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Journal of Theoretical Biology 233 (2005) 287–300
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A theoretical study on effect of the initial redox state of
cytochrome b559 on maximal chlorophyll fluorescence level (FM):
implications for photoinhibition of photosystem II
Dušan Lazára,, Petr Ilı́ka, Jerzy Krukb, Kazimierz Strza"kab, Jan Nauša
a
Laboratory of Biophysics, Department of Experimental Physics, Faculty of Science, Palacký University, tř. Svobody 26, 771 46 Olomouc,
Czech Republic
b
Department of Plant Physiology and Biochemistry, Faculty of Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland
Received 18 August 2004; received in revised form 6 October 2004; accepted 8 October 2004
Available online 1 December 2004
Abstract
In this work, we extended the reversible radical pair model which describes energy utilization and electron transfer up to the first
quinone electron acceptor (QA) in photosystem II (PSII), by redox reactions involving cytochrome (cyt) b559. In the model, cyt b559
accepts electrons from the reduced primary electron acceptor in PSII, pheophytin, and donates electrons to the oxidized primary
electron donor in PSII (P680+). Theoretical simulations of chlorophyll fluorescence rise based on the model show that the maximal
fluorescence, FM, increases with an increasing amount of initially reduced cyt b559. In this work we applied, the first to our
knowledge, metabolic control analysis (MCA) to a model of reactions in PSII. The MCA was used to determine to what extent the
reactions occurring in the model control the FM level and how this control depends on the initial redox state of cyt b559. The
simulations also revealed that increasing the amount of initially reduced cyt b559 could protect PSII against photoinhibition. Also
experimental data, which might be used to validate our theory, are presented and discussed.
r 2004 Elsevier Ltd. All rights reserved.
Keywords: Chlorophyll fluorescence induction; Cytochrome b559; DCMU; Photoinhibition; Reversible radical pair model
1. Introduction
Although cytochrome (cyt) b559 is an essential part of
the functional photosystem II (PSII) reaction centre, the
Abbreviations: Cyt, cytochrome; DCMU, 3-(30 , 40 -dichlorophenyl)1, 1-dimethylurea; F0, minimal fluorescence; FM, maximal fluorescence;
FV/FM, maximal quantum yield of photosystem II photochemistry;
FLR, fluorescence rise; HP, high potential; LP, low potential; MCA,
metabolic control analysis; OEC, oxygen evolving complex; P680,
primary electron donor in photosystem II; Pheo, (pheophytin) primary
electron acceptor in photosystem II; PQ, plastoquinone; PSII,
photosystem II; QA, the first quinone electron acceptor in photosystem
II; QB, the second quinone electron acceptor in photosystem II; RCII,
reaction centre of photosystem II; RRP, reversible radical pair; 3P680,
triplet state of primary electron donor in photosystem II
Corresponding author. Tel.: +420 58 5225737;
fax: +420 58 5634153.
E-mail address: [email protected] (D. Lazár).
0022-5193/$ - see front matter r 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jtbi.2004.10.015
function of cyt b559 is not yet fully understood (see the
latest reviews on cyt b559 by Cramer et al. (1993);
Shuvalov (1994); Whitmarsh et al. (1994); Whitmarsh
and Pakrasi (1996); Stewart and Brudvig (1998)).
Different results on the cyt b559 function are based on
the fact that cyt b559 was identified in four redox forms
in dependence on the measurement conditions and
sample preparation: the extra low potential (LP) form
(Em=–45 mV), the LP form (Em=+30 to +80 mV), the
intermediary potential form (Em=+150 mV), and the
high potential (HP) form (Em=+350 mV). The
function of cyt b559 is mainly ascribed to its protective
role against photoinhibition of PSII as it arises from
numerous in vitro studies. In donor side photoinhibition,
when oxygen evolving complex (OEC) cannot effectively
pass through its S-states and does not thereby effectively
donate electrons to primary electron donor in photo-
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system II (P680+), cyt b559 substitutes for the role of
OEC and donates electrons to P680+. In such a way cyt
b559 prevents P680+ accumulation which could initiate
oxidation of nearby chromophores or residues leading
to PSII degradation (Thompson and Brudvig, 1988;
Blubaugh et al., 1991; Buser et al., 1992; van der Bolt
and Vermaas, 1992). Under acceptor side photoinhibition, when electron transfer from Q
A (reduced the first
quinone electron acceptor in PSII) to QB (the second
quinone electron acceptor in PSII) is disabled, cyt b559
can accept electrons from the primary reduced electron
acceptor in PSII, pheophytin (Pheo), directly (Nedbal
et al., 1992; Barber and De Las Rivas, 1993; Poulson et
al., 1995) or via an additional plastoquinone (PQ)
molecule in PSII (Kruk and Strzalka, 2001) to prevent a
recombination of Pheo with P680+. This recombination can result in the formation of a triplet state of P680
(3P680) which could initiate the formation of reactive
singlet oxygen causing PSII degradation once again
(Styring et al., 1990; Vass et al., 1992). In addition to the
acceptor and donor side photoinhibitions, the unifying
model for the photoinhibition of PSII occurring in vivo
in a steady state was proposed which depends on the
generation and maintenance of an increased concentration of P680+Pheo radical pair and where the damage
of PSII was suggested to be caused by P680+ rather
than by singlet oxygen (Anderson et al., 1998). The role
of cyt b559 in the unifying model for the photoinhibition
of PSII has not been described as yet.
In order to access the cyt b559 function, one can use
the measurement of chlorophyll (chl) a fluorescence rise
(FLR) as cyt b559 is a part of PSII and FLR is assumed
to come exclusively from PSII (see the latest reviews on
the FLR by Krause and Weis (1991); Govindjee (1995);
Strasser et al. (1995); and Lazár (1999)). An increase in
the chl fluorescence level reflects accumulation of the
reduced primary quinone electron acceptor in PSII (Q
A)
as was originally suggested by Duysens and Sweers
(1963). When the FLR is measured at high intensity of
exciting light with an untreated sample, it is characterized by typical O2J2I2P transient. The J step is
explained by transient accumulation of only reduced QA
and hence the O–J transient is called the photochemical
phase of the FLR (Neubauer and Schreiber, 1987;
Strasser et al., 1995; Tomek et al., 2001; Lazár, 2003).
The J–I–P transient is called the thermal phase and, in
addition to QA reduction, probably reflects reduction of
secondary quinone electron acceptor in PSII (QB) and
PQ pool (Neubauer and Schreiber, 1987; Lazár et al.,
1997; Stirbet et al., 1998; Tomek et al., 2001). Other
processes can also be involved in this phase of FLR
(Schreiber et al., 1989; Schreiber and Krieger, 1996;
Koblı́žek et al., 2001; Pospı́šil and Dau, 2002; Bukhov
et al., 2003; Hsu and Leu, 2003; Heredia and De Las
Rivas, 2003; Schansker et al., 2003; Vredenberg and
Bulychev, 2003). However, even if significant progress in
understanding the FLR has occurred, the O–J–I–P
transient is still not fully understood at present. Therefore, an inhibitor of electron transfer between QA and
QB (Oettmeier and Soll, 1983; Trebst and Draber, 1986;
Trebst, 1987; Shigematsu et al., 1989), 3-(30 ,40 -dichlorophenyl)-1,1-dimethylurea (DCMU), is often used to
make the reactions in PSII simpler. When a sample is
treated with DCMU, the FLR measured at high
intensity of exciting light is characterized by a steep
fluorescence increase reaching the maximal saturation
level at approximately the position of the J step
measured with the sample without DCMU (Strasser
et al., 1995; Lazár et al., 1998; Lazár and Pospı́šil, 1999).
Mathematical modelling is at present an important
tool in better understanding the explored processes. This
is also valid in understanding plant metabolic pathways
(for reviews see Giersch, 2000; Morgan and Rhodes,
2002) as well as the FLR (for review see Lazár, 1999).
Excitation energy utilization and photochemical reactions in the reaction centre of PSII (RCII), which is also
the case when whole PSII (thylakoid membranes) is
treated by DCMU, can be well described by the
reversible radical pair (RRP) model, also called the
exciton radical pair equilibrium model and first proposed by Breton (1983) (see also van Grondelle, 1985;
Schatz et al., 1988; Leibl et al., 1989; Roelofs et al., 1992;
Dau, 1994). The RRP model and its derivatives have
already been used for simulation or fitting of the FLR
measured both with and without DCMU (Baake and
Schlöder, 1992; Trissl et al., 1993; Trissl and Lavergne,
1995; Lavergne and Trissl, 1995; Lazár and Pospı́šil,
1999; Lazár, 2003). Although a modified RRP model
was used for studying PSII photoinhibition occurring in
DCMU-treated thylakoid membranes (Vavilin et al.,
1998), cyt b559 has not yet been included in the RRP
model. The contribution of cyt b559 to the FLR and the
protective function of cyt b559 against PSII photoinhibition have also not yet been studied with the help of
theoretical simulations.
With respect to known electron transport pathways in
which cyt b559 is involved (see above) and the formulation of the RRP model, cyt b559 can be included into the
RRP model as the electron acceptor from Pheo and/or
electron donor to P680+. Each of these electron
pathways separately, or considered together as one
pathway enabling a cycle around PSII (Heber et al.,
1979; Falkowski et al., 1986; Arnon and Tang, 1988;
Prasil et al., 1996), should protect PSII against photoinhibition. Moreover, addition of a new component
(cyt b559) to the RRP model should lead to a change in
fluorescence properties of the RRP model.
A powerful tool in theoretical studying of explored
phenomenon is the metabolic control analysis (MCA;
for reviews see Fell, 1992; Visser and Heijnen, 2002). In
MCA, in addition to other coefficients, the control
coefficient C is calculated which exactly quantifies to
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D. Lazár et al. / Journal of Theoretical Biology 233 (2005) 287–300
what extent particular model parameter (concentration
or activity or rate constant) controls selected variable of
the model (concentration or flux). MCA has already
been used for exploration of several plant metabolic
pathways, e.g. Calvin cycle (Poolman et al., 2000) or the
malate valve (Fridlyand et al., 1998) but it has never
been used for exploration of the FLR.
In this work, we extended the RRP model by redox
reactions involving cyt b559. Firstly, using this model, we
focused on the effect of the initial redox state of cyt b559
on the maximal fluorescence level FM in the presence of
DCMU. Further, we performed an analysis of fluxes in
our model (MCA) in order to discover to what
extent the reactions occurring in the model control the
FM level and how this control depends on the initial
redox state of cyt b559. Additionally, we used theoretical
simulations to explore whether changes in the initial
redox state of cyt b559 can protect PSII against
photoinhibition. Finally, we presented and discussed
experimental results, which might be used to validate
our theory.
2. Material and methods
2.1. Plant material and preparation of thylakoid
membranes
Pea seedlings (Pissum sativum) were cultivated in
perlite for 14 days at 22 1C. The seedlings were watered
regularly and exposed to a periodic light–dark cycle of
16 h of white light (90 mmol photons of photosynthetically active radiation m2 s1) and 8 h of dark. Detached
pea leaves were used for the thylakoid membrane
preparation.
Thylakoid membranes were isolated according to Dau
et al. (1995) with a slight modification. The pea leaves
were mixed with a chilled grinding buffer (0.4 M sucrose,
0.4 M NaCl, 35 mM HEPES (pH 7.5), 4 mM MgCl2,
1 mM EDTA, 5 mM ascorbic acid and 2 g l1 BSA) and
homogenized three times for 5 s by T25 Basic homogenizer (IKA Labortechnik, Staufen, Germany). The
homogenate was filtered through miracloth and centrifuged for 6 min at 5000g. The chloroplast pellet was
resuspended in a buffer containing 25 mM HEPES (pH
7.5), 150 mM NaCl, 8 mM MgCl2 and then centrifuged
for 10 min at 5000g. The resulting pellet consisting of
intact thylakoid membranes was resuspended in a small
amount of the buffer containing 50 mM MES (pH 6.0),
15 mM NaCl, and 10 mM MgCl2. All steps were
performed under dim green light at 4 1C. The chl
content was determined spectrophotometrically in 80%
acetone according to Lichtenthaler (1987). Thylakoid
membranes were diluted in the last mentioned buffer to
the chl concentration of 50 mg ml1 prior to the
fluorescence or spectroscopic measurements.
289
2.2. Measurement of FLR curves
FLR curves were measured with 300 ml of thylakoid
membranes in a standard 0.5 0.5 cm glass cuvette
using a Plant Efficiency Analyser (Hansatech, Norfolk,
United Kingdom). LEDs producing strong red light
(l ¼ 650 nm) of an intensity of 12,000 mmol photons
m2 s1 were used for excitation and fluorescence
emission was detected by PIN diode above 700 nm.
The FRs were recorded for up to 1 s starting at 10 ms
after the onset of irradiation. A fluorescence signal at
50 ms was considered as minimal fluorescence F0
(Strasser et al., 1995). Before each measurement,
thylakoid membranes were treated with DCMU
(20 mM, 6 min in dark) or additionally with FeCy
(20 mM, 6 min in dark) or Asc (5 mM, 6 min in dark).
A higher volume of thylakoid membrane suspension
with a chl concentration of 50 mg ml1 was always
prepared. 300 ml of the suspension was chemically
treated as described above and used for the measurements and another 300 ml of the suspension was not
treated at all and used for measurement of the control
(O2J2I2P) FLR. FM level of the control FRs
subsistent to the DCMU, DCMU+FeCy, and
DCMU+Asc FRs were normalized to the same level
and appropriate DCMU, DCMU+FeCy, and
DCMU+Asc FRs were then multiplied by the same
normalization factors as the subsistent control FRs.
This procedure was used in order to be able to present
the FLR curves as they are shown in Fig. 5 and to avoid
differences in the fluorescence signal caused by a
different chl concentration in the measuring cuvette
due to possibly incorrect pipetting. Each measurement
(DCMU, DCMU+FeCy, DCMU+Asc) was performed four times with a new thylakoid membrane
preparation each time with the mean value7standard
deviation from all the measurements presented in the
text. The light intensity was measured with a Quantum
Radiometer LI-189 (LI-COR, Lincoln, USA).
2.3. Determination of the cyt b559 redox state
The amount of particular redox forms of cyt b559 in
the DCMU-treated thylakoid membranes was determined with a AMINCO DW-2000 UV/VIS spectrophotometer (SLM Instruments, Urbana, USA) using a
split beam mode. Thylakoid membranes were treated
with DCMU (20 mM, 6 min in dark), FeCy (20 mM,
6 min in dark), hydroquinone (HQ; 4 mM, 6 min in
dark), and Asc (5 mM, 6 min in dark), step by step, and
followed by the measurement of absorption spectrum in
an interval of 520–590 nm. Relative amounts of particular redox forms of cyt b559 in thylakoid membranes
were determined from absorbances at 559 nm related to
the background absorbances at 540 nm in difference
spectra obtained as follows: reduced HP cyt b559
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290
(spectrum 2–spectrum 1), oxidized HP cyt b559
(spectrum 3–spectrum 2), and oxidized LP cyt b559
(spectrum 4–spectrum 3). The spectra 1–4 were
obtained as a difference of spectra of the sample and
the reference (both with thylakoid membranes) treated
in the following way: DCMU in both the sample and
reference (spectrum 1), DCMU in the sample and
DCMU and FeCy in the reference (spectrum 2), DCMU
and HQ in the sample and DCMU and FeCy in the
reference (spectrum 3), and DCMU, HQ, and Asc in the
sample and DCMU and FeCy in the reference (spectrum
4). The contribution of cyt f to the absorbance at 559 nm
was negligible. The amount of cyt b559 was measured
four times with new thylakoid membrane preparation
each time and with a mean value7standard deviation
from all the measurements presented in the text.
2.4. Theoretical simulations
The FLR curves were simulated using Gepasi software version 3.21 (P. Mendes, The University of Wales,
Aberystwyth, United Kingdom) that was designed for
simulations of chemical and biochemical kinetics
(Mendes, 1993, 1997). Gepasi uses routine LSODA
(Livermore Solver of Ordinary Differential Equations)
for solution of a set of differential equations. LSODA is
a sophisticated algorithm that measures the stiffness of
equations and switches the integration method dynamically according to the measure of stiffness.
As we made use of Gepasi software for our
simulations, the expression of differential equations
describing the time derivations of particular states in the
model is not necessary. The model is entered into the
software directly as chemical reactions as presented in
Scheme 1.
2.5. Metabolic control analysis (MCA)
In MCA, in addition to other parameters, the control
coefficient is defined as a relative change in steady-state
variable Xj upon a relative change in the steady-state
rate vi,
J
CX
vi ¼
qX j =X j q ln X j
¼
:
qvi =vi
q ln vi
In our case, the steady-state variable Xj is the FM level
and the steady-state rate vi is the rate of selected reaction
in the theoretical model. As the fluorescence signal in the
FLR is defined in our model as a deactivation of excited
states via the rate constant of fluorescence emission (it is
a product of the rate constant of fluorescence emission
and the concentration of all excited states; see Theory),
fluorescence emission amounts to flux. Therefore, the
control coefficients calculated by us amount to the flux
control coefficients. The Gepasi software (see above)
calculated the control coefficients. For this purpose, any
reversible reaction in the model was entered into the
software as two irreversible reactions in order to obtain
the flux control coefficients of the reaction in both
directions. If a particular reaction occurred more than
once in the model (e.g. a charge separation in open
RCII; see Scheme 1), the overall flux control coefficient
of this reaction was calculated as the sum of all the
particular flux control coefficients. The correctness of
the MCA results was checked by the calculation of the
sum of all the flux control coefficients determined for the
given amount of initially reduced cyt b559. According to
the theory of MCA, the sum of all flux control
coefficients is equal to unity (for reviews on MCA see
Fell, 1992; Visser and Heijnen, 2002). As the simulated
fluorescence is in our case the sum of the four fluxes
(see Theory), the sum of all the flux control coefficients
is 4 and not unity. This was verified as true for
all the amounts of initially reduced cyt b559 used in
our calculations.
3. Theory
While the original RRP model describes charge
separation, recombination and stabilization when QA
is both oxidized and reduced (Breton, 1983; van
Grondelle, 1985; Schatz et al., 1988; Leibl et al., 1989;
Roelofs et al., 1992; for a review see Dau, 1994), we
previously extended it (Lazár and Pospı́šil, 1999) by
addition of the reduction of P680+ by electrons from
OEC in the donor side of PSII. The model employed
here for simulations of FLR curves in the presence of
DCMU is based on the extended RRP model, which was
further extended in this work by the addition of redox
reactions involving cyt b559. We considered in our model
the cyt b559, which accepts electrons from Pheo and
donates electrons to P680+ (see Scheme 1). This cyt b559
electron pathway is only hypothetical but it is consistent
with facts known from literature (see Introduction) and
resulting theoretical simulations are in agreement with our
experimental results (see Discussion). The initial conditions and values of the rate constants used in the
simulations are summarized in the legend to Scheme 1
and Table 1, respectively. All the reactions in the model
are assumed to be of the first order with respect to one
component and follow the mass action theory. Fluorescence signal at time t in the FLR is defined to be
proportional to the sum of the forms with excited chl a
multiplied by the rate constant of chl a fluorescence, i.e. as
F ðtÞ ¼ kF fððL PÞ IAÞðtÞ þ ððL PÞ I AÞðtÞ
þ ððL PÞ IA ÞðtÞ þ ððL PÞ I A ÞðtÞg:
See Scheme 1 for the meaning of the symbols.
We had to estimate the value of the rate constant of
cyt b559 reduction by Pheo (kCred; see Scheme 1), while
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Scheme 1. A scheme for all the reactions in our model used for simulations of the FLR curves in the presence of DCMU. Part A shows the main
reaction scheme where numbers 1, 2, and 3 next to the arrows mean P+ reduction, C+ reduction, and excited states deactivation, respectively, as
described in detail in part B. The reactions described in part B occur at any time when a redox state of form(s) in the top left part of each particular
reaction scheme is formed and unrelated to, but conserving, the redox state of other forms. L, P, I, A, C, YZ, and Si mean all chlorophylls from PSII
light harvesting antennae, P680, pheophytin (Pheo), QA, cyt b559, tyrosine Z, and the S-states of OEC, respectively. The asterisk indicates an excited
P
state. The rate constants mean: kL—excited states formation; kLHA
HD —overall non-radiative loss of excited states in light harvesting antennae; kHD—
(non-radiative) heat dissipation of excited states by P680+ (the fluorescence quenching by P680+); kPQ
HD—(non-radiative) heat dissipation of excited
states by PQ molecules (fluorescence quenching by PQ molecules); kUU—excited states transfer between RCIIs; kF—fluorescence; koðcÞ
1 —electron
+
transfer from P680 to Pheo (the charge separation) in the open (closed) RCIIs; koðcÞ
1 —backward electron transfer from Pheo to P680 (the charge
recombination) in open (closed) RCIIs leading to formation of excited-state P680*; ko2 —electron transfer from Pheo to QA (the charge stabilization)
in open RCIIs; kc2 —backward electron transfer from Pheo to P680+ (non-radiative charge recombination) in closed RCIIs leading to the ground
*
+
reduction by YZ;
state of P680; kPArec —charge recombination between P680+ and Q
A leading to formation of excited-state P680 ; kPred —P680
kPox—backward electron transfers from P680 to Y þ
in
the
previous
reactions;
k
—electron
donation
from
the
S
state
of
OEC
to Y þ
12
1
Z
Z during the
S1 ! S2 transition; kCox—electron donation from cyt b559 to P680+; and kCred—electron donation from Pheo to cyt b559. The values of the rate
constants used in all the simulations are compiled in Table 1 and the initial conditions were as follows: [(LP)IA]0=1 (i.e. there is no excitation
formed, P680 is reduced and Pheo and QA are oxidized in the dark); [YZ]0=1 (i.e. Y Z is reduced in the dark); ½S1 0 ¼ 1 (i.e. all OEC is initially in the
S1 state; Messinger and Renger, 1993); ½PQ0 ¼ 7 (i.e. there are 7 PQ molecules in the PQ pool per PSII and all are oxidized in the dark; McCauley
and Melis, 1986); ½C0 and ½C þ 0 were changed from 0 to 1 (½C0 þ ½C þ 0 ¼ 1) to simulate the effect of the initial redox state of cyt b559 on the FLR
curve; all other forms in the model have initially a zero relative concentration in the simulations.
the values of all the other rate constants in the
model were known from literature (see Table 1).
The time constant of this cyt b559 reduction was
roughly estimated by Satoh et al. (1990) to be in the
submicrosecond range. As discussed by Whitmarsh et al.
(1994), the rate of electron flow from Pheo to cyt b559
should be very high. In order to obtain an approximate
value of this rate constant, we simulated the FLR
curves in the presence of DCMU and with initially fully
oxidized cyt b559 for the rate constant in the range of
107–1010 s1. We discovered that only when kCred was
about the same or higher than the rate constant of
electron transfer from Pheo to QA (k02; 2.3 109 s1),
the theoretical FLR curves resembled the experimentally
measured FLR curves in the presence of DCMU (data
not shown). For lower kCred the simulated FRs had a
local maximum at the position of the J step (see
Introduction) thus differing from the experimental
FLR measured with DCMU-treated samples (see
Fig. 5). Therefore, we used in all our simulations of
FLR curves in the presence of DCMU kCred equalled to
the value of k02 (see Table 1). In addition, only when
kCred was above 109 s1 and cyt b559 fully initially
oxidized in our model, a significantly lower accumulation of stable Pheo (P680PheoQ
A state) occurred (to
protect against the acceptor side photoinhibition of
PSII; see Nedbal et al., 1992) in comparison with the
case at which cyt b559 is not included in the model
(data not shown). See also discussion on kCred in the
Discussion.
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292
Table 1
Values of the rate constants used in the simulations
Rate constant
Used value (s1)
Value in literature (s1)
kL
kF
kLHA
HD
kPHD
4000
6.7 107
5 108
1 109
5.6 107
4000–5500a
6.7 107b
3–10 108c
1 109d
5.6 107e
1 109
3 109
3 108
2.3 109
4.8 108
3.4 108
1 109
10000
2.9 107
5.8 106
10000
667
2.3 109
1–6 109f
1.9–9.3 109c
3–20 108c
2–2.3 109c
4.8–15 108c
3.4–30 108c
3–10 108c
5000–10000g
2–5 107h
1.1–8.3 106i
7150–25000j
500–667k
See the theory
kPQ
HD
kUU
ko1
ko1
ko2
kc1
K c1
K c2
kPArec
kPred
kPox
K 12
kCox
kCred
For the meaning of the rate constants see the legend to Scheme 1.
a
For the light intensity used in our measurement, see Lazár and
Pospı́šil (1999); Tomek et al. (2001) and Lazár (2003).
b
Rabinovich and Govindjee (1969).
c
Reviewed by Dau (1994).
d
Bruce et al. (1997).
e
Lazár (2003).
f
Lavergne and Trissl (1995) and Trissl and Lavergne (1995).
g
Haveman and Mathis (1976) and Renger and Wolff (1976).
h
Brettel et al. (1984) and Meyer et al. (1989).
i
For the equilibrium constant K ¼ kPred =kPox 3; see Brettel et al.
(1984).
j
Summarized by Razeghifard et al. (1997).
k
Satoh et al. (1990) and Havaux (1998).
4. Results
4.1. Theoretical FLR curves for different amounts of
initially reduced cyt b559
In our theoretical model, we hypothesize that cyt b559
can accept electrons from Pheo and donate them to
P680+. We first tested how the initial redox state of such
cyt b559 can affect and control the FLR. In addition, as
the function of cyt b559 in PSII is mainly connected with
its protective role against photoinhibition of PSII (see
Introduction), we also tested if changes in the initial
redox state of cyt b559 can protect PSII against its
photoinhibition.
The FLR curves in the presence of DCMU simulated
for different amounts of initially reduced cyt b559 by our
model are shown in Fig. 1. These curves indicate that
both the F0 and the FM increase with an increasing
amount of initially reduced cyt b559. The FM/F0 ratio is
about 2.1–2.7 in the simulations which is close to about
2.5–3.1 found for the FM/F0 ratio for experimental FLR
Fig. 1. FLR curves simulated on the basis of our model for different
amounts of initially reduced cyt b559 as indicated above the simulated
curves. The vertical dotted line at 50 ms indicates the time at which the
F0 level is determined in the experimental FRs (see Fig. 5). Inset A
shows the difference in simulated F0 levels obtained for different
amounts of initially reduced cyt b559 in more detail. Inset B shows the
dependence of FM of the simulated FLR curves (FM of the 100% curve
is set as 100%) on the initial redox state of cyt b559.
curves (see Fig. 5) but it is lower than 4.5–5 found for
the FM/F0 ratio of FLR curves measured without any
treatment (Lazár et al., 1998; Lazár, 2003). The low FM/
F0 ratio, in case when DCMU is present (Fig. 5) or
appropriate FLR curves are simulated (Fig. 1), is due to
an increase of the F0 level and a decrease of the FM level.
This is caused by backward electron flow from Q
B to QA
preceding DCMV binding and by the oxidized PQ pool
quenching, respectively.
As described and discussed in detail in Lazár (2003),
F0 in the FLR simulated by the RRP-based model is
established at about 3 ns after the beginning of irradiation and lasts to about 1 ms. This simulated F0 level
reflects accumulation of chl excited states when all
electron acceptors in the acceptor side of PSII are
oxidized, which means the accumulation of P680*PheoQA state in our case. An increased accumulation of this
state is the only reason (data not shown) why the F0
level with an increasing amount of initially reduced cyt
b559 increases (inset A in Fig. 1). This increase is caused
by a shift of equilibrium among all occurring reactions
in the model in favour of accumulation of the
P680*PheoQA state when cyt b559 is initially reduced to
a greater extent.
Inset B in Fig. 1 clearly shows that the FM level in the
simulated FLRs increases with an increasing amount of
initially reduced cyt b559. Simulated time courses of
particular excited states in the model revealed (data not
shown) that the main reason of the increase of FM is in
an increase of the maximal (i.e. steady state) concentration of P680*PheoQ
A state when cyt b559 is initially
reduced to a greater extent. This increase is caused, as in
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case of P680*PheoQA described above, by a shift of
equilibrium among all occurring reactions.
According to the original suggestion by Duysens and
Sweers (1963), an increase in the FM level should reflect
an increased accumulation of Q
A. This is also valid in
our case, as shown in Fig. 2, where the time courses of
the sum of all the reduced QA in the model are simulated
in dependence on the initial amount of reduced cyt b559.
Fig. 2 also shows that for only 100% of cyt b559 initially
reduced, the maximal accumulation of Q
A reaches 1, in
other words, only in this case the FM level actually has
the meaning of fluorescence intensity coming from fully
closed RCIIs. For lower amounts of initially reduced cyt
b559 not all RCIIs are closed and the corresponding FM
level does not have its definition meaning. This is
surprising as the value of the rate constant of excited
states formation (kL ; see Scheme 1) was calculated for
high (=saturating) intensity of exciting light (see Lazár
and Pospı́šil, 1999 for the calculation) which is used for
measurements of the experimental FRs (see Fig. 5).
Therefore, the presence of a certain amount of initially
oxidized cyt b559 can prevent, even at very high intensity
of exciting light, the reaching of the real FM level (in the
sense of a definition) thus leading to the incorrect
determination of the maximal quantum yield of PSII
photochemistry which is expressed as the FV/FM
(=(FMF0)/FM) ratio (Kitajama and Butler, 1975).
Thus, a different amount of initially reduced cyt b559
reflecting the use of different buffers during preparation
of chloroplasts or thylakoid membranes could be the
reason why the measured values of fluorescence parameters (F0, FM, FV/FM) are different. Special attention
should be paid to this fact particularly when the results
293
with one sample preparation are compared with the
results obtained with another sample preparation.
As electrons can be transferred from Pheo not only
to QA but also to cyt b559 in our model, the steady-state
level of reduced cyt b559 should reflect changes in
maximal Q
A accumulation and changes in FM level as
described above. The inset in Fig. 2, however, reveals
that the FM level (and also the maximal accumulation of
Q
A; data not shown) linearly increases with the steadystate amount of reduced cyt b559 only up to about 80%
of cyt b559 initially reduced. When more than about 80%
of cyt b559 is initially reduced, more than 99.9% of cyt
b559 is reduced in the steady state and a further increase
in the amount of initially reduced cyt b559 does not lead
to a proportional increase in the steady-state amount of
reduced cyt b559. In the latter case however, the FM level
(and also the maximal accumulation of Q
A) continually
increases. This indicates that when cyt b559 is initially
more reduced than a certain amount (80% in our case),
changes in the FM level do not reflect changes in the
steady-state amount of reduced cyt b559.
A question arises as to what is a mechanistic
explanation for the changes in FM level and maximal
steady-state accumulations of reduced cyt b559 and QA
in dependence on the initial redox state of cyt b559 as
shown in Figs. 1 and 2. The changes can be easily
explained by the fact that there is one more electron in
PSII when cyt b559 is initially reduced when compared to
the case when cyt b559 is initially oxidized. The presence
or absence of the ‘‘additional’’ electron either stored or
not initially in cyt b559 results in different equilibrium in
steady state: PSII is more reduced (more reduced cyt
b559 and QA in the steady state leading to high FM level)
when cyt b559 is initially reduced and, vice versa, PSII is
less reduced (less reduced cyt b559 and QA in the steady
state leading to low FM level) when cyt b559 is initially
oxidized.
4.2. Control of the FM level
Fig. 2. Time courses of the sum of all Q
A (all states in the model which
have Q
A) simulated on the basis of our model for different amounts of
initially reduced cyt b559 (0%, 20%, 40%, 60%, 80%, and 100%). The
inset shows the dependence of the simulated FM level (data from Fig.
1) on the maximal (i.e. steady-state) accumulation of reduced cyt b559
as resulted from the simulations for different amounts of initially
reduced cyt b559 (indicated in the inset).
In order to understand the control of the FM level in
more detail, we applied the so-called MCA to our
model. MCA is a sophisticated tool which substitutes an
analysis of a model made using the time courses of
reactant concentrations at different initial conditions.
To the best of our knowledge, we are the first to apply
MCA to a model describing electron transfer reactions
X
in PSII. In MCA, the control coefficient C vi j ; describing
to what extent a particular reaction in a model
(characterized by its steady-state rate vi) controls a
selected steady-state variable Xj of the model (the FM
level in our case), is defined (see Material and methods).
The positive C FviM indicates that an increase in the
steady-state rate vi results in an increase in the FM level
(i.e. stimulation of fluorescence emission) and, on the
other hand, the negative C FviM means that an increase in
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the steady-state rate vi results in a decrease in the FM
level (i.e. fluorescence quenching). A higher absolute
value for the C FviM indicates a higher control of the FM
level by the reaction characterized by its steady-state
rate vi.
The results of the MCA for our model are presented
in Fig. 3. Some reactions in the model only control the
FM level in a minor way (Fig. 3A) whereas other
reactions have a high control on the FM level (Fig. 3B).
As for the 100% initial reduction of cyt b559, the control
coefficients for reactions involving redox changes of cyt
b559, i.e. its oxidation by P680+ (vCox) and of cyt b559
reduction by Pheo (vCred), are almost zero (0.009) and
zero, respectively, the values of the other control
coefficients provide information as to how particular
reactions in the model control the FM level in the case
when cyt b559 is not considered in the model. This case is
worth mentioning as the simulations of the FLR in the
presence of DCMU was reported several times in
literature but always without MCA (Trissl et al., 1993;
Lavergne and Trissl, 1995; Trissl and Lavergne, 1995;
Vavilin et al., 1998; Lazár and Pospı́šil, 1999; Lazár,
2003). In our case, in addition to excitation formation
Fig. 3. The dependence of the flux control coefficients C FviM , by which
the rate vi (i.e. flux) of the particular reaction in our model controls the
FM level of the simulated FLR curves, on the amount of initially
reduced cyt b559 in the simulations. The subscripts and superscripts in
the denotation of the rates have the same meaning as those in the
denotation of the rate constants used in our model shown in Scheme 1.
(vL) and fluorescence emission (vF), the radiative charge
recombination between P680+ and Q
A (vPArec) and the
charge separation in the closed RCII (vc1 ) contribute the
most to fluorescence emission in the FM level. On the
other hand, overall non-radiative loss of excited states in
light harvesting antennae (vLHA
HD ), non-radiative charge
recombination between Pheo and P680+ in the closed
RCIIs (vc2 ), the fluorescence quenching by oxidized PQ
molecules (vPQ
HD), the charge separation between P680
and Pheo in the open RCIIs (vo1 ), and the charge
stabilization between Pheo and QA (vo2 ) contribute the
most to the quenching of the FM level. As the nonradiative charge recombination between Pheo and
P680+ in the closed RCIIs (vc2 ) and overall non-radiative
loss of excited states in light harvesting antennae (vLHA
HD )
quench the FM level by about the same extent (for the
values of the rate constants used in our simulations),
MCA cannot finally judge if the quenching of the FM
level is mainly located in the RCIIs or in the light
harvesting antennae.
A particularly interesting result of the MCA is that
the initial redox state of cyt b559 strongly affects the
extent in which particular reactions in the model control
the FM level. While the control coefficients of certain
reactions are almost unaffected by the initial redox state
of cyt b559 (Fig. 3; excited states transfer between RCIIs
(vUU), electron donation from the S1 state of the OEC to
Yþ
Z during the S1-S2 transition (v12 ), vF ), some
reactions lose their control on the FM level when cyt
b559 is initially more reduced (Fig. 3; electron donation
from Pheo to cyt b559 (vCred), the fluorescence quenching by P680+ (vPHD), vo2 ) whereas some reactions first
significantly increase and then decrease their control on
the FM level when cyt b559 is initially reduced more (Fig.
3; backward electron transfer from Pheo to P680+ in
the closed RCIIs (vc1 ), vo1 ; vL ; electron transfer from
P680 to Pheo in the closed RCIIs (vc1 ), P680+ reduction
by YZ (vPred), backward electron transfers from P680 to
+
Yþ
Z (vPox), electron donation from cyt b559 to P680
LHA
c
(vCox), vHD , v2 ). In the latter case, maximum control of
the FM level by the reactions is reached at about 77% of
the initially reduced cyt b559. Therefore, the FM level is
extremely sensitive to changes in the rates of the abovementioned reactions at around 77% initial reduction of
cyt b559. Furthermore, the 77% of initially reduced cyt
b559 corresponds to the roughly determined 80% initial
reduction of cyt b559 representing the break point in
linear dependence between the steady-state amount of
reduced cyt b559 and the FM level (see the inset in Fig. 2).
The ‘‘critical’’ amount of 77% of initially reduced cyt
b559 for the control of the FM level need not be exactly
77% as found here but can vary in dependence on the
values of the rate constants used in the model.
It is also worth noting that for up to about 95% of
initially reduced cyt b559, the non-radiative charge
recombination between Pheo and P680+ in the closed
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295
RCIIs (vc2 ), i.e. fluorescence quenching located in the
RCII, mainly controls the lowering of the FM level. On
the other hand when more than about 95% of cyt b559 is
initially reduced, the FM level is mainly quenched by the
overall non-radiative dissipation of excited states in light
harvesting antennae (vLHA
HD ), i.e. by fluorescence quenching located in the light harvesting antennae (Fig. 3).
Although in O2 evolving samples (leaves, chloroplasts,
thylakoid membranes, PSII membranes) most of cyt b559
is initially reduced (Hiller et al., 1971; Heber et al., 1976;
Ortega et al., 1988; Thompson et al., 1989; Shinohara et
al., 1992; Mizusawa et al., 1995; McNamara and
Gounaris, 1995), cyt b559 has never been reported in
literature to be fully reduced initially. Therefore, the
fluorescence quenching by a non-radiative charge
recombination between P680+ and Pheo in closed
RCII seems to be the main process in PSII, which
quenches the FM level.
4.3. Consequences for PSII photoinhibition
With respect to the previous theoretical results, one
can summarize that the initial redox state of cyt b559 can
control the FM level and fine-tune the contribution of
particular reactions in PSII to this control. Therefore
and because the function of cyt b559 is mainly connected
to the protection of PSII against its photoinhibition (see
Introduction), we further explored, through theoretical
simulations whether the initial redox state of cyt b559 can
control and protect PSII against photoinhibition. Here
it is important to note that, when DCMU is applied to a
sample or an appropriate theoretical model is used for
simulations as it is in our case (see also Theory and
Introduction), electrons cannot be transferred from Q
A
to QB in the acceptor side of PSII and the acceptor side
PSII photoinhibition can be triggered (see Introduction). When DCMU is applied, only two electrons can
be stored in the electron acceptors of PSII (one on Pheo
and one on QA) resulting in the impossibility of OEC to
continuously pass through its S-states. This situation
leads to the donor side photoinhibition of PSII (see
Introduction). Therefore, the FRs measured with a
DCMU-treated sample or simulated with an appropriate theoretical model represent a suitable system for
studying the protective role of cyt b559 against photoinhibition of PSII.
In the donor or acceptor side photoinhibition of PSII,
accumulation of P680+ or 3P680, respectively, is the
starting process occurring in PSII leading to its damage
(see Introduction). As our model does not include the
formation of the 3P680, we assumed in agreement with
literature (Booth et al., 1990; van Mieghem et al., 1995;
Pospı́šil, 1997) that the amount of formed 3P680 is
proportional to the amount of forms with charged P680
and Pheo in both the open and closed RCII, i.e. to the
sum of P680+PheoQA and P680+PheoQ
A forms in
Fig. 4. Time courses of the sum of all P680+ (all states in the model
which have P680+; part A) and of the sum of all P680+Pheo (all
states in the model which have P680+Pheo; part B) simulated on the
basis of our model for different amounts of initially reduced cyt b559
(0%, 20%, 40%, 60%, 80%, and 100%).
our model. The time courses of all P680+ and
P680+Pheo accumulations, simulated for different
amounts of initially reduced cyt b559 in our model, are
shown in panels A and B, respectively, of Fig. 4. The
simulated curves clearly show that there is a decrease in
steady-state concentrations of both P680+ and
P680+Pheo states with an increasing amount of
initially reduced cyt b559. This means that an increase
in the amount of initially reduced cyt b559 can protect
PSII against its photodamage with respect to both the
donor and acceptor side photoinhibitions of PSII.
PSII photoinhibition in vivo was found to be a light
dosage event and single hit process (Park et al., 1995,
1996, 1997; Sinclair et al., 1996; Anderson et al., 1998)
implying, in the sense of our model concept, that after
absorption of a certain light dose by a photosynthetic
apparatus, its electron transfer chain is saturated (i.e.
clogged) and PSII behaves like that treated with
DCMU. Absorption of the last quantum of light then
triggers the photodamage of PSII. We can also, therefore, use our model to test whether the initial redox state
of cyt b559 can control and protect against PSII
photoinhibition with respect to the unifying model of
PSII photoinhibition in vivo in the steady state, which
depends on the generation and maintenance of
P680+Pheo radical pair (Anderson et al., 1998). In
this case, we can use once again the simulations shown
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D. Lazár et al. / Journal of Theoretical Biology 233 (2005) 287–300
in panel B of Fig. 4 where now we take the accumulation
of all P680+Pheo states in the model, not as a measure
of 3P680 accumulation, but as a measure of the radical
pair accumulation per se. The simulations in panel B of
Fig. 4, showing that the increasing amount of initially
reduced cyt b559 leads to a decrease of P680+Pheo
states at steady-state level, demonstrate that the initial
reduction of cyt b559 can protect PSII against its
photodamage also with respect to the unifying model
of PSII photoinhibition. In agreement with the protective role of reduced cyt b559 against PSII photoinhibition
in vivo is also the fact that in O2 evolving samples most
of cyt b559 is initially reduced (Hiller et al., 1971; Heber
et al., 1976; Ortega et al., 1988; Thompson et al., 1989;
Shinohara et al., 1992; Mizusawa et al., 1995; McNamara and Gounaris, 1995).
It is also important to note that according to Fig. 4,
the steady-state maximal accumulation of all P680+
(panel A) is by 4–5 orders of magnitude higher than of
all P680+Pheo (panel B). The accumulation of P680+
is mainly caused by accumulation of P680+PheoQ
A and
P680+PheoQA states in the model and not by
+
P680+PheoQ
A and P680 Pheo QA states (data not
shown). Therefore, if we take accumulation of all
P680+Pheo states in the model again as a measure of
3
P680 accumulation (see above), the results in Fig. 4
suggest that photoinhibition of PSII is more likely to be
caused by accumulation of P680+ than by accumulation
of 3P680. This finding is in agreement with the statement
by Anderson et al. (1998) who suggested in the
framework of their unifying model of PSII photoinhibition that damage of PSII in vivo occurs rather via
generation of P680+ than via generation of singlet
oxygen caused by the 3P680 formation.
5. Discussion
Although theoretical simulations presented above are
based on a model with hypothetical function of cyt b559,
we present below experimental data, which might be used
as a support for the correctness of our model. Fig. 5
shows typical FLR curves measured with pea thylakoid
membranes at a high intensity of exciting red light
(12,000 mmol photons m2 s1) in the presence of DCMU
(20 mM) alone, DCMU with Asc (5 mM), and DCMU
with FeCy (20 mM). Considering redox changes of
electron carriers in PSII, the addition of Asc or FeCy in
the used concentrations before the FLR measurements
should lead to reduction or oxidation, respectively, of cyt
b559 only. Therefore, the experimental DCMU-FLR
curves can be directly compared with theoretical FRs
simulated for different initial redox state of cyt b559 as
shown in Fig. 1. When the FM level determined from the
experimental DCMU-FRs is plotted in dependence on
the amount of initially reduced cyt b559 (the inset in
Fig. 5. Typical FLR curves measured at room temperature upon red
light (l ¼ 650 nm) excitation of an intensity of 12,000 mmol photons m2 s1 with pea thylakoid membranes in the presence of DCMU.
The thylakoid membranes were also treated with Asc (the
DCMU+Asc curve) or with FeCy (the DCMU+FeCy curve) before
the measurement to initially reduce or oxidize cyt b559, respectively.
The inset shows the dependence of FM of the measured FLR curves
(FM of the DCMU+Asc curve is set as 100%) on the initial redox state
of cyt b559 determined spectroscopically (the bars indicate standard
deviations).
Fig. 5) and compared with the same plot determined
from theoretical FRs (the inset B in Fig. 1), there is an
almost numerical agreement between the simulated and
experimental changes in the FM levels.
However, it can be argued that the Asc and FeCy
treatment could also lead to processes, which are different
to changes in the initial redox state of cyt b559. For
example in the case of the FM level, the Asc and FeCy
driven changes in the redox state of the PQ pool leading
to different fluorescence quenching by the oxidized PQ
pool (Vernotte et al., 1979; Hsu and Lee, 1995; Kramer
et al., 1995; Kurreck et al., 2000; Pospı́šil and Dau, 2000;
Koblı́žek et al., 2001) could be mentioned. In this case,
however, the strong exciting light employed in our
experiments also excites photosystem I, which in
DCMU-treated thylakoids should effectively oxidize the
PQ pool. Therefore, the changes in the FM level caused by
Asc or FeCy treatment are probably not caused by a
different PQ-based fluorescence quenching. Further, it
can be argued that the FM level could be under the direct
control of QA/Asc and QA/FeCy redox pairs during the
FLR measurement. This suggestion also seems to be
improbable as we used in all the experiments a very high
intensity of exciting red light (12,000 mmol photons m2 s1; l ¼ 650 nm) which is thought to be saturating
for Q
A reduction even for samples untreated with
DCMU. Therefore, the different FM levels presented in
Fig. 5 are probably caused by a different Q
A accumulation which is driven by Asc- or FeCy-induced difference
in the initial redox state of cyt b559 as it also implies from
our theoretical simulations shown in Fig. 2.
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The question arises as to which form of cyt b559 can
accept electrons from Pheo and donate them to P680+
as it is assumed in our model. Here we can use again the
experimental results of Fig. 5 but also facts know from
the literature for a help. As we found that in our
thylakoid membranes treated with DCMU, both the HP
and LP forms of cyt b559 were present (60.679.8% of
reduced HP form, 24.673.1% of oxidized HP form, and
14.778.9% of oxidized LP form; mean7standard
deviation), both the forms could be involved in the
observed changes in the FM level. As the LP form,
however, of cyt b559 was present only in the oxidized
state, it should not be involved in the change of the FM
level caused by FeCy treatment. On the other hand,
both the HP and LP forms of cyt b559 could be involved
in the changes in the FM level caused by Asc treatment.
Therefore, from the experiments, cyt b559, which can
accept electrons from Pheo and donate them to P680+
seems to be the HP form. Further, to protect PSII
against damage with respect to the unifying model of
photoinhibition (see Introduction), cyt b559, which
accepts electrons from Pheo and donates electrons to
P680+ would provide the best protection. This statement is in line with the suggestion that cyt b559
participates in cyclic electron transfer around PSII
(Heber et al., 1979; Falkowski et al., 1986; Arnon and
Tang, 1988; Prasil et al., 1996). Moreover, cyt b559,
which accepts electrons from Pheo and donates
electrons to P680+ was suggested to have taken part
in electron transfer reactions in PSII during photoactivation of PSII to protect it against photoinhibition
(Magnuson et al., 1999). This cyt should be the HP form
as the HP form is restored from the LP form during the
photoactivation of PSII (Mizusawa et al., 1995, 1997).
With respect to a high rate of electron transfer
from Pheo to cyt b559 assumed in our model (kCred; see
Table 1) describing the case when DCMU is present, one
can argue that when DCMU is not present, assumed
high rate of electron transfer from Pheo to cyt b559 can
result in a decrease of quantum yield of PSII photochemistry and therefore in a decrease of overall rate of
linear electron transport because electrons flow to cyt
b559 instead of QA and further. But it seems, it is not
true: using a model of Lazár (2003) that completely
describes electron transfer reactions in PSII extended by
the cyt b559 reactions described in this work, we found
(data not shown) that only the height of J step decreases
with increasing amount of initially oxidized cyt b559 but
F0 and FM are always the same in all three cases when
cyt b559 is either initially fully reduced or fully oxidized
or not considered at all in the model. As F0 and FM are
not changed, the maximal quantum yield of PSII
photochemistry expressed by FV/FM ratio (=(FM–F0)/
FM; Kitajma and Butler, 1975) and therefore also the
overall rate of linear electron transport are not
decreased by electron flow to cyt b559. Therefore, high
297
rate of electron flow from Pheo to cyt b559 assumed in
this work does not anyhow decrease functionality of
PSII when DCMU is not present.
Simulations also revealed that insensitivity of F0 and
FM to no consideration of cyt b559 at all in the model of
Lazár (2003) or to the initial redox state of cyt b559 in
this model is caused (data not shown) by a small rate of
electron transfer from reduced cyt b559 to P680+ (kCox;
see Table 1). In other words, even if electrons are
transferred fast from Pheo to cyt b559, the small rate of
electron transfer from reduced cyt b559 to P680+ results
in only a small electron flux through cyt b559 leading to
no effect on electron flux from Pheo to QA and further
in the case when DCMU is not present. Therefore, the
fast electron transfer from Pheo to cyt b559 assumed in
this work seems to have no effect on quantum yield of
PSII photochemistry when DCMU is not present but it
can protect PSII against photoinhibition in the case
when PSII behaves like if DCMU is present (see also the
Consequences for PSII photoinhibition section).
6. Conclusions
We extended the RRP model by cyt b559, which
accepts electrons from Pheo and donates electrons to
P680+. Our theoretical simulations revealed that an
increased amount of initially reduced cyt b559 leads to an
increase in the FM level. MCA of our model revealed
that the initial redox state of cyt b559 strongly affects the
extent in which particular reactions in the model control
the FM level. The simulations also demonstrated that
increasing the amount of initially reduced cyt b559 can
protect PSII against its degradation with respect to the
donor and acceptor side photoinhibitions and also with
respect to the unifying model of photoinhibition of PSII.
Even if we assumed a high rate of electron transfer from
Pheo to cyt b559, it seems, it has no effect on the
quantum yield of PSII photochemistry of fully functional PSII (when electron transfer from Q
A to further
electron acceptors is not disabled, e.g. by long lasting
high light exposure or DCMU application) but it can
ensure protection of PSII against photoinhibition when
electron transfer from Q
A to further electron acceptors
is disabled (i.e. when PSII behaves like when DCMU is
present). Therefore, this work contributed to a better
understanding of the ‘‘enigmatic’’ cyt b559 function and
also showed the power of theoretical simulations and
their benefits for further research.
Acknowledgements
This work was supported by the Ministry of Education of the Czech Republic (Grant No. MSM
153100010) and by a project cofinanced by a Centre of
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Excellence grant from the European Union (contract no.
BIER ICA1-CT-2000-70012) and a Polish Committee
for Scientific Research Grant (No. 158/E-338/SPUB-M/
5 PR UE/DZ 9/2001-2003). D.L. would like to thank
the Grant Agency of the Czech Republic for their
financial support (Grant No. 204/02/P071).
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