 49.70 · 103 48.90 · 103 64.30 · 103

TF33 Cl OH H 121.10 · 103 127.10 · 103 149.37 · 103 180.44 · 103 152.7 · 103

QA7 H OCH3 H 4.40 · 103 5.10 · 103 4.60 · 103 7.14 · 103 8.50 · 103

QA6 H H H 11.40 · 103 10.6 · 103 13.20 · 103 17.00 · 103 25.20 · 103

QA5 H OH H 13.30 · 103 17.90 · 103 24.50 · 103 28.30 · 103 29.60 · 103

QA4 H OCOCH3 H 30.90 · 103 36.70 · 103 46.30 · 103 49.30 · 103 48.40 · 103

R3, R9, R10 are the substituent groups. The data are mean values from 5 to 7 independent experiments. The standard errors are from ±0.03 · 103 to

±0.09 · 103.

2

4

6

8

10

1,4-anthraquinones

TF33

QA4

QA5QA6TF29

KSV

x10-3

[M-1]

Fig. 1. Stern–Volmer quenching constants (KSV) for 1,4-anthraqui-

none, QA6, and the substituted 1,4-anthraquinones, QA4, QA5, TF29

and TF33, in thylakoid membranes from barley wt (�) and Chlorina f2

mutant (�). Fluorescence emission bands: for barley wt at 685 nm and

for Chlorina f2 mutant at 695 nm, respectively. The concentration of

1,4-AQ in the reaction medium is 200 lM. The parameter Fm/F0m

increases linearly with increasing the quinone concentration to 250 lM.

118 E. Apostolova et al. / Journal of Photochemistry and Photobiology B: Biology 78 (2005) 115–123

Chlorina f2 (Fig. 1). These results concerning the

quenching efficiency of 1,4-AQ in thylakoid membranes

with different organization from a number of plant types

studied in this work confirm our previous data for an-

other variety of pea [13].

Besides the amount of the LHCII, its organizationalso affects the degree of quenching. Ksv has bigger val-

ues for membranes with higher degree of LHCII oligo-

merization (Table 2). The smallest Ksv values for all

1,4-AQ, with the exception of 1,4-anthraquinone

(QA6) and of 3-bromo-9-acetoxy-1,4-anthraquinone

(TF23), are observed for thylakoid membranes from

the mutant Chlorotica XV/1422, which is characterized

by the lowest oligomer to monomer LHCII ratio. Thesmallest Ksv values for QA6 and TF23 are found for

Auralia wt, which has a slightly bigger LHCIIo/LHCIImratio than that of Chlorotica XV/1422. Therefore, the

higher ratio of oligomeric to monomeric LHCII

(LHCIIo/LHCIIm) is related to bigger quenching effi-

ciency of the quinones (Table 2). The observation that

Ksv has significantly higher value for barley wild type

than for Chlorina f2 thylakoid membranes (Fig. 1) sup-

ports this finding.

3.3. Photochemical activity of thylakoid membranes in the

presence of 1,4-anthraquinones

1,4-Anthraquinones affect significantly the photo-

chemical activity of the thylakoid membranes as well

(Table 3).

In the presence of all mono-substituted 1,4-AQ (QA4,

QA5, QA6, QA7) the photochemical activity (A/A0) of

thylakoid membranes from Borec wild type and its mu-tants (Costata 2/133 and Coeruleovireus 2/16) is bigger

than that of thylakoids from Auralia wild type and

Chlorotica XV/1422 (Table 3). The opposite effect is ob-

served for the di-substituted 1,4-AQ (TF11, TF23,

TF29, TF33, TF34) (Table 3). The strongest inhibition

of the PSII activity was observed in the presence of

chlore substituted 1,4-anthraquinones. Therefore, the

degree of inhibition of the PSII activity by mono- anddi-substituted 1,4-anthraquinones depends on the

amount of the supramolecular complex LHCII–PSII,

which is smaller in thylakoids from Auralia wt and its

mutant than that from Borec wt and its mutants (Table

1). Differences in the structural organization of this

complex might be responsible for the different extent

of contacts between PSII and mono- and di-substituted

1,4-AQ.

3.4. Surface electric properties of thylakoid membranes in

the presence of 1,4-anthraquinones

As mentioned above, there is a correlation between

the LHCIIo/LHCIIm ratio (Table 1) and the values of

the permanent and induced dipole moments (Table 4).

Table 3

Photochemical activities of PS II (A/A0) of pea thylakoid membranes with different organization in presence of 200 lM of 1,4-anthraquionone and

substituted 1,4-anthraquinones

1,4-AQ Substituent group Chlorotica XV/1422 Auralia wt Costata 2/133 Borec wt Coerul. 2/16

R3 R9 R10

TF11 H OH Cl 0.496 0.486 0.453 0.382 0.404

TF33 Cl OH H 0.502 0.493 0.425 0.467 0.451

TF29 Cl OCOCH3 H 0.539 0.569 0.461 0.474 0.485

TF23 Br OCOCH3 H 0.583 0.585 0.598 0.512 0.535

TF34 Br OCOCH3 H 0.605 0.599 0.590 0.545 0.526

QA4 H OCOCH3 H 0.610 0.591 0.686 0.736 0.608

QA5 H OH H 0.657 0.669 0.694 0.848 0.813

QA6 H H H 0.795 0.826 0.833 0.879 0.843

QA7 H OCH3 H 0.835 0.840 0.898 0.896 0.897

A and A0 are the photochemical activities with and without 1,4-AQ, respectively. The data are mean values from 5 to 7 independent experiments. The

standard errors are from ±0.034 to ±0.069.

Table 4

Values of the permanent dipole moment (p^) and the electric polarizability per unit surface area (ci/S) of thylakoid membranes from pea wild types

(Borec and Auralia) and the mutants (Chlorotica XV/1422, Costata 2/133 and Coeruleovireus 2/16) in presence of three selected substituted 1,4-

anthraquinones

1,4-AQ Chlorotica XV/1422 Auralia wt Costata 2/133 Borec wt Coeruleovireus 2/16

p^ · 1024 [Cm]

Without 0.613 0.582 0.446 0.350 0.270

QA5 0.662 0.856 0.691 0.525 0.651

TF11 1.018 0.656 0.571 0.616 0.327

TF33 0.340 0.395 0.268 0.153 0.205

ci/S · 1016 [F]

Without 1.287 0.853 0.730 0.380 0.280

QA5 1.596 2.039 1.905 0.749 0.549

TF11 3.501 0.876 0.947 0.669 0.449

TF33 0.580 0.620 0.504 0.315 0.173

The standard errors are: for p^ – from ±0.031 · 10�24 to ±0.058 · 10�24 and for ci/S are from ±0.072 · 10�16 to ±0.11 · 10�16.

E. Apostolova et al. / Journal of Photochemistry and Photobiology B: Biology 78 (2005) 115–123 119

A detailed analysis of the orientational behaviour of

thylakoid membranes in an external electric field before

and after interaction with 1,4-AQ shows strong varia-

tion of p^ and ci (data are shown for the di-substituted1,4-anthraquinones TF11, TF33 and the mono-substi-

tuted QA5; Fig. 2).

The orientational behaviour of thylakoid membranes

in an external a.c. electric field is determined by their

permanent and induced electric dipole moments (details

on the electro-optic phenomena can be found in Ref.

[17]). The dependence of the electro-optic effect a on

the frequency of the applied a.c. electric pulses, a/lgm,gives information about both electric moments (Fig.

2(a)), as well as on the dynamics of the charges respon-

sible for the creation of the induced dipole moment. At

low frequencies (below the plateau of the frequency

dependence) the effect a decreases with the frequency de-

crease (Fig. 2(a)), which is usually related to the pres-

ence of a permanent dipole moment along the minor

axis of the particles and to orientation dominated bythe permanent dipole [22]. The vertical line shown in

Fig. 2 (a) denotes the frequency at which the a/E2 depen-

dences (Fig. 2(b)) are recorded and that in Fig. 2(b) the

electric field strength at which the frequency depen-

dences of a are obtained (Fig. 2(a)), E being in the linear

part of a/E2 dependence, where a scales linearly with E2

and U/kT�1.The electro-optic effect a of the thylakoid membranes

from Borec wt in presence of the mono-substituted 9-hy-

droxy-1,4-anthraquinone (QA5) is bigger than that

without quinone in the entire frequency range (Fig.

2(a)). On the contrary the magnitude of the effect a in

presence of di-substituted TF33 is reduced (Fig. 2(a)).

The difference in the magnitude of the effect a in the

kHz range, where the orientation of the thylakoid mem-branes is determined by the motion of charge(s) in the

diffuse electric double layer (i.e., by the electric polariz-

ability ci), corresponds to the differences in the initial

slopes of the a/E2 dependence (Fig. 2(b)) between the

thylakoid membranes without and with 1,4-AQ (results

shown for QA5, TF11, TF33; Fig. 2(b)). Therefore, the

interaction of 1,4-AQ with thylakoid membranes leads

to changes in the values of both dipole moments (Table4). This reflects the variation of the transmembrane

charge asymmetry and the ion composition of the dif-

fuse electric double layer related to the interaction of

0 1 2 3 4 5

νc

νc*

α %

α %

lg ν (Hz)

0.2 0.4 0.6 0.8 1.00

6

12

18

24

30

36

6

12

18

24

30

36

E2 x 10-8 (V/m)2

(a)

(b)

Fig. 2. (a) Frequency dependence of the electro-optic effect a in an

external electric field of strength E = 5.8 · 103 V/m for thylakoid

membranes from Borec wt without (�) and in presence of the

substituted 1,4-anthraquinones: QA5 (h), TF11 (n) and TF33 (.). (b)

Dependence of the electro-optic effect a on the square of the a.c.

electric field strength (frequency = 1 kHz), all other details as in (a).

The dotted vertical lines are drawn at: (a) 1 kHz – the frequency at

which the a/E2 dependences are recorded; (b) the field strength E at

which the frequency dependences of a are obtained, E being in the

linear part of a/E2 dependence, where a scales linearly with E2 and U/

kT� 1. The arrows show the critical relaxation frequency for

thylakoid membranes in the presence of QA5 (mc*) and TF33 (mc),respectively.

120 E. Apostolova et al. / Journal of Photochemistry and Photobiology B: Biology 78 (2005) 115–123

thylakoids with 1,4-AQ (Table 4). The high frequency

part of the a/lgm dependence is connected with the

dynamics of the charges responsible for the creation of

the electric polarizability. The shift of the critical relax-ation frequency (mc) for the thylakoid membranes of

Borec wt toward lower frequency in the presence of

QA5 and TF11, compared with TF 33 and untreated

membranes, indicates a reduced charge mobility. The

relaxation frequency for both QA5 (mc*) and TF33 (mc)are shown with arrows (Fig. 2(b)). The changes of the

charge dynamics can be related to the altered conforma-

tion upon interaction of 1,4-AQ with the membranesurface.

The values of the electric moments, p^ and ci, of thy-lakoid membranes from Auralia, Borec and their

mutants, increase in the presence of 9-hydroxy-1,4-

anthraquinone (Table 4). The same tendency is observed

upon additional substitution with Cl at position 10 in

the molecule of 9-hydroxy-1,4-anthraquinone (TF11),

while additional substitution with Cl at position 3

(TF33) results in opposite changes of the electric dipole

moments (Table 4). This indicates that the position of

the Cl atom as a substituent in the molecule of 9-hydro-xy-1,4-anthraquinone (position 10 or 3), which plays a

significant role in the quinone interaction with thylakoid

membranes, is important for the strong changes in the

membrane surface electric properties and charge dynam-

ics. The changes of the membrane surface electric state

could be attributed to alteration of the exposure of dif-

ferent charged groups of the polypeptides on the mem-

brane surface as a consequence of conformationalchanges induced by the interaction of the membranes

with 1,4-anthraquinones (Table 4).

The experimental fact concerning the role of the posi-

tion of substituent Cl atom in the quinone molecule on

the surface electric properties would explain the ob-

served difference in the quenching efficiencies of the

1,4-AQ associated with the position of the Cl-atom.

4. Discussion

In our previous study we found a stronger quinone-

induced quenching of the chlorophyll fluorescence than

quinone-induced changes in the activity of the photosys-

tem II after interaction of thylakoid membranes with

substituted 1,4-anthraquinones [13]. It was shown thatthis strong quenching effect is due rather to the interac-

tion of 1,4-AQ and chlorophyll molecules than to an

indirect effect caused by stimulation of the photochemis-

itry [13]. The present work demonstrates that the inter-

action of 1,4-AQ with thylakoid membranes results in

strong changes of the surface electric properties of the

membranes (Table 4). The variation of the transmem-

brane charge asymmetry (p^) and the structure of theelectric double layer (ci) might be associated with

changes in the conformation of the membrane proteins

and/or in the exposure of the polypeptides on the mem-

brane surface after interaction with 1,4-AQ. We have

also shown earlier that the electric properties of the

membranes correlate with the structural organization

of the LHCII [18,20]. The structural organization of

the LHCII, which is the main photosynthetic antennacomplex in thylakoid membranes of higher plants

[23,24], strongly affects its optical and functional proper-

ties [25].

The main results of the present experimental finding

are summarized in Table 5. It is demonstrated that the

increase of the oligomerization of LHCII, which medi-

ates the reduction of the electric moments, p^ and ci,correlates with increase of the quenching efficiency.The biggest quenching effect of all anthraquinones

was observed for Coeruleovireus 2/16 which has the

Table 5

Summary of the effects of 1,4-AQ on the quenching of chlorophyll fluorescence, PSII activity and electric properties (p^ and ci/S) of thylakoidmembranes with different stoichiometry of chlorophyll-protein complexes

Characteristics of thylakoid membranes Parameters

Quenching of

chlorophyll fluorescence

Inhibition of PSII activity p^ and ci/Sa

High amount of LHCII Strong effect No dependence No dependence

Small amount of LHCII Smaller effect No dependence No dependence

High ratio of LHCIIo/LHCIIm Strong effect No dependence No dependence

Small ratio of LHCIIo/LHCIIm Smaller effect No dependence No dependence

High amount of LHCII–PSII No dependence Higher effect for mono-substituted than di-substituted No dependence

Small amount of LHCII–PSII No dependence Smaller effect for mono-substituted than di-substituted No dependence

a The changes of p^ and ci/S depend on the chemical structure of 1,4-AQ.

Fig. 3. Sketch illustrating the correlation between the changes in the

thylakoid membrane electric dipole moments (permanent dipole

moment (p^) and electric polarizability per unit surface area (ci/S))and the extent of fluorescence quenching induced by the interaction of

membranes with 1,4-AQ. A simplified model of thylakoid membrane is

given (only part of the granal thylakoid is drawn, where the PSII and

LHCII complexes are located predominantly). The white and grey

ellipsoids represent the LHCII-oligomers and LHCII-trimers, respec-

tively; the black ellipsoids refer to the PSII-complexes. Other proteins,

pigment–protein complexes and lipid molecules are omitted for

simplicity and clearer representation of the model. The charge

distribution on the membrane surfaces should not be taken as real, it

is used to visualize the changes in charge distribution as a consequence

of the interaction of thylakoid membrane with 1,4-AQ. The changes in

the magnitude of the dipole moments are represented by longer and

shorter arrows, respectively. The scheme emphasizes that the 1,4-AQ-

induced increase and decrease in the dipole moments correlate (i) with

small and high accessibility of the chlorophyll molecules to 1,4-AQ,

and consequently (ii) with high and low fluorescence quenching.

E. Apostolova et al. / Journal of Photochemistry and Photobiology B: Biology 78 (2005) 115–123 121

lowestvalues of both the transmembrane charge asym-

metry and the electric polarizability among all the

variety of pea studied [18,20] (without 1,4-AQ) (Table

4). Therefore, the organization of the LHCII and the

electric properties of the membrane are important fac-

tors determining the accessibility of the chlorophyll

molecules to the substituted 1,4-anthraquinones. Dif-

ferences in the packing of the chlorophyll moleculesand in the exposure of polypeptide on the membrane

surface, related to variations in the LHCII organiza-

tion and the electric moments, respectively, might be

responsible for the different degree of the chlorophyll

quenching by substituted 1,4-AQ in thylakoid mem-

branes from different plant species. The relationship

between the variations of the electric dipole moments

and fluorescence quenching is illustrated in Fig. 3,where we have sketched the thylakoid membranes

(an oversimplified model) with the permanent dipole

moment perpendicular to the membrane plane and

the longitudinal electric polarizability. The arrows

show the magnitude of the dipole moments in presence

and without 1,4-AQ.

3-Chloro-9-hydroxy-1,4-anthraquinone (TF33) has

the highest quenching affinity among all substituted1,4-athraquinones and leads to the strongest reduction

of the membrane electric dipole moments (Table 4) for

all samples studied (wild types and mutants). It is to

be noted that in presence of TF33 the smallest values

of p^ and ci are observed for thylakoids from the mutant

Coeruleovireus 2/16 having the biggest quenching con-

stant Ksv (as already mentioned). The good correlation

between the quenching efficiency of 1,4-AQ and theorganization of LHCII, respectively, with the electric

parameters led to the proposal that the interaction and

probable penetration of the anthraquinones within the

membrane bilayer might be controlled by the surface

electric properties of the membrane (Fig. 3). The elec-

tro-optic data support the hypothesis of Karukstis and

Monell [26] that addition and/or removal of quinones

from their interaction site alter the conformation of thatregion. The conformational changes evidently depend

on the structure of the quinone, i.e., on the substituent

and its position in the molecule of 1,4-anthraquinone

(Table 4).

The degree of inhibition of PSII depends on the total

amount of the supramolecular complex LHCII–PSII aswell as on the number of substituting groups in the 1,4-

AQ. The latter reflects different sites of interaction of

mono- and di-substituted 1,4-AQ.

In conclusion, the quenching of the chlorophyll

fluorescence induced by 1,4-AQ strongly depends on

the organization of the thylakoid membranes, particu-

larly on the organization of LHCII, related to the

122 E. Apostolova et al. / Journal of Photochemistry and Photobiology B: Biology 78 (2005) 115–123

membrane surface electric properties. This is proved

for thylakoid membranes, characterized with different

stoichiometry of pigment–protein complexes and orga-

nization of the light-harvesting chlorophyll a/b protein

complex of photosystem II, from pea, barley and their

mutants.

5. Abbreviations

1,4-AQ 1,4-anthraquinones

QA4 9-acetoxy-1,4-anthraquinone

QA5 9-hydroxy-1,4-anthraquinone

QA6 1,4-anthraquinoneQA7 9-methoxy-1,4-anthraquinone

TF11 10-chloro-9-hydroxy-1,4-anthraquinone

TF23 9-acetoxy-3-bromo-1,4-anthraquinone

TF33 3-chloro-9-hydroxy-1,4-anthraquinone

TF34 3-bromo-9-hydroxy-1,4-anthraquinone

DCMU 3-(3-4-dichlorophenyl)-1,1-dimethylurea

KSV Stern–Volmer quenching constant

LHCII light-harvesting chlorophyll a/b protein com-plex of photosystem II

LHCIIo LHCII oligomer

LHCIIm LHCII monomer

PSII photosystem II

ci electric polarizability

p^ permanent dipole moment

mc critical relaxation frequency

Acknowledgements

The work was supported by contract K-1301/03 with

Bulgarian National Fund, Science Research. The

authors are grateful to Dr. N. Naydenova from the

Institute of Genetics, Bulgarian Academy of Sciences,for the donation of seeds of pea chlorophyll mutants.

References

[1] B.L. Trumpower (Ed.), Function of Quinones in Energy

Conserving Systems, Academic Press, New York, London,

1982.

[2] N. Fisher, P.R. Rich, A motif for quinone binding sites in

respiratory and photosynthetic systems, J. Mol. Biol. 296 (2000)

1153–1162.

[3] J. Ames, D.C. Fork, Quenching of chlorophyll fluorescence by

quinones in algae and chloroplasts, Biochim. Biophys. Acta 143

(1967) 97–107.

[4] N.G. Bukov, G. Sridhram, E.A. Egorov, R. Carpentier, Interac-

tion of exogenous quinones with membranes of higher plant

chloroplasts: modulation of quinone capacities as photochemical

and non-photochemical quenchers of energy in Photosystem II

during light–dark transitions, Biochim. Biophys. Acta 1604 (2003)

115–123.

[5] N.-U. Frigaard, S. Tokita, K. Matsuura, Exogenous quinones

inhibit photosynthetic electron transfer in Chloroflexus aurantia-

cus by specific quenching of the excited bacteriochlorophyll c

antenna, Biochim. Biophys. Acta 1413 (1999) 108–116.

[6] K.K. Karukstis, S.C. Boegeman, J.A. Fruetel, S.M. Gruber,

M.H. Terris, Multivariate analysis of photosystem II fluorescence

quenching by substituted benzoquinones and naphthoquinones,

Biochim. Biophys. Acta 891 (1987) 256–264.

[7] K.K. Karukstis, S.M. Gruber, J.A. Fruetel, S.C. Boegeman,

Quenching of chlorophyll fluorescence by substituted anthraqui-

nones, Biochim. Biophys. Acta 932 (1988) 84–90.

[8] M. Kitajima, W.L. Butler, Quenching of chlorophyll fluorescence

and primary photochemistry in chloroplasts by dibromothymo-

quinone, Biochim. Biophys. Acta 376 (1975) 105–115.

[9] J.W. Lee, W. Zipfel, T.G. Owens, Quenching of chlorophyll

excited states in photosystem I by quinones. Stern–Volmer

analysis of fluorescence and photochemical yield, J. Lumin. 51

(1992) 79–89.

[10] S. Tokita, N.-U. Frigaard, M. Hirota, K. Shimada, K. Matsuura,

Quenching of bacteriochlorophyll fluorescence in chlorosomes

from Chloroflexus aurantiacus by exogenous quinones, Photo-

chem. Photobiol. 72 (2000) 345–350.

[11] S. Vasil�ev, S. Wiebe, D. Bruce, Non-photochemical quenching of

chlorophyll fluorescence in photosynthesis. 5-Hydroxy-1,4-naph-

thoquinone in spinach thylakoids as a model for antenna based

quenching mechanisms, Biochim. Biophys. Acta 1363 (1998) 147–

156.

[12] K.K. Karukstis, M.A. Berliner, C.J. Jewell, K.T. Kuwata,

Chlorophyll fluorescence measurements to assess the competition

of substituted anthraquinones for the Qb binding site, Biochim.

Biophys. Acta 1020 (1990) 163–168.

[13] E. Apostolova, Tz. Markova, Tz. Filipova, M.T. Molina, S.G.

Taneva, Influence of substituted 1,4-anthraquinones on the

chlorophyll fluorescence and photochemical activity of pea

thylakoid membranes, J. Photochem. Photobiol. B 70 (2003) 75–

80.

[14] K.E. Steinback, J.J. Burke, C.J. Arntzen, Evidence for the role of

surface-exposed segments of the light-harvesting complex in

cation-mediated control of chloroplast structure and function,

Arch. Biochem. Biophys. 195 (1979) 546–557.

[15] A. Dobrikova, A. Ivanov, R.M. Morgan, I. Petkanchin, S.G.

Taneva, Contribution of LHC II complex to the electric properties

of thylakoid membranes: an electric light scattering study of Chl

b-less barley mutant, J. Photochem. Photobiol. B 57 (2000) 33–40.

[16] H.K. Lichtenthaler, Chlorophylls and carotenoids: pigments of

photosynthetic biomembranes, Methods Enzym. 148 (1987) 350–

383.

[17] S.G. Taneva, I. Petkanchin, Surface electric properties of biolog-

ical systems, Trends Photochem. Photobiol. 6 (1999) 113–139.

[18] A. Dobrikova, R.M. Morgan, A. Ivanov, E. Apostolova, I.

Petkanchin, N.P.A Huner, S.G. Taneva, Electric properties of

thylakoid membranes from pea mutants with modified carotenoid

and chlorophyll-protein complexes composition, Photosynth. Res.

65 (2000) 165–174.

[19] S.G. Taneva, M. Busheva, I. Petkanchin, Electric parameters of

Photosystem II particles – electric light scatering study, J.

Photochem. Photobiol. B 10 (1991) 315–327.

[20] A. Dobrikova, A.G. Ivanov, E. Apostolova, N. Naydenova, I.

Petkanchin, S.G. Taneva, Contribution of LHCII complex to the

electric properties of thylakoid membranes, in: N. Gozukirmizi

(Ed.), Plants of the Balkan Peninsula: Into the Next Millenium,

vol. 2, 2001, pp. 75–80.

[21] K.D. Allen, L.A. Staehelin, Resolution of 16 to 20 chlorophyll-

protein complexes using a low ionic strength native green gel

system, Anal. Biochem. 194 (1991) 214–222.

[22] S.P. Stoylov, Colloid Electrooptics, Academic Press, London,

1991.

E. Apostolova et al. / Journal of Photochemistry and Photobiology B: Biology 78 (2005) 115–123 123

[23] S. Jansson, The light-harvesting chlorophyll a/b-binding protein,

Biochim. Biophys. Acta 1184 (1994) 1–19.

[24] H. Paulsen, Chloropyll a/b binding proteins, Photochem. Photo-

biol. 62 (1995) 367–382.

[25] S. Nussberger, J.P. Dekker, W. Kuhlbrandt, B.M. van Bolhuis,

R. van Grondelle, H. van Amerongen, Spectroscopic character-

ization of three different monomeric form of the main chlorophyll

a/b binding protein from chloroplast membranes, Biochemistry 33

(1994) 14775–14783.

[26] K.K. Karukstis, C.R. Monell, Reversal of quinone-induced

chlorophyll fluorescence quenching, Biochim. Biophys. Acta 973

(1989) 124–130.