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org/pccp | Physical Chemistry Chemical Physics

Characterization of the water oxidizing complex of photosystem II of the

Chl d-containing cyanobacterium Acaryochloris marina via its reactivity
towards endogenous electron donors and acceptors
Dmitriy Shevela,a Birgit Nöring,a Hann-Jörg Eckert,b Johannes Messinger*a and
Gernot Renger*b
Received 27th March 2006, Accepted 2nd June 2006
First published as an Advance Article on the web 20th June 2006
DOI: 10.1039/b604389e

Acaroychloris (A.) marina is a unique oxygen evolving organism that contains a large amount of
chlorophyll d (Chl d) and only very few Chl a molecules. This feature raises questions on the
nature of the photoactive pigment, which supports light-induced oxidative water splitting in
Photosystem II (PS II). In this study, flash-induced oxygen evolution patterns (FIOPs) were
measured to address the question whether the Si state transition probabilities and/or the redox-
potentials of the water oxidizing complex (WOC) in its different Si states are altered in A. marina
cells compared to that of spinach thylakoids. The analysis of the obtained data within the
framework of different versions of the Kok model reveals that in light activated A. marina cells
the miss probability is similar compared to spinach thylakoids. This finding indicates that the
redox-potentials and kinetics within the WOC, of the reaction center (P680) and of YZ are
virtually the same for both organisms. This conclusion is strongly supported by lifetime
measurements of the S2 and S3 states. Virtually identical time constants were obtained for the
slow phase of deactivation. Kinetic differences in the fast phase of S2 and S3 decay between A.
marina cells and spinach thylakoids reflect a shift of the Em of YD/Yox D to lower values in the
former compared to the latter organisms, as revealed by the observation of an opposite change in
the kinetics of S0 oxidation to S1 by Yox
D . A slightly increased double hit probability in A. marina
cells is indicative of a faster QA
to QB electron transfer in these cells compared to spinach
thylakoids.

Introduction Oxidative water splitting takes place via a sequence of four

The essential steps of photosynthetic water-splitting take place
in a multimeric pigment protein complex of more than 20
subunits,1 referred to as photosystem II (PS II). This complex,
which acts as waterplastoquinone-oxidoreductase, is anisotro-
pically incorporated into the thylakoid membrane.2,3 The
overall process comprises three types of reaction sequences:
(a) light-induced charge separation leading to the stabilized
 
radical ion pair P6801 QA
4; (b) oxidative water-splitting to

molecular oxygen and four protons, with P6801 as the driving

force; and (c) plastoquinol (PQH2) formation with QA
as
reductant. Among these reactions, the formation of the

strongly oxidizing cation radical P6801 and the oxidative
water splitting are unique for oxygenic photosynthesis. The

cation radical P6801 is one of the most oxidizing species in
redox steps (Kok cycle, see ref. 5) at the catalytic Mn4OxCa
cluster of the water oxidizing complex (WOC). The WOC is

functionally coupled to P6801 via the redox active tyrosine
residue YZ (for reviews see ref. 6–10).
In PS II, the cofactors of light-induced charge separation
are chlorophyll a (Chl a), pheophytin a (Pheo a) and a special
noncovalently bound plastoquinone molecule QA.4 Recently, a
cyanobacterium, Acaryochloris (A.) marina, was found that
contains a large fraction of Chl d and only a few Chl a
molecules.11–15 In this organism, the photoactive pigment of
PS I was shown to be a Chl d–Chl d 0 dimer. This pigment
exhibits a peak of 740 nm in the reduced minus oxidized
difference spectrum and is therefore denoted P740. The mid-

point potential of P740/P7401 was found to be þ335 mV.16
This value is about 100 mV lower than the Em of P700/P7001


biological systems, characterized by a midpoint potential of
about þ1.25 V, which is achieved by a special protein en-
vironment.4
a
Max-Planck-Institut für Bioanorganische Chemie, Stiftstrasse 32-34,
D-45470, Mülheim an der Ruhr, Germany. E-mail: messinger@mpi-
muelheim.mpg.de; Fax: þ49 208 306 3951; Tel: þ49 208 306 3865
b
Max-Volmer-Laboratorium der TU Berlin, Strasse des 17. Juni 135,
D-10623, Berlin, Germany. E-mail: rengsbbc@mailbox.tu-berlin.de;
Fax: þ49 30 31421122; Tel: þ49 30 314 22794
of the usual Chl a containing PS I, which varies between þ420
mV and þ470 mV for different organisms.17,18 The nature of
the photoactive pigment of PS II in A. marina is a matter of
controversy. If one assumes that replacement of Chl a by Chl d
would lead to an analogous downshift of Em, the delicate
energetic balance of oxidative water cleavage could be ser-
iously affected. Therefore, based on the observation of a long-
lived 15 ns-fluorescence component at 685 nm, Chl a was
assumed to be the constituent of the photoactive pigment of

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PS II.12,19,20 This idea is in line with a study on electron
transfer kinetics of the PS II donor side of A. marina, but
the results cannot rule out the alternative of Chl d forming the
photoactive pigment complex.21 Furthermore, a recent study
of the fluorescence dynamics in A. marina indicates that the
long-lived 685 nm fluorescence cannot be assigned to a delayed
Chl a fluorescence, and therefore cannot be used as an argu-
ment for the hypothesis that the primary donor of PS II
contains Chl a.22 In the case of Chl d being the photoactive
pigment of PS II, the protein environment should be changed
to establish a sufficiently high Em value.
Apart from the particular problem of identifying the photo-
active pigment and characterizing its redox properties, questions
arise whether or not the properties of the WOC in A. marina are
modified in comparison to the Chl a containing organisms.
Important information on the properties of photosynthetic
water oxidation can be gathered from flash-induced oxygen
evolution patterns (FIOPs). In these experiments, the samples
are excited with a train of single turnover flashes. The FIOPs of
dark-adapted samples are characterized by a pronounced max-
imum of oxygen yield induced by the 3rd flash of the sequence
due to the dominant population of S1.23,24 The damping of this
pattern originates from the intrinsic probability of misses a (see
ref. 25 and references therein) and double hits b. The b para-

meter depends on the kinetics of the QA
oxidation26 and the
10,25
flash profile. The magnitude of the a-value is expected to
depend on redox equilibria of the donor and acceptor side of

PS II.27–29 Furthermore, the kinetics of P6801 reduction by YZ
30
determines the value of a. As a consequence, the parameter a
depends on the redox state Si of the WOC. However, the precise
dependence of the a- and b-values on the Si states is difficult to
measure and cannot be gathered from the FIOPs in a straight-
forward manner.
In a recent report, the properties of the WOCs of thylakoids
from the thermophilic cyanobacterium Thermosynechococcus
(T.) elongatus and spinach thylakoids were compared and
significant differences were observed in the kinetics of the
reactions of S0, S2 and S3 with the redox couple YD/Yox D and
other endogenous acceptors.31
The present study describes an investigation based on FIOPs
measurements performed in whole cells of A. marina and thyla-
koids from spinach in order to unravel possible peculiarities of
the WOC in the Chl d-containing organism. The results obtained
reveal that the WOCs in both sample types are very similar, but
exhibit a pronounced difference in the kinetics of S0 oxidation
and S2/S3 reduction by Yox D and YD, respectively. The implica-
tions of these findings are discussed.
Materials and methods
Sample preparation
The cyanobacterium A. marina was grown as described in ref.
15 in artificial sea water32 at 28 1C under an illumination
intensity of 20 mE m
2 s
1 (Osram TLD 18W-25 fluorescent
tubes) and continuous aeration. The cells were harvested 1–2
days before the measurements and transferred into the growth
medium containing 2.5 mM of bicarbonate (pH 7.4). Before
the measurements, the A. marina cells were diluted to about
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c the Owner Societies 2006
0.4 mg Chl ml
1 and dark-adapted. Spinach thylakoids were
isolated from market spinach as previously described.33,34
After the final isolation step, thylakoids were resuspended to
a chlorophyll concentration of about 8 mg ml
1. Samples were
frozen in small aliquots in liquid nitrogen and then stored at

70 1C until used. Before the measurements, the thylakoid
beads were thawed in the dark on ice and diluted to [Chl] =
1 mg ml
1 with MCMH buffer (400 mM mannitol, 20 mM
CaCl2, 10 mM MgCl2, and 50 mM HEPES/NaOH at pH 7.4).
Measurements of flash-induced oxygen evolution patterns
(FIOPs)
The FIOPs of A. marina cells and spinach thylakoids were
obtained in the absence of exogenous electron acceptors with a
home-built Joliot type (bare platinum) electrode35,36 that
keeps the temperature of the electrode constant within
0.3 1C. The measurements of this study were performed at
an electrode temperature of 20 1C. Sample aliquots of 10 mL
were transferred to the electrode in very dim green light. The
polarization voltage of
750 mV was switched on 30 s before
excitation with a train (2 Hz) of saturating xenon flashes
(EG&G, model PS 302, light pack FY-604). The amplified
signals were recorded with a personal computer.
If not stated otherwise, light-activation of A. marina cells
was performed by exposing them for 30 min to room light.
Thereafter, they were dark-adapted at room temperature and
used for several hours. S1Yox D samples of A. marina were
obtained from light-activated cells by one saturating preflash
directly on the Joliot electrode, followed by 10 min dark-
adaptation. A similar treatment (without light-activation) was
used to populate the S1YoxD state in spinach thylakoids.
For S2 and S3 lifetime measurements, spinach thylakoids
and activated A. marina cells were first sedimented on the
Joliot electrode for 1 min. Afterwards, the samples were
illuminated by one (S2) or two (S3) preflashes followed, after
dark-incubation times of 0.5 to 180 s, by a train of 20 flashes.
For measurements of the kinetics of S0 oxidation to S1 by
Yox ox
D spinach thylakoids and light activated S1YD A. marina,
ox
cells enriched in the S1YD state were preflashed on the Joliot
electrode with three flashes and, after dark-times varying
between 0.5 to 60 min, FIOPs were recorded.
Analysis of FIOPs
The O2 yields of the first 16 flashes were analyzed using an
Excel spreadsheet program based on an extended Kok model,
which includes (i) the reduced S
1 state, (ii) an activity
parameter d, that compensates for changes in the number of
active PS II centers during the flash train and (iii) the reactions
of the S2 and S3 states with YD.31,34
In addition, for some fits we considered the possibility of Si
state dependent misses:
2 3 2 3 2 3
½S
1 n a
1 0 0 0 0 ½S
1 n
1
6 ½S0 n 7 6 g
1 g3 7 6 7
6 7 6 a0 0 b 7 6 ½S0 n
1 7
6 ½S1  7 ¼ 6 b g0 a1 0 b7  6 ½S1  7
n
1 7 d ð1Þ
6 n 7 6 7 6
4 ½S2  5 4 0 b g1 a2 0 5 4 ½S2 n
1 5
n
½S3 n 0 0 b g2 a3 ½S3 n
1
where, for example, g
1 = 1 – a
1
b.
Phys. Chem. Chem. Phys., 2006, 8, 3460–3466 | 3461
 The O2-yield of the nth flash, Ynfit, was calculated by
eqn (2):
Yfit
n = (1
a3)[S3]n
1 þ b[S2]n
1 (2)
The program is minimizing the expression
" , !#2
XF X
F XF
2 exp fit exp fit
dyn ¼ Yn
Yn Yn Yn ð3Þ
n¼1 n¼1 n¼1

where, Yexp
n is the relative O2 yield of the nth flash and F equals
the number of analyzed flashes (F = 16 in this study). The
normalization is given by
X
3
½Si  ¼ 1 ð4Þ
i¼
1

The fit quality is calculated according to eqn (5):

dy2n
fq ¼ ð5Þ
F
P
where P is the number of free parameters used in the fit.
The Si state lifetime findings were analyzed taking into
account the fast reductions of S2 and S3 by YD between flashes
(see ref. 31).

Rate constant and half-time calculations

The rate constants for the fast (kfi ) and slow (ksi ) S2 and S3
decay were calculated using eqn (6):
f s
Si ðtÞ ¼ Ai e
ki t þ Bi e
ki t ð6Þ
where i = 2 or 3, and t is the dark-time between the last
preflash and the first flash of the detecting flash train. Ai and Bi
are the relative amplitudes of fast and slow decay, respectively.
For the reoxidation of S0 to S1 by Yox D , a simple one
exponential decay was assumed:
S0 ðtÞ ¼ S0 ðt ¼ 0Þ e
k0 t ð7Þ
Half-times were calculated according to
t1/2 = (ln 2)/k (8)
Results
Flash-induced oxygen oscillation patterns (FIOPs)
Fig. 1 shows FIOPs measured in whole cells of A. marina
either after long dark-adaptation (1–2 days; trace a) or after
light activation (30 min room light) followed by 10 min dark-
incubation and preflashing (one flash plus 10 min dark-adap-
tation; trace b). Trace c shows for comparison a FIOP of
spinach thylakoids that were pre-illuminated by one flash and
subsequently 10 min dark-incubated. Inspection of this data
shows that the oscillation patterns of light activated A. marina
cells and of thylakoids are quite similar and very pronounced,
whereas A. marina cells after long dark-adaptation exhibit a
markedly different pattern of oxygen evolution. Under these
conditions, the first O2 yield is measured after the 3rd flash and
thereafter the yield increases from flash to flash with virtually
no oscillation. This ‘photo-activation’ is reminiscent of similar
Fig. 1 Original traces of the FIOPs of A. marina cells (a, b) and
spinach thylakoids (c) at 20 1C and pH 7.4. Trace a was obtained with
long-term dark-adapted (2 days) A. marina cells, while trace b was
recorded from the same batch of cells after light activation (30 min
room light plus 10 min dark-adaptation) and preflashing into the
S1Yox
D state (one flash plus 10 min dark-adaptation). Trace c depicts a
FIOP obtained with S1Yox D thylakoids of spinach. No exogenous
electron acceptors were added. The flash frequency was 2 Hz. Chloro-
phyll concentrations of A. marina cells and spinach thylakoids during
measurements were 0.4 mg ml
1 and 1.0 mg ml
1, respectively.
features observed in several Synechocystis PCC 6803 mutants,
where in certain regions the large lumal loop E of CP47 was
truncated by deletions of 4–8 amino acids. These mutants were
shown to reach an inactivated state of the WOC and full
oxygen evolution could be achieved only after illumination
with numerous flashes.37,38 In the case of A. marina cells, only
a few flashes (B20) are required to obtain fully active systems.
Therefore, the underlying mechanism seems to be different and
may be related to a reduced plastoquinone pool in long-term
dark-adapted samples that is oxidized during the light-activa-
tion procedure.
Light-activated and than preflashed (S1Yox D ) A. marina cells
exhibit the conventional four period oscillation (similar to that
obtained in the presence of exogenous oxidants in ref. 39) that
is characteristic for a functionally fully competent WOC (Fig.
1b). A comparison to the corresponding patterns of thylakoids
(Fig. 1c), PS II membrane fragments and Inside-Out (ISO)
vesicles from spinach reveals that the PQ-pool of A. marina
cells is of virtually the same capacity as in the thylakoids26,28
from higher plants and samples reconstituted with PQ.40 In

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Table 1 Fits of the flash-induced oxygen evolution patterns (FIOPs) of preflashed S1Yox D Acaryochloris marina cells and spinach thylakoids. The
FIOPs were obtained at 20 1C and pH 7.4 (Fig. 1b and c) and fit using different approaches. For fits A–C an extended Kok model (for details, see
Materials and Methods) with Si state-independent miss and double-hit parameters was used. In fits D1–D4 the miss parameters of the S2 - S3 and
S3 - S0 transitions (a23 and a30, respectively) were used as free parameters; in fits E1-E4 they were forced to be equal, while those for the S0 - S1
and S1 - S2 transitions (a01 and a12, respectively) were fixed to 0. Dashes indicate that parameters were excluded from the fit, while stars mark
values that were fixed during the optimization. The first 16 flash-induced oxygen yields of each FIOP were analyzed. The fit quality (fq) values were
calculated by using eqn (5)

Fit parameters (%)

Sample Fit a a23 a30 b S2 S1 S0 S
1 YD fq  10
6
Acaryochloris marina A1 14.6 – – 3.3 – 100* – – 0* 7.2
B1 13.1 – – 3.2 – 91.1 8.9 – 0* 2.3
C1 13.0 – – 3.0 0.3 89.9 8.2 1.6 0* 2.4
D1 – 26.1 21.1 2.5 – 100* – – 0* 1.6
E1 – 23.7 23.7 2.5 – 100* – – 0* 1.5

A2 14.3 – – 3.3 – 100* – – 10* 6.7

B2 12.8 – – 3.2 – 90.9 9.1 – 10* 2.2
C2 12.7 – – 3.0 0.3 89.9 8.2 1.6 10* 2.3
D2 – 25.7 20.7 2.5 – 100* – – 10* 1.5
E2 – 23.2 23.2 2.5 – 100* – – 10* 1.6

Spinach thylakoids A3 12.3 – – 2.4 – 100* – – 0* 7.0

B3 12.1 – – 2.4 – 98.6 1.4 – 0* 8.1
C3 11.9 – – 2.1 0.0 96.4 0.3 3.3 0* 7.5
D3 – 5.9 33.1 1.6 – 100* – – 0* 9.1
E3 – 19.1 19.1 1.9 – 100* – – 0* 10.2

A4 11.6 – – 2.3 – 100* – – 10* 6.9

B4 11.6 – – 2.3 – 100 0.0 – 10* 8.1
C4 11.3 – – 2.1 0.0 97.2 0.0 2.8 10* 8.0
D4 – 19.1 19.1 1.9 – 100* – – 10* 11.7
E4 – 3.8 32.9 1.6 – 100* – – 10* 10.7

order to obtain more detailed information on the properties of
the WOC of A. marina, FIOPs of both sample types (trace b
and c in Fig. 1) were fit by different versions of the Kok model
(see Materials and Methods). In the most simple and widely
used fit procedure, the values of misses (a) and double hits (b)
were set to be equal in each flash-induced Si state transition,
i.e. independent of the Si state and acceptor site redox equili-
bria (Table 1, fit approaches A–C). In a more elaborate
approach, the a-value depends on the Si state. In this case,
for the sake of simplicity, the a-values for the transitions Yox
Z S0
- YZS1 and Yox Z S1 - YZS2 are assumed to be negligibly small
(a01 = a12 = 0) and those of Yox Z S2 - YZS3 and YZ S3 -
ox
1
YZS0 þ O2 þ nH were free running parameters and either
independent (approach D) or fixed to be equal, i.e. a23 = a30
(approach E).31 Based on the obtained fit qualities, the oscilla-
tion patterns of both samples can be satisfactorily described by
all these approaches (see Table 1). With the most simple
approach, A, the FIOPs of both sample types can be fit equally
well. These fits reveal that the miss parameter is only slightly
lower for spinach thylakoids (fit A3; a = 12.3%) compared to
whole cells of A. marina (fit A1; a = 14.6%). In addition, the
double hit parameter is slightly smaller (2.4%) in spinach
compared to A. marina (3.3%). Both factors together account
for the visually lower damping in the FIOP of spinach (Fig. 1c)
compared to A. marina (Fig. 1b). For spinach thylakoids, no
improvement of the fit quality was obtained with the more
elaborate fit approaches (B–E). In contrast, for A. marina cells
the best fit quality was obtained with a23 = a30 E 23% (fit
E1). As expected for well preflashed samples, the inclusion of
10% YD in the fits did not improve the fit quality (fits A2–E2
and A4–E4). In spite of the differences described above, the
values obtained for a, b and S1 population can generally be
considered to be similar for A. marina cells and spinach
thylakoids.
Decay rates of S2 and S3
The kinetics of the S2 and S3 decay were determinated by
preillumination of the samples with one and two single turn-
over flashes, respectively, and monitoring the FIOPs at differ-
ent times after the preflash(es), as described in ref. 31 and 40.
Fig. 2 shows the calculated values of the S2(t) and S3(t)
population as a function of dark-time t between the preflashes
and the flash sequence for FIOPs detection for whole A.
marina cells (closed symbols) and spinach thylakoids (open
symbols). Both sample types exhibit the characteristic biphasic
behaviour of the decay kinetics, which originates from the
reduction of S2 and S3 by YD acting as electron donor (fast
kinetics) and by the acceptor side (slow kinetics). At first
glance, the data of Fig. 2 reveal that the decay kinetics of
both S2 and S3 are slower for A. marina cells than for spinach
thylakoids.
The numerical fit by biexponential kinetics leads to the
results summarized in Table 2. A comparison of these numbers
leads to the interesting finding that the difference between A.
marina cells and spinach thylakoids is entirely related to the
reactivity with YD. The half-lifetimes of the fast kinetics of A.
marina are larger by a factor of 2.5–3 for both S2 and S3, while
the rates for the slow S2/S3 decay are virtually identical for
both organisms.

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 Fig. 3 The kinetics of S0 state oxidation by Yox
D as a function of dark-
time between three preflashes and the main train of saturating single
turnover flashes for activated A. marina cells (closed symbols) and
spinach thylakoids (open symbols). The lines represent monoex-
ponential fits (Table 2). The measurements were performed at 20 1C
and pH 7.4.

koids. A fit of the data points to monoexponential kinetics

Fig. 2 The kinetics of S2 (A) and S3 (B) state decay as a function of
dark-time between the preflashe(s) (one or two, respectively) and the
main flash train for activated A. marina cells (closed symbols) and
spinach thylakoids (open symbols). The lines represent biexponential
fits (Table 2). The lifetime measurements were performed at 20 1C
and pH 7.4.
S0 oxidation by Yox
D
After repetitive excitation of PS II, the WOC attains a
normalized steady state population of [S0] = [S1] = [S2] =
[S3] = 0.25. In a following dark period, S2 and S3 relax via
reduction into the S1 state while S0 becomes slowly oxidized to
S1 by Yox
D (for review see ref. 41). The kinetics of S0 oxidation
can be best studied by giving three flashes to activated (in case
of A. marina) and preflashed samples and then varying the
dark-time to the flash train between 1–60 min.24 The results
obtained for A. marina cells (filled circles) and spinach
thylakoids (open circles) are depicted in Fig. 3. Inspection
of this data shows that the oxidation of S0 by Yox
D is
significantly faster in A. marina compared to spinach thyla-
leads to the values shown in Table 2. A comparison of these
numbers reveals that the reaction is faster by a factor of
almost five in A. marina.
Discussion
The results obtained in this study lead to two conclusions:
(a) the WOC of the Chl d-containing A. marina closely
resembles that of Chl a containing cyanobacteria and higher
plants, and (b) the only significant difference of the WOC
properties between A. marina and spinach thylakoids is
the interaction of the states S2/S3 and S0 with the redox
couple YD/Yox D.
The first conclusion is based on the numerical analysis of the
FIOPs, which reveals that the misses (a), double hits (b) and
the dark-state population of redox state S1 are similar for both
species. A similarity of the a-values is indicative for (i) similar
kinetics of Yox
Z formation by the cation radical of the photo-
active pigment and (ii) similar redox-potential differences

between P680/P6801 , YZ/Yox Z and the Si states. This finding

of a practically unchanged redox-potential of P6801 does,
however, not permit any straightforward conclusions on the

Table 2 Rate constants (k), half-times (t1/2) and amplitudes (% of total decay) for S0 state oxidation by Yox
D and for the S2 and S3 state reduction
by YD (fast phase) and the acceptor side (slow phase) for A. marina cells and spinach thylakoids at 20 1C and pH 7.4

A. marina cells Spinach thylakoids

1
k/min t1/2/s k/min
1 t1/2/s
S0 0.330 125 0.074 580

Slow phase Fast phase Slow phase Fast phase

1
1
1
k/s t1/2/s % k/s t1/2/s % k/s t1/2/s % k/s
1 t1/2/s %
S2 0.0079 88 49 0.13 5.5 51 0.0080 87 20 0.35 2.0 80
S3 0.0068 102 30 0.13 5.2 70 0.0068 102 14 0.42 1.7 86

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chemical nature of the primary donor (Chl a and/or Chl d
content), since changes in the protein environment that tune
Chl d molecules to the same potential cannot be excluded.
The b-values of A. marina are somewhat higher than those
of spinach thylakoids (see Table 1). It was recently shown that

the extent of b is linearly related to the rate of QA
reoxida-
tion (see appendix of ref. 26). Therefore, it can be concluded

that the reoxidation of QA
is slightly faster in A. marina than
in spinach thylakoids. A similar phenomenon has been found
recently for the Chl a-containing thermophilic cyanobacterium
T. elongatus.31 Based on this finding, it is concluded that a
 
slightly faster electron transfer from QA
to QB (QB
) is
likely to be a property of cyanobacteria rather than due to
replacement of Chl a by Chl d in PS II of A. marina.
The most interesting finding of our study is the observation
of changes in the rates of S2 and S3 reduction by YD and of S0
oxidation by Yox
D . A retardation of the former reactions by
factors of 2.5–3 and an acceleration of the latter reaction by a
factor of almost 5 can be explained in a straightforward
manner by the assumption of a small Em shift of the redox
couple YD/Yox
D relative to the values of S0/S1, and in the
opposite direction relative to Em of S1/S2 and S2/S3. Based
on the findings that the kinetics of the slow S2 and S3 decay are
the same in A. marina cells and spinach thylakoids, it appears
most reasonable to assume that the energetics of the redox
transitions in the WOC are virtually identical in both organ-
isms, i.e. the slight Em shift relates to YD/Yox D . This idea is
strongly supported by a recent comparative study on T.
elongatus and spinach thylakoids. It was shown that, in this
case, the S2 and S3 reduction by YD was faster by a factor of
2–3 in T. elongatus compared with spinach thylakoid, while
the S0 oxidation by Yox D was slower by a factor of 4 in the
former organism. These are just the opposite shifts to that
observed for A. marina cells versus spinach thylakoids. The
phenomenon per se can be explained by the same mechanism,
i.e. Em shift of YD/YoxD . However, in T. elongatus this Em shift
is opposite in direction.
These findings also show that the phenomenon of an Em
(YD/Yox
D ) shift is not a unique property of Chl d-containing
cyanobacteria, but appears to be a common feature also
among Chl a-containing PS II. As a consequence, it is
concluded that the WOC in the Chl d-containing A. marina
is the same as in all the other oxygen evolving organisms.
Therefore, the replacement of a large number of Chl a by
Chl d in PS II has virtually no effect on the properties of
the WOC.
This observation, however, is restricted to the WOC and
cannot offer any straightforward conclusion on the nature of
the photoactive pigment of PS II. All considerations on this
point should take into account that in Chl a-containing
organisms, P680 is a multimeric Chla4 Pheox complex (x =
0, 1 or 2) as discussed in ref. 4. It is therefore difficult to assign
the possible presence of only two Chl a molecules in PS II13 to
a definite structure of the photoactive pigment.
Acknowledgements
The authors would like to thank M. Weß for excellent
technical work in cultivating the A. marina cells. They also
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gratefully acknowledge the financial support by the DFG (Sfb
429, TP A1 and Me 1629/2-3) and the Max-Planck-
Gesellschaft. DS wishes to thank the DAAD (Deutscher
Akademischer Austausch Dienst) for his fellowship.
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