Photosynth Res (2008) 98:251–260

DOI 10.1007/s11120-008-9364-4

REGULAR PAPER

Effects of methanol on the Si-state transitions in photosynthetic

water-splitting
Birgit Nöring Æ Dmitriy Shevela Æ Gernot Renger Æ
Johannes Messinger

Received: 13 June 2008 / Accepted: 4 September 2008 / Published online: 26 September 2008

The Author(s) 2008. This article is published with open access at Springerlink.com

Abstract From a chemical point of view methanol is one Introduction

of the closest analogues of water. Consistent with this idea
EPR spectroscopy studies have shown that methanol binds In all oxygen-evolving photosynthetic organisms (cyano-
at—or at least very close to—the Mn4OxCa cluster of bacteria, green algae, and higher plants) water-splitting
photosystem II (PSII). In contrast, Clark-type oxygen rate takes place at the l-oxo bridged tetra-manganese calcium
measurements demonstrate that the O2 evolving activity of cluster (Mn4OxCa, with 4 B x B 8) of photosystem II
PSII is surprisingly unaffected by methanol concentrations (PSII). This process is energetically driven by strongly
of up to 10%. Here we study for the first time in detail the oxidizing equivalents that are generated one at a time
effect of methanol on photosynthetic water-splitting by following light absorption in the chlorophyll-containing
employing a Joliot-type bare platinum electrode. We reaction center of PSII (Renger and Holzwarth 2005). In
demonstrate a linear dependence of the miss parameter for this way the Mn4OxCa cluster is oxidized stepwise and
Si state advancement on the methanol concentrations in the cycles through five oxidation states that are referred to as Si
range of 0–10% (v/v). This finding is consistent with the states (Kok et al. 1970). The index (i = 0,…,4) signifies
idea that methanol binds in PSII with similar affinity as the number of stored oxidizing equivalents. After the
water to one or both substrate binding sites at the Mn4OxCa accumulation of four oxidizing equivalents the highly
cluster. The possibility is discussed that the two substrate reactive S4 state is reached, which decays under the release
water molecules bind at different stages of the cycle, one of molecular oxygen and rebinding of new substrate water
during the S4 ? S0 and the other during the S2 ? S3 into the S0 state. After dark-adaptation the predominant
transition. state is the S1 state, since S0, S2, and S3 are meta-stabile
and react to S1 via different pathways (for details see,
Keywords Photosystem II
Manganese cluster
Hillier and Messinger 2005; Messinger and Renger 2008).
Water-splitting
Oxygen evolution
Methanol This kinetic scheme (Kok cycle) explains the periodicity of
four that is observed when dark-adapted thylakoids are
illuminated with a train of saturating single turn-over fla-
shes (Joliot et al. 1969).
B. Nöring
D. Shevela
J. Messinger The Si state turnovers are coupled to miss (a) and double
Max-Planck-Institut für Bioanorganische Chemie, Stiftstrasse hit (b) probabilities. Therefore the period-four oscillation
34-36, 45470 Mülheim an der Ruhr, Germany and damps out within a couple of cycles. The double hit
parameter (probability for Si ? Si?2 transitions) mostly
Present Address:
D. Shevela
J. Messinger (&) originates from double turnovers on the acceptor side and
Department of Chemistry, Umeå University, Umeå, Sweden its magnitude is dependent on the half-width of the
e-mail: [email protected] employed flashes. The miss parameter (probability that no
Si state advancement occurs) maybe affected by all redox-
G. Renger
Max-Volmer Laboratorium für Biophysikalische Chemie, TU equilibria and kinetics within PSII and is therefore a very
Berlin, Strasse des 17. Juni 135, Berlin, Germany sensitive probe for changes within PSII. The magnitude of

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252 Photosynth Res (2008) 98:251–260

a strongly depends on pH and temperature (Messinger and

Renger 1994; Messinger et al. 1993), and is also affected
by H/D isotope exchange (Christen et al. 1999).
Due to recent progress important details are known
about the structure of the Mn4OxCa cluster and its ligands
in the dark-stable S1 state (Ferreira et al. 2004; Loll et al.
2005; Yano et al. 2006) and some information is also
available on the number of substrate water molecules and
their binding modes in the various Si states (Chu et al.
2000; Evans et al. 2004; Hansson et al. 1986; Hillier and
Wydrzynski 2000; Kimura et al. 2005a, b; Messinger et al.
1995; Noguchi et al. 1995; Noguchi and Sugiura 2000,
2002; Radmer and Ollinger 1986; Su et al. 2008; Turconi
et al. 1997). However, due to the limited resolution and to
radiation damage during the collection of the X-ray dif-
fraction pattern (Yano et al. 2005), the substrate binding
sites and the Si state dependent protonation states of the
substrate ‘water’ molecules are still a matter of specula-
tion—a fact that currently diminishes the chances of
deriving the correct mechanism of photosynthetic water-
splitting seriously.
Water is not only the substrate for oxygen evolution, but
it is also likely involved in functionally important H-
Scheme 1 Molecules structurally related to water. Top left to bottom
bonding networks that are required for the structural and right: hydrogensulfate (H2S), methanol (CH3OH), ammonia (NH3),
functional integrity of PSII (Kaminskaya et al. 2003; water (H2O), hydrogenperoxide (H2O2), hydroxylamine (NH2OH),
Noguchi and Sugiura 2002). Therefore, it is not possible to and hydrazine (NH2NH2)
gain direct information on substrate binding by varying the
water concentration. Accordingly, isotope labeling, partly review see, Haddy 2007) cannot be observed in presence of
in connection with time-resolved methods, has been methanol or ethanol, while in PSII membrane fragments
employed to gather information on water-binding sites and from higher plants the S0 multiline signal can only be
modes at the Mn4OxCa cluster (for summary see, Hillier resolved after methanol addition (Messinger et al. 1997a).
and Messinger 2005). An alternative approach is to utilize An ESEEM study employing 2H-labeled methanol clearly
substrate analogs. Prominent examples for molecules showed that methanol (and ethanol) binds at—or at least
structurally related to water are hydrogen sulfide, metha- very close to—the Mn4OxCa cluster (Force et al. 1998).
nol, ammonia, hydrogen peroxide, hydroxylamine, and Consistent with this result it was recently demonstrated by
hydrazine (Scheme 1). molecular mechanics simulations that an access channel
Water and methanol differ structurally by the replace- exists that is big enough in diameter to allow methanol (and
ment of one H-atom by a CH3-group (Scheme 1). While of cause the smaller water) to diffuse from the lumen to the
this exchange affects (i) molecular properties like polarity catalytic Mn4OxCa cluster (Ho and Styring 2008; see, also
and H-bonding capacity, and (ii) bulk properties, such as Murray and Barber 2007).
the boiling point, it leaves one end of the molecule struc- In case of ammonia the oxygen of water is exchanged
turally identical and increases the overall size of the against the isoelectronic NH-group (Scheme 1). Several
molecule only to a relatively small extent. These compar- EPR and FTIR studies suggest that ammonia has one or two
atively conservative differences between methanol and binding sites at the Mn4OxCa cluster (for review see, Debus
water are reflected by the fact that methanol and water are 1992; Hillier and Messinger 2005). At high enough con-
completely mixable at all molar ratios. Because of these centrations ammonia inhibits O2 evolution and alters the S2
properties methanol can be considered to be one of the state EPR multiline signal (Haddy 2007). The possible
closest analogs of water. competition between ammonia and methanol for binding
In previous EPR-based studies it was noticed that most sites at the water-splitting Mn4OxCa cluster of photosystem
EPR signals of the Mn4OxCa cluster are affected by the II has been studied by two groups. Based on FTIR mea-
addition of 1–5% methanol. For example, the S3 state surements Chu and co-workers concluded that methanol is
parallel mode EPR signal (Matsukawa et al. 1999) and the unable to replace ammonia from its binding site at the
g = 4.1 perpendicular mode S2 state EPR signal (for Mn4OxCa cluster (Fang et al. 2005), whereas for the reverse

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Photosynth Res (2008) 98:251–260 253

exchange experiment Evans et al. found employing elec- slight modifications outlined in Messinger and Renger
tron-spin echo envelope modulation (ESEEM)- (1993). After the final isolation step, thylakoids were fro-
spectroscopy for detection that ammonia can displace zen as small aliquots in liquid nitrogen and then stored at -
methanol from its binding site (Evans et al. 2005). These 80C until used. These samples were manipulated as
data indicate that ammonia binds more strongly to the described below to populate the reduced or/and oxidized
Mn4OxCa cluster than methanol, and that these two water form of tyrosine D (YD and YDox, respectively). Before the
analogs have at least one common binding site. measurements of flash-induced oxygen-evolution patterns
The question remains whether one or more of these sites (FIOPs) were performed, the thylakoids were thawed in the
are substrate water binding sites. If that were the case one dark on ice and diluted to [Chl] = 1 mg/ml with medium
would expect that increasing methanol concentrations A (0.4 M sucrose, 15 mM NaCl, 5 mM MgCl2, 5 mM
should affect the efficiency of oxygen-evolution and pos- CaCl2, and 50 mM MES/NaOH at pH 6.0). Methanol was
sibly also oxidation products of these water-analogs maybe added to give final concentrations of 0, 3, 5, 7, and 10% (v/
created due to side reactions. The effect of methanol on the v) about 5 min before the samples were applied onto the
oxygen-evolving activity of PSII has so far only been Joliot-type electrode (see below).
probed by measurements with Clark-type oxygen elec- PSII membrane fragments of ‘BBY’ type were prepared
trodes. In such experiments suspensions of PSII samples from fresh thylakoid membranes according to the method
are illuminated for about 1 min with strong white light in of Berthold et al. (1981) with minor modifications (Ono
the presence of artificial electron acceptors. These kinds of and Inoue 1983).
measurements have been performed by several groups and
consistently did not reveal any inhibitory effects of meth- Preparation of ‘S1YD’- and ‘S1YDox’-thylakoids
anol at concentrations of up to 5–10% (v/v) (Bernat et al.
2002; Deak et al. 1999; Force et al. 1998). This finding (i) S1YD thylakoids contain a high percentage (C80%) of
raises the question as to whether or not methanol really acts the reduced form of tyrosine D (YD). These prepara-
as substrate analog in PSII. However, one other study tions were obtained by long-term (several months)
(Frasch et al. 1988) reports that methanol can be oxidized dark-storage at -80C (Messinger and Renger 1990,
by the Mn4OxCa cluster in the presence of H2O2—a result 1993; Vass et al. 1990b).
consistent with methanol binding at a substrate water-
(ii) S1YDox thylakoids with YD oxidized in about 90% of
binding site.
the centers were obtained by excitation of S1YD
A third group of molecules structurally related to water
samples (pH 6.0; 20C) with one saturating flash and
is formed by hydrogen peroxide, hydroxylamine, and
subsequent 5 min dark-incubation (Messinger and
hydrazine (Scheme 1, lower row). These molecules are,
Renger 1990).
however, not isoelectronic to water. Instead, they are
redox-active and known to reduce stepwise the Mn4OxCa
cluster (see, for example Frasch and Mei 1987; Mano et al. Measurements of flash-induced O2-evolution patterns
1987; Messinger et al. 1991). While it is still possible that (FIOPs)
these molecules react via binding to a substrate water-
binding site at the Mn4OxCa cluster, hydrogen peroxide, FIOPs were obtained in the absence of exogenous elec-
hydrazine, and hydroxylamine do not appear to bind for tron acceptors with an unmodulated home-built Joliot-
long periods of time at the Mn4OxCa cluster. Hydrogen type (bare platinum) electrode (Joliot 1972; Messinger
sulfide has been reported to react in a similar fashion with 1993) at 20C (±0.3C). Sample aliquots of 10 ll were
PSII as hydroxylamine (Sivaraja et al. 1988). transferred to the electrode in very dim green light. Prior
In this study we employ flash-induced oxygen-evolution to starting the measurements thylakoids were kept for
measurements to probe in detail the effects of small (0– 3 min on the electrode at given temperature to ensure
10%, v/v) concentrations of methanol on the efficiency of complete sedimentation and temperature equilibration.
photosynthetic water-splitting. The pH of the flow buffer, which also contained the
indicated methanol concentrations, was adjusted to the
value of 6.0. The polarization voltage of -750 mV was
Materials and methods switched on 40 s before flash excitation. Saturating fla-
shes at a frequency of 2 Hz were provided through a
Sample preparation polished plexi-glass rod by a xenon flash lamp (Perkin
Elmer, LS-1130-4). The amplified amperometric signals
Spinach thylakoids were isolated from leaves of market were recorded with a personal computer at a sampling
spinach as described previously (Winget et al. 1965) with rate of 3 ms/point.

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254 Photosynth Res (2008) 98:251–260

S2/S3 lifetime measurements dy2n

fq ¼ ð6Þ
FP
The S2 and S3 state lifetimes were measured with the Jo-
liot-type electrode. Dark-adapted S1YD-tylakoids were where, P is the number of free parameters used in the fit. In
excited with one (S2 state formation) or two (S3 state for- a more advanced approach, the fit program also includes (i)
mation) pre-flash(es). After various dark times, td (0.5 to the reduced S-1 state and (ii) Si state dependent misses
120 s) the O2-yields induced by flash train were recorded (Isgandarova et al. 2003; Messinger et al. 1997b; Shevela
and deconvoluted into Si state populations (Isgandarova et al. 2006). This extended Kok model is summarized in
et al. 2003; Shevela et al. 2006, 2007). Eq. 7:
2 3 2 3
½S1 n a1 0 0 0 0
6 ½S0 n 7 6 c1;n a0 0 bn c3;n 7
6 7 6 7
Analysis of FIOPs 6 ½S1  7 ¼ 6 bn c0;n a1 0 bn 7
6 n 7 6 7
4 ½S2  5 4 0 bn c1;n a2 0 5
n
The first 16 flashes of each FIOP were analyzed within the ½S3 n 0 3 bn c2;n a3
20
framework of different extended Kok models using Excel ½S1 n1
spreadsheet programs (Kebekus et al. 1995; Messinger 6 ½S0 n1 7
6 7
et al. 1991, 1997b). All fit programs are based on the 6 7
6 ½S1 n1 7  d ð7Þ
formulas: 4 ½S2  5
n1
Sn ¼ KSn1 d ð1Þ ½S3 n1

and where, for example, c1,n = 1 - a1 - bn is the Si state and

flash number (n) dependent single-hit probability. In case
Yfit
n ¼ ð1  aÞ½S3 n1 þb½S2 n1 ð2Þ of Si state dependent misses, for the sake of simplicity, the
where, Sn-1 and Sn are vectors of the Si state populations a-values for the transitions S0 ? S1 and S1 ? S2 were
before and after nth flash of the train, K is the matrix fixed to 0 (a1 = a2 = 0) since they are assumed to be
containing the Kok parameters a (miss probability) and b insignificantly small. The a-values of S2 ? S3 and
(double hit probability), d is an activity parameter that S3 ? S0 were set as free running parameters, but fixed to
compensates for changes in the number of active PSII be equal, i.e., a2 = a3 (Isgandarova et al. 2003; Shevela
centers during the flash train, Yfit et al. 2006). For bn it is assumed that the double hit of the
n is the oxygen yield due to
the nth flash, and [S3]n-1 and [S2]n-1 are the normalized S3 first flash (b1) is greater or equal to the value of b in the
and S2 state populations immediately before the nth flash, remaining flashes, which are assumed to then cause
respectively. In the most commonly used fit procedure, identical double hits; i.e., b1 C b2 = b3…. Accordingly,
misses and double hits are assumed to be Si state the value of Ynfit was calculated using Eq. 8:
independent and the S vectors contain only redox states Yfit
n ¼ ð1  a3 Þ½S3 n1 þb2 ½S2 n1 ð8Þ
from S0 to S3, i.e.
2 3 2 3 The Si state lifetime data were analyzed by further
½S0 n a 0 b c extending the Kok model by also taking into account the
6 ½S1 n 7 6 c a 0 b7
½Sn ¼ 6 7 6
4 ½S2  5 and K ¼ 4 b c a 0 5
7 ð3Þ fast reduction of S2 and S3 by YD that can occur during the
n 500 ms dark-times between flashes of a flash train
½S3 n 0 b c a
(Messinger and Renger 1993; Vass et al. 1990a). The
where, c is the single hit probability (c = 1 - a - b).The required first order rate constants (kf2, kf3) and the percent-
computer program minimizes the expression age of YD in the samples were determined from lifetime
" , !#2 measurements in an iterative process that is described in
X
F XF XF
detail in (Isgandarova et al. 2003).
dy2n ¼ Yexp
n  Yn
fit
Yexp
n Yfit
n ð4Þ
n¼1 n¼1 n¼1
Rate measurements of photosynthetic O2 evolution
where, Yexp
n is the relative O2 yield of the nth flash and F
equals the number of analyzed flashes. The normalization The oxygen-evolving activity of PSII membrane fragments
is given by was measured with a Clark-type oxygen electrode
X
3 (Hansatech) at 25C under continuous actinic illumination
½Si  ¼ 1 ð5Þ (250 W halogen lamp) at the saturating intensity through a
i¼0
Schott GG 455 (2 mm) and Schott KG 3 (2 mm) filters,
The fit quality is calculated according to Eq. 6: 8-cm water filter, and 2-cm CuSO4 solution (5%) as a heat

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Photosynth Res (2008) 98:251–260 255

filter. Measurements were done at 20 lg Chl/ml in 1-ml in Table 1) are visualized for each condition as solid line in
chamber filled with buffer A containing the designated the lower part of Fig. 2 (for comparison the dashed line
MeOH concentrations, and 200 lM PPBQ and 500 lM shows pattern A). Although this fit approach (A1–A3,
K3[Fe(CN)6] as artificial electron acceptors. The electrode Table 1) comprises several simplifying assumptions (see
was calibrated using air-saturated water. ‘‘Materials and methods’’) it still allows a quite good
description of the data (compare solid lines with symbols).
While the double hit parameter is hardly affected by
Results methanol (see also Fig. 3b), the miss parameter increases
significantly from 9.7% in the control to 19.4% at a meth-
Previous reports showed that methanol concentrations up to anol concentration of 10%. Figure 3a (closed symbols)
10% (v/v; 2.5 M) do not affect the oxygen-evolution rates includes the results of several intermediate concentrations
(Bernat et al. 2002; Deak et al. 1999; Force et al. 1998), and shows that the increase in the miss parameter is prac-
while at 20% (v/v; 5 M) a small, reversible decline was tically linearly related to the methanol concentration.
reported (Force et al. 1998). The data in Fig. 1 confirms Importantly, the open symbols in Fig. 3a show that the
this finding for PSII membrane (BBY) preparations under increased miss probability can be practically fully reversed
our experimental conditions. to normal values by washing the samples in methanol free
The top row of Fig. 2 displays original FIOPs obtained buffer A. The enhanced miss probability is therefore not due
with dark-adapted S1YDox thylakoids (spinach) that contain to for example, an irreversible extraction of pigments or
none (A), 3% (B), or 10% (C) methanol. The control sample denaturation.
without methanol (A) exhibits a pronounced period-four A significant improvement of the fit quality can be
oscillation with a maximum at the 3rd, 7th, 11th, and 15th achieved if the initial S1 state population is not fixed to
flash. Inspection of this data readily reveals that with 100% and the states S0 and S-1 are included (fits B1–B3).
increasing methanol concentration this oscillation becomes While in this case the miss parameter is lower, the overall
less pronounced. These raw data also show that the overall trend remains the same. A similar good fit quality can be
oxygen yields (steady state yields) of the thylakoids reached if the miss parameter is assumed to be zero for the
decrease only slightly at higher methanol concentrations. S0 ? S1 and S1 ? S2 state transitions, and the S2 ? S3
To quantify the increased damping caused by the pres- and S3 ? (S4) ? S0 transitions are connected with larger
ence of methanol the FIOPs were analyzed by various miss parameters (fits C1–C3). For simplicity it is assumed
extended Kok models as described in ‘‘Materials and that a23 = a30. Since it is unlikely that the preflashed
methods.’’ The obtained fit parameters are listed in Table 1. (S1YDox) samples contain centers in the S0 and S-1 states,
The fits assuming equal miss probabilities (A1, A2, and A3 we favor fit approach C over B. For fits C1–C3, the values
a23 and a30 are higher than the average miss a in the other
two approaches. This is expected since the overall miss
factor of the cycle is distributed over only two instead of
four Si state transitions (if divided by two a very similar
value is obtained as for A and B). Also with fits C1–C3 a
linear increase of the miss parameter with the methanol
concentration is observed (data not shown in Fig. 3).
The above data were obtained with pre-flashed samples,
in which most centers are in the state S1YDox. Therefore, it
is unlikely that fast S2 or S3 state decay can explain the
observed increase in the miss parameter. However, it
cannot be fully excluded that methanol affects the dark-
stability of the YDox radical, which normally is stable for
several hours at the employed conditions. In such a sce-
nario both the amplitude and the rate of fast S2 or S3 decay,
which are ascribed to the reactions S2YD ? S1YDox and
Fig. 1 Dependence of oxygen-evolving activity on concentrations of
methanol in BBY samples as measured by Clark-type electrode after S3YD ? S2YDox, might be functions of the methanol
2-min incubation of the samples in medium A with different concentration. To test these possibilities S2 and S3 state
concentrations of methanol at pH 6.0 and 25C under continuous lifetime measurements were performed with samples
actinic illumination. The assays were performed in the presence of
enriched in either YD or YDox. The results of the S2/3YD
200 lM PPBQ and 500 lM K3[Fe(CN)6] as electron acceptors. The
rate of oxygen evolution of control samples (0% of methanol) was measurements show that only small changes in the rates of
440 ± 30 lmol O2 (mg Chl)-1 h-1 fast S2/S3 decay can be discerned (Table 2). The

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256 Photosynth Res (2008) 98:251–260

Fig. 2 Normalized FIOPs of spinach thylakoids (S1YDox) measured represent FIOPs prior to the addition of MeOH (the same as in a). The
before (a), after an addition of 3% (b), and 10% (c) of methanol on ice inserts show the original, unnormalized FIOPs that were recorded
at pH 6.0. Methanol was added to the samples about 3–5 min before with a flash-frequency of 2 Hz at pH 6.0 and 20C. No exogenous
FIOP measurements. The measurements were performed without electron acceptors were added. Normalization of the FIOPs was
removal of MeOH from the samples. Solid lines correspond to fits A performed by dividing each flash-induced O2-yield by the O2-yield
of Table 1, while symbols show experimental values of FIOPs induced by 3rd flash in ‘control’ sample (a)
obtained. Broken gray lines with open symbols in (b) and (c)

Table 1 Fits of the FIOPs of preflashed S1YDox spinach thylakoids in the presence of 0% (FIOP A), 3% (FIOP B), and 10% (FIOP C) of
methanol (% v/v) (revealed in Fig. 1)a
Sample Fit parameters (%) fq 9 106
Fit a a23 a30 b S1 S0 S-1

0% MeOH A1 9.7 – – 2.4 100* – – 22.6

(FIOP A) B1 7.9 – – 1.9 86.6 8.0 5.4 6.5
C1 – 16.4 16.4 2.0 100* – – 8.0
3% MeOH A2 11.3 – – 2.3 100* – – 20.4
(FIOP B) B2 9.4 – – 1.8 85.6 8.8 5.3 5.6
C2 – 19.0 19.0 1.8 100* – – 5.2
10% MeOH A3 19.4 – – 2.5 100* – – 25.7
(FIOP C) B3 16.2 – – 1.5 81.0 9.4 9.6 9.4
*
C3 – 30.6 30.6 1.4 100 – – 7.5
a
The FIOPs were obtained at 20C and pH 6.0 (Fig. 1) and fit using different approaches. For fits A and B an extended Kok model (for details,
see, ‘‘Materials and methods’’) with Si state-independent miss and double-hit parameters was used. In fits C1–C3 the miss parameters of the
S2 ? S3 and S3 ? S0 transitions (a23 and a30, respectively) were used as free parameters (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 quality of a fit is represented by the fq parameter. Smaller fq values indicate a better fit. The
first 16 flash-induced oxygen yields of each FIOP were analyzed

experiments performed with the pre-flashed (YDox) samples This is also confirmed basically by the full reversibility of
confirmed that the rates of fast and slow S2 decay are the described methanol effect by washing the samples with
virtually unchanged in presence of 3% methanol (data not methanol-free buffer (see Fig. 3)—a treatment that is
shown in Table 2). Interestingly, they also revealed a small unable to re-oxidize YD.
increase of the amplitude of the fast phase from 20%
(control) to 36% (3% methanol). Calculations with our fit
program show (data not presented) that inclusion of the fast Discussion
decay into the fitting of the FIOPs only insignificantly
reduces the above described differences in the miss In this report we describe a surprisingly strong linear
parameters of control and methanol containing samples. relationship between the methanol concentration in the

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Photosynth Res (2008) 98:251–260 257

The magnitude of the a-value depends on equilibria of

both the donor and the acceptor sides of PSII (Renger and
Hanssum 1988; Shinkarev and Wraight 1993; Shinkarev
1996, 2005). The almost complete invariance of the double
hit parameter b to methanol addition (see Fig. 3b) reveals
that acceptor-side effects are negligible. Therefore, the
increase of a is ascribed to donor-side effects. It seems
most plausible to assume that methanol acts as an effective
competitor of water for its binding site that suppresses Si-
state advancement when bound. The results also show that
methanol exchanges among different PSII complexes dur-
ing the flash sequence. We have previously used the FIOPs
technique to study the exchange rates of herbicides at the
QB-site (Vermaas et al. 1984). The present study reveals an
exchange rate of methanol that is fast when compared to
the dark-time between the flashes (0.5 s), but slow when
compared to the lifetime of YZox (order of 100 ls). At 10%
(v/v) methanol the molar ratio of methanol to water is
about 5% and gives rise to an increase of a from 9.7% in
the control to 19.4% (see Fig. 3a). One could expect an
increase of a of about 5% provided that functional water
and methanol exhibit the same binding affinity to the
Mn4OxCa cluster and if all clusters containing one bound
methanol molecule are temporarily blocked in the Si-state
advancement. In fact, our data show an increase of about
Fig. 3 Dependence of the miss (top, a) and double hit (bottom, b)
probabilities on the presence of different methanol concentrations (% 10% thus suggesting that methanol binds somewhat
v/v) in S1YDox (spinach) thylakoids (closed symbols). Parameters stronger than water (factor of about 2). The proposal of
obtained after removal of methanol (by washing the samples twice in direct binding of methanol to Mn is consistent with two
methanol free buffer A) from the samples are represented by open
recent ESEEM studies employing isotopically labeled
symbols. The measurements were recorded at 20C and pH 6.0. The
Si state independent misses (a) and double hits (b) were calculated (CD3–OH) methanol (Åhrling et al. 2006; Force et al.
from FIOPs using the extended Kok model described in ‘‘Material and 1998).
methods.’’ Symbols and error bars represent the average of 2 or 3 While the above interpretation is very tempting, further
independent measurements. All other conditions are as described in
studies are required to probe these ideas. At this stage of
Fig. 1
the investigation other specific or unspecific interactions of
methanol with PSII cannot be fully ruled out.
Table 2 S2 and S3 state decay in S1YD-thylakoids in the absence and
the presence of 3% of methanola An increase of a by about 10% is expected to give rise to
a decrease of the average oxygen yield per single turnover
No MeOH 3% MeOH
flash of a similar extent (Messinger and Renger 1994;
k (s ) t1/2 (s) A (%) k (s-1) t1/2 (s) A (%)
-1
Christen et al. 1999). Indeed a small decrease of oxygen
yield is observed in the FIOPs displayed in Fig. 2. A
Fast phase S2 0.05 14.1 96 0.08 8.6 89
similar effect cannot be seen up to 10% (v/v; or 2.5 mM)
S3 0.07 10.0 94 0.09 7.4 89
when measuring the oxygen rate under excitation with
Slow phase S2 0.007 107 4 0.007 105 11
saturation continuing with white light (Fig. 1, Force et al.
S3 0.006 120 6 0.008 92 11
1998), as in this case the activity of PSII is limited by the
a
Rate constants (k), half-times (t1/2), and amplitudes (A, % of total turnover at the acceptor side. Even if present, an effect of
decay) for the S2 and S3 state reduction by YD (fast phase) and the 10% would be within the error of Clark-type measure-
acceptor side (slow phase) in spinach thylakoids in medium A. The
FIOPs measurements were performed at 20C and pH 6.0 either in the ments. Therefore, the results of Figs. 1 and 2 are not in
absence or presence of 3% methanol. The fit error for the values is contradiction with theoretical expectations.
*10% Recently the first 17O-HYSCORE detection of bound
(substrate) water was reported (Su et al. 2008). The results
sample and the miss parameter of photosynthetic water- obtained resolved only one type of bound water and the
splitting. This methanol induced increase of a is fully possibility was discussed that the presence of 3% methanol
reversible at concentrations of up to at least 10% methanol. may prevent the observation of the second substrate

123
258 Photosynth Res (2008) 98:251–260

binding site. On the basis of the data from the present study Boussac A, Rutherford AW, Styring S (1990) Interaction of ammonia
this scenario can be ruled out, since only about 6% of the with the water splitting enzyme of photosystem II. Biochemistry
29:24–32. doi:10.1021/bi00453a003
centers would have been undetectable due to methanol Christen G, Seeliger A, Renger G (1999) P680? reduction kinetics
binding to a substrate site. Therefore, the more likely and redox transition probability of the water oxidizing complex
option is that there exists only one type of 17O-exchange- as a function of pH and H/D isotope exchange in spinach
able substrate water-binding site in the S1 and S2 states that thylakoids. Biochemistry 38:6082–6092. doi:10.1021/bi9827520
Chu H-A, Sackett H, Babcock GT (2000) Identification of a Mn–O–
can be detected under the conditions of the 17O-HYSCORE Mn cluster vibrational mode of the oxygen evolving complex in
technique. On the other hand, results of time-resolved mass photosystem II by low-frequency FTIR spectroscopy. Biochem-
spectrometric H216O/H18 2 O-substrate water exchange istry 39:14371–14376. doi:10.1021/bi001751g
experiments suggest that both substrate water molecules Dau H, Iuzzolino L, Dittmer J (2001) The tetra-manganese complex
of photosystem II during its redox cycle: X-ray absorption results
are already bound in the S2 state (Hendry and Wydrzynski and mechanistic implications. Biochim Biophys Acta 1503:24–
2002). However, the reported rate for the fast exchange in 39. doi:10.1016/S0005-2728(00)00230-9
S2 is close to the limit of the time resolution of the Deak Z, Peterson S, Geijer P, Ahrling KA, Styring S (1999) Methanol
experiment in which structurally perturbed samples (two modification of the electron paramagnetic resonance signals
from the S0 and S2 states of the water-oxidizing complex of
extrinsic proteins were removed) have been employed. photosystem II. Biochim Biophys Acta 1412:240–249. doi:
Therefore, at present the possibility cannot be fully 10.1016/S0005-2728(99)00064-X
excluded that the two substrate water molecules bind in the Debus RJ (1992) The manganese and calcium ions of photosynthetic
Kok cycle at different redox levels Si of the water oxidizing oxygen evolution. Biochim Biophys Acta 1102:269–352. doi:
10.1016/0005-2728(92)90133-M
complex (WOC): the first one during the S4 ? S0 transi- Evans MCW, Nugent JHA, Ball RJ, Muhiuddin I, Pace RJ (2004)
tion and the second one may bind during the S2 ? S3 Evidence for a direct manganese-oxygen ligand in water binding
transition that is known to be coupled with significant to the S2 state of the photosynthetic water oxidation complex.
structural changes (Boussac and Rutherford 1988; Boussac Biochemistry 43:989–994. doi:10.1021/bi035489y
Evans MCW, Ball RJ, Nugent JHA (2005) Ammonia displaces
et al. 1990; Dau et al. 2001; Guiles et al. 1990; Haumann methanol bound to the water oxidizing complex of photosystem II
et al. 2005; Liang et al. 2000; Messinger et al. 1991; in the S2 state. FEBS Lett 579:3081–3084. doi:10.1016/j.febslet.
Noguchi and Sugiura 2002; Pushkar et al. 2008; Renger 2005.04.066
and Hanssum 1992; Siegbahn 2008). To decide between Fang CH, Chiang KA, Hung CH, Chang KJ, Ke SC, Chu HA (2005)
Effects of ethylene glycol and methanol on ammonia-induced
both possibilities further advanced EPR, FTIR/Raman, and structural changes of the oxygen-evolving complex in photosys-
time-resolved membrane-inlet mass spectrometry mea- tem II. Biochemistry 44:9758–9765. doi:10.1021/bi050030k
surements will be required. Ferreira KN, Iverson TM, Maghlaoui K, Barber J, Iwata S (2004)
Architecture of the photosynthetic oxygen-evolving center.
Acknowledgments Financial support by the Max-Planck-Gesell- Science 303:1831–1838. doi:10.1126/science.1093087
schaft (to Johannes Messinger) and Deutsche Forschungsgemeinschaft Force DA, Randall DW, Lorigan GA, Clemens KL, Britt RD (1998)
(to Gernot Renger, Sfb 429 TPA1) is gratefully acknowledged. ESEEM studies of alcohol binding to the manganese cluster of
the oxygen evolving complex of photosystem II. J Am Chem Soc
Open Access This article is distributed under the terms of the 120:13321–13333. doi:10.1021/ja982713b
Creative Commons Attribution Noncommercial License which per- Frasch WD, Mei R (1987) Hydrogen peroxide as an alternate
mits any noncommercial use, distribution, and reproduction in any substrate for the oxygen evolving complex. Biochim Biophys
medium, provided the original author(s) and source are credited. Acta 891:8–14. doi:10.1016/0005-2728(87)90077-6
Frasch WD, Mei R, Sanders MA (1988) Oxidation of alcohols
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