bicarbonate-induced redox tuning in photosystem ii for regulation … · 2016-10-08 · 1 1...
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1
Bicarbonate-induced redox tuning in 1
Photosystem II for regulation and protection 2
Katharina Brinkerta, Sven De Causmaecker
a, Anja Kieger-Liszkay
b, Andrea Fantuzzi
a* and 3
A.William Rutherforda* 4
5
aDepartment of Life Sciences, Imperial College London, London SW7 2AZ, UK 6
bInstitute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ Paris-Sud, Université 7
Paris-Saclay, F-91198, Gif-sur-Yvette, cedex, France 8
9
Running Title: Bicarbonate redox tuning of QA in Photosystem II 10
11
12
Supporting Information 13
14
1 Materials and methods 15
16
Chemicals: The redox mediators anthraquinone-2-sulfonate (Em = -195 mV, pH 6.5), 2-17
hydroxy-1,4-naphthoquinone (Em = -100 mV, pH 6.5) and N,N,N',N'-tetramethyl-p-18
phenylenediamine (TMPD, Em = +300 mV, pH 6.5) and additional chemicals were purchased 19
from Sigma Aldrich. 20
21
Spectroelectrochemical redox titrations 22
Spectroelectrochemical redox titrations of plant PSII-enriched membranes were carried out at 23
15 °C in an optically transparent thin-layer (spectro)electrochemical cell (optical path-length 24
0.1 mm, Als Co., Ltd.). The working electrode (100 mesh, gold gauze, Als Co., Ltd.) was 25
cleaned prior to each measurement by exposing it to an oxygen plasma (Emitech K1050X 26
Plasma Asher). The clean electrode was modified by incubation, under a stream of argon, in a 27
1 mM aqueous solution of 4,4’-dithiodipyridine (Sigma Aldrich) (1). The electrode was 28
carefully rinsed with water before use. The reference electrode was a Ag/AgCl (3 M KCl, 29
diameter 6 mm) electrode and the counter electrode was a platinum electrode (80 mesh, 30
platinum gauze, Als Co., Ltd.). The electrode potential was controlled by a potentiostat 31
2
(Metrohm/Eco Chemie Autolab potentiostat/galvanostat, electrochemical analyser, 32
PGSTAT12). Redox titrations were carried out in an anaerobic glove box using conditions 33
reported earlier by Shibamoto et al. (2, 3). The redox state of QA- was monitored by 34
measuring chlorophyll a fluorescence. Photosystem II was diluted to a [Chl] = 150 μg/mL in 35
a buffer containing 50 mM MES-NaOH (pH 6.5), 0.2 M KCl, 0.1 % dodecyl-β-D-maltoside, 36
1 M glycine-betaine and 1% taurine. A combination of redox mediators was added: 100 μM 37
anthraquinone-2-sulfonate, 100 μM 2-hydroxy-1,4-naphthoquinone and 200 µM TMPD. The 38
sample was kept in the electrochemical cell in complete darkness except when chlorophyll a 39
fluorescence was excited for ~5 s every ~10 min to monitor the change in fluorescence and to 40
evaluate the electrochemical equilibrium. The excitation was carried out with a DUAL-PAM-41
100 P700 & Chlorophyll Fluorescence Measuring System (Heinz WALZ GmbH), using the 42
pulse amplitude modulation (PAM) method and a very weak monochromatic beam (460 nm, 43
3 μE m-2
s-1
). The emission (> 625 nm, RG665 filter) was detected in line with the measuring 44
beam using a Photomultiplier-Detector Unit (DUAL-DPM). The fluorescence intensity 45
changes were plotted against the applied potential and the data fitted to the Nernst equation. 46
All of the potential are expressed versus SHE. 47
48
Influence of freeze-thawing on bicarbonate binding 49
With samples that were frozen and thawed titrations often became less reversible, in that the 50
return from reducing potentials showed indications of higher potential transitions. Good 51
reversible titrations were found with samples that had not been frozen and thawed. One 52
freeze-thaw cycle was apparently enough to decrease bicarbonate binding, while a second 53
freeze-thaw cycle led to relatively easy loss of the bicarbonate. In kinetic experiments the 54
addition of bicarbonate showed varying degrees of acceleration of the rate of forward 55
electron transfer in frozen and thawed samples. These effects where not characterised in a 56
systematic way, but the data shown in this work were done with sample that were either 57
unfrozen or had undergone one freeze-thaw cycle (where indicated). It is of note Shibamoto 58
et al (3) specified that their titrations required unfrozen samples. We suggest that this effect 59
could be due to changes in access to HCO3- binding site. 60
61
Connectivity determination 62
Due to antenna connectivity the exciton visiting a closed centre has a significant probability 63
of continuing its random walk and eventually hitting an open trap (4, 5). A consequence is 64
3
that the fluorescence yield F depends on the fraction of closed centres, c, in a hyperbolic 65
rather than linear way, according to the equation: 66
67
68
𝐹(𝑐) = 𝑐
1+𝐽−𝐽𝑐 (SE1) 69
70
71
The parameter J expresses the antenna connectivity. The non-linear relationship between the 72
fluorescent yield F and the concentration of reduced QA has consequences on the correct 73
determination of the fraction of reduced QA as a function of the redox potential. This effect 74
can be corrected using a calibration of the F(c) relationship, where c(t) can be determined 75
directly from the fluorescence (6). A weak sub-saturating flash that excites approximately 76
one sixth of the centres (fluorescence amplitude 15% of the Fmax with saturating flash) elicits 77
a fluorescence decay that can be taken as the true c(t) decay (6). 78
79
80
81
Figure S1 The fluorescence intensity elicited from a weak flash (wf) (5% of the Fmax) plotted as a 82
function of that using a saturated flash (sat). The data is fitted the equation SE1. (see text for details). 83
84
4
Thus the time course of the fluorescence decay was followed with a Joliot-type 85
spectrophotometer, JTS-10 from Biologique using i) a saturating and ii) a flash attenuated to 86
the point where it gave approximately 15% of the maximum fluorescence change measured 87
with the saturating flash. Figure S1 shows the plot of fluorescence with the weak flash (“real” 88
QA- concentration) as a function of the fluorescence seen on the saturating flash. Using 89
equation SE1 and substituting the measured values of fluorescence to obtain c, the mole 90
fraction of QA-, the resulting curve was fitted. A connectivity parameter J of 1.6 was obtained 91
and this was used to determine the concentration of QA- as a function of the electrode 92
potential in the redox titrations. In these experiments the saturating actinic flash (5 ns, 93
690 nm, 1.5 mJ) was provided by a dye laser (6 mM LDS 698 (CAS 89846-19-5) in DMSO), 94
using a Minidye accessory (GSI Group France) pumped by a frequency-doubled Nd:YAG 95
laser (532 nm; Minilite II, Continuum). The fluorescence emission was probed by weak 96
monochromatic flashes (450 nm) from the JTS-10. 97
98
Chlorophyll a fluorescence decay measurements. 99
The flash-induced increase and subsequent decay of chlorophyll fluorescence yield 100
were measured by a double fluorometer FL3000 (PSI Instruments, Brno, Czech Republic). A 101
sequence of 5 actinic flashes spaced by 1 second was used. The sample concentration was 5 102
μg Chl/ml. Ferricyanide at a final concentration of 250 μM was added to all samples prior to 103
the measurement in order maintain the plastoquinone pool in its oxidised form. 104
To verify HCO3--depletion according to the method (7), the sample (on ice under Ar), 105
was transferred in the dark with a gas-tight syringe into a sealed cuvette previously flushed 106
with Ar. Ferricyanide was added to the sample from a degassed and sealed stock to a final 107
concentration of 250 μM using a gas-tight syringe. The solution was allowed to equilibrate 108
for 1 minute before starting the flash sequence. Measurements were carried out at room 109
temperature (+20°C) in 40 mM MES pH 6.5, 15 mM CaCl2, 15 mM MgCl2. 110
To test the light-dependent dissociation of HCO3- the PSII sample was placed into a 111
sealed cuvette and degassed by bubbling argon for 15 minutes at +15 °C. The sample was 112
maintained at +15 °C and then illuminated for 4 minutes through a 590 nm cut-off filter and a 113
light intensity of 250 μmol photons m-2
s-1
(measured with a QRT-1 light meter from 114
Hansatech). The sample in the sealed cuvette was then transferred into the spectrofluorometer 115
where ferricyanide was added to the sample and the flash fluorescence measured were 116
performed as described in the previous paragraph. 117
118
5
Singlet oxygen measurements. 119
Singlet oxygen was trapped using the water soluble spin-probe 2,2,6,6-tetramethyl-4-120
piperidone (TEMPD) hydrochloride (11) and measured with a Bruker e-scanTM
(Bruker 121
Biospin, Rheinstetten, Germany). HCO3--depleted samples were diluted in non-degassed 122
buffer (0.3 M sucrose, 10 mM NaCl, 20 mM MES pH 6.5) containing 100 mM TEMPD and 123
shaken vigorously in room light to allow air to enter the solution and measured immediately. 124
Samples (10 µg Chl ml-1
) were illuminated for 2 min with 500 µmol quanta m-2
s-1
red light 125
(RG 630). 126
127
128
6
129
2 Additional Data 130
131
A) Redox titration data showing reconstitution of bicarbonate and recovery of the 132
lower potential Em values. 133
134
Fig. S2 Redox titration curves of QA in Mn-containing (A) and Mn-depleted (B) PSII 135
membranes in HCO3--depleted PSII (triangles) and (10mM) HCO3
- reconstituted PSII 136
(cirlces), measured as fluorescence yield. Filled symbols, oxidative titration, open symbols, 137
reductive titration. The concentration of reduced QA was determined by correcting the 138
fluorescence values for connectivity according to equation SE1 considering excitation energy 139
transfer between two PSII unit with J = 1.6. The data points were fitted to the Nernst equation 140
with n=1. The error bars indicate the standard deviation of three independent redox titrations. 141
The calculated mean Em values were -124 mV ± 3 mV and -60 mV ± 2 mV (A) and -22 mV ± 142
2 mV and +64 mV ± 3 mV (B), respectively 143
7
B) Redox titration data prior to correction for connectivity 144
145
i)Effect of depletion of depletion of bicarbonate on intact and Mn dpeleted PSII 146
147
148
149
Fig. S3 Redox titration curves of fluorescence yield levels uncorrected for connectivity in 150
intact (A) and Mn-depleted (B) PSII core complexes from PSII-membranes in the presence 151
(circles) and absence (triangles) of the HCO3- ligand measured as fluorescence yield. Filled 152
symbols, oxidative titration, open symbols, reductive titration. The data points were fitted to 153
the Nernst equation with n=1. The error bars indicate the standard deviation of three 154
independent redox titrations. The calculated mean Em values were -138 mV ± 2 mV and -61 155
mV ± 3 mV (A) and -23 mV ± 3 mV and +65 mV ± 3 mV (B), respectively. These are the 156
same data as shown in Figure 2A and B in the main text but prior to correction for 157
connectivity. 158
8
ii) Effect of bicabonate depeletion using formate 159
160
161
162
163
164
Fig. S4 Redox titration curves of fluorecence yield in intact PSII membranes in the presence 165
of the HCO3- ligand (circles) and upon replacement of the HCO3
- ligand by CHO2
- (triangles) 166
measured as fluorescence yield uncorrected for connectivity. Filled symbols, oxidative 167
titration, open symbols, reductive titration. The data points were fitted to the Nernst equation 168
with n=1. The error bars indicate the standard deviation of three independent redox titrations. 169
The calculated mean Em values were -138 mV ± 2 mV and -95 mV ± 2 mV, respectively. 170
171
9
iii) Effect of illumination on the redox titration 172
173
174
175
Fig. S5 Redox titration curves of fluorescence yield (uncorrected for connectivity) in intact 176
PSII membranes measured in the presence of the HCO3- ligand (circles and red triangle) in 177
the dark and and after illumination. The following protocol (triangles): a potential of -58 mV 178
was applied in the dark for 12 min and the fluorescence value recorded (red triangle), the 179
potential was then switched off for 10min in the dark, followed by QA reduction induced by 180
illuminating the sample for 4 min with a 620 nm actinic light (273 μEm-2
s-1
). The light was 181
switched off for 10 min to allow QA- to re-oxidize, after which a potential of -58 mV was 182
applied for 8 min in the dark and the fluorescence yield was measured. A reductive titration 183
was performed up to -150 mV followed by an oxidative titration. Filled symbols, oxidative 184
titration, open symbols, reductive titration. The data points were fitted to the Nernst equation 185
with n=1. The error bars indicate the standard deviation of three independent redox titrations. 186
The calculated mean Em values were -138 mV ± 2 mV and -61 mV ± 4 mV respectively. 187
188
189
190
191
10
iv) Effect of bicarbonate reconstitution on bicarbonate-depleted PSII 192
193
194
195
196
Fig. S6 Redox titration curves of fluorescence yield (uncorrected for connectivity) in Mn-197
containing (A) and Mn-depleted (B) PSII membranes in HCO3--depleted PSII (triangles) and 198
(10 mM) HCO3- reconstituted PSII (cirlces). Filled symbols, oxidative titration, open 199
symbols, reductive titration. The data points were fitted to the Nernst equation with n=1. The 200
error bars indicate the standard deviation of three independent redox titrations. The 201
calculated mean Em values were -126 mV ± 4 mV and -61 mV ± 3 mV (A) and -20 mV ± 3 202
mV and +65 mV ± 3 mV (B), respectively. 203
204
11
D) The thermodynamic cycle showing the relationship between Em and HCO3- binding. 205
206
207
208
Fig. S7 Thermodynamic cycle for the intact (A) and Mn-depleted (B) PSII linking QA/QA-· 209
reduction potential and HCO3- dissociation. The HCO3
- dissociation constant with QA 210
reduced (in red) was calculated based on the ΔΔG beween the reduction reaction with HCO3- 211
bound and that with HCO3- dissociated. 212
213
214
215
12
E) Results of EPR spin trapping experiments 216
217
218
219
sample EPR signal size
control 91%
91%
88%
Control + CO2 100%
-CO2 54%
59%
58%
-CO2 + 1 mM
NaHCO3
78%
99%
98%
220
221
Table S1 Results of the EPR spin-trapping measurements. Two different PSII enriched 222
membrane preps were used which were from a frozen sample. The control sample + 1 mM 223
NaHCO3 was set to 100%. 224
225
226
227
228
229
230
231
232
13
F) The Em values measured here compared to those discussed from the literature 233
234
235
Table S2 QA/QA- reduction potential values from spinach obtained in this work compared to 236
relevant literature values (Krieger et al 1995 (8); Shibamoto et al 2010 (2); Allakhverdiev et 237
al 2011 (9)). The four states of PSII are indicated by the colored squares for the data 238
presented here: intact with bicarbonate (blue), intact without bicarbonate (red), Mn-depleted 239
with bicarbonate (green) and Mn-depleted without bicarbonate (orange). The shading 240
represents the interpretation made in this work of the literature data. It is possible to do this 241
for the dozens of values in the literature but we chose to focus on the limited set of values 242
discussed in the main text. We consider the new values to have only a small error. Some 243
literature values have much greater error however the breadth of the shading does not 244
represent that error it is merely a means of illustrating the new interpretation of the data. 245
246
247
248
249
250
14
3 Additional Discussion 251
252
a) Relevance to donor-side effects of bicarbonate: further discussion 253
Of additional relevance to the donor-side debate is the observation made here that the 254
size of the QA/QA-• Em shift is greater in Mn-depleted PSII than in intact PSII. This seems to 255
indicate an influence of the donor-side on the acceptor-side. Donor-side effects on the 256
electron acceptor-side have been reported and discussed before (8, 10-14). In the present 257
studies we had the impression that when the HCO3- was lost, it was easier to lose the Mn 258
cluster. This needs to be verified by systematic study but it could fit with the reported 259
susceptibility to Mn loss that occurred in material that we now consider to have been depleted 260
of HCO3- (8,10,11). The bigger effect of HCO3
- binding on the Em of QA in Mn depleted PSII 261
and the consequent bigger shift in the dissociation constant of HCO3- when QA
- is present 262
could have specific importance during the process of photoassembly, where two QA redox 263
tuning events may occur, one involving Ca binding and the other bicarbonate binding. The 264
sequence and mechanistic significance of these events is worth investigating. 265
In a recent study Kahn et al (15) questioned whether Ca2+
/Sr2+
ion binding in the Mn 266
cluster at the donor-side of PSII is in fact responsible for the decrease in the Em of QA 267
described in the introduction. Instead they suggest that Ca2+
/Sr2+
ions bind to the glutamate 268
patch close to QA and that this is responsible for the changes in the Em of QA reported earlier 269
(15). This suggestion is contradicted by the data. Just as in earlier work, all the redox data 270
presented here, including those with the large positive shift in redox potential in PSII when 271
the Mn4O5Ca was removed, were obtained with identical cation concentrations and without 272
divalent cations. In some earlier work low potential values were obtained when reducing the 273
sample but high potential values were obtained in the same sample when reoxidising after 274
reduction because the Mn (and hence Ca) ions had been released due to reduction and loss of 275
the Mn cluster : thus the cation concentration barely changed under these conditions only the 276
integrity of the cluster changed (8). The redox titration work using Sr2+
was due to a single 277
Sr2+
ion per PSII shown spectroscopically to be located in the Ca2+
site in the Mn cluster and 278
the redox titrations comparing the influence of Sr2+
exchange were done in the presence of 279
identical concentrations of Ca2+
(13). It seems clear that the changes in Em described cannot 280
be attributed to changes in divalent cation binding to the electron acceptor side. Nevertheless, 281
given the proximity of the glutamate patch to QA (16), it does seem likely that binding of 282
cations to this site could influence the potential of QA. However, if this were the case the Em 283
15
of QA/QA- would be expected to increase (due to an electrostatic effect) rather than decrease, 284
and so far titration studies have not shown such effects. 285
286
b) On the relevance of the new model to published literature 287
It is worth considering if the new H3CO--mediated regulation mechanism presented here can 288
explain observations in the literature. Given the extensive literature, we provide only one 289
recent example. In recent work the effects of illumination regimes on CO2-limited 290
cyanobacterial cells were investigated in cyanobacteria under CO2 restricted conditions (17). 291
A series of observations related to photosynthetic electron transfer (Chl fluorescence, O2 292
evolution, cytochrome f, P700+, NADP
+) were reported leading the authors to postulate that 293
the over-reduced PQ pool prevents the cytochrome b6f complex from functioning after a 294
pulse of strong light. This reduced-pool mechanism of electron transfer inhibition could 295
indeed explain the phenomena reported, however the new HCO3--mediated regulatory 296
mechanism reported here could account for the observations made. Given the current model it 297
should be possible to distinguish the two mechanisms experimentally. 298
299
c) On the occurrence of the HCO3--mediated regulation of PSII in vivo 300
A wide range of strategies have evolved to deal with pressures associated with CO2 limitation 301
when encountered by different species in different environments. Several types of alternative 302
electron transfer mechanisms have evolved to alleviate problems encountered with 303
photosynthetic electron transfer under CO2-limiting conditions, including O2 reduction 304
(involving flavo-di-iron proteins, plastid alternative oxidase, photorespiration etc.) and cyclic 305
electron flow around PS I (e.g. 18-21). Similarly different mechanisms have evolved to 306
mitigate CO2 limitation, (CO2 concentrating mechanisms, CAM and C4 carbon fixation) and 307
these will have different influences on the stromal HCO3- concentrations (22-23). It thus 308
seems likely that the importance of HCO3--mediated regulation of PSII in vivo could vary 309
greatly depending on the species. These considerations will have to be taken into account in 310
future efforts to demonstrate its role in vivo. 311
312
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