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Exchange of chloride by bromide in the manganese photosystem-II complex studied by cw- and pulsed-EPR
~i'
Chemical Physics
I'
ELSEVIER
Chemical Physics 194 (1995) 409-418
Exchange of chloride by bromide in the manganese Photosystem-II complex studied by cw- and pulsed-EPR Alain Boussac Sert'ice de Bio~nerg~tique (URA CNRS 1290), DBCM, CEA Saclay, 91191 Gif sur Yt~etteCedex, France Received 16 September 1994
Abstract
C1- is an essential cofactor in Photosystem-II (PS-II) oxygen evolution. By using electron spin echo envelope spectroscopy it is shown that, in some circumstances, magnetic couplings between chloride and the Mn cluster can be observed. In order to extract only the electron nuclear couplings between the Mn cluster, in the S2-state, and those of the halide, a comparison was done between chloride- and bromide-reconstituted samples. In salt-washed, CaC12- or CaBr2-reconstituted Photosystem-II membranes, frequencies similar to the Larmor frequency of bromide and chloride were detected. When CI- and Br- reconstituted PS-I1 membranes are compared to membranes that have also been cae+-depleted, and EGTA-treated, these features in the frequency spectra are absent. After chloride depletion at high pH in the presence of sulfate, no difference could be observed in the frequency spectra of bromide- and chloride-reconstituted samples. These results are discussed in view of possible different C1- binding sites in PS-II.
Photosystem-lI catalyzes light-driven water oxidation resulting in oxygen evolution. A cluster of 4 Mn located in the reaction centre of PS-II probably acts both as the active site and as a charge accumulating device of the water-splitting enzyme ([1,2] for reviews). During the enzyme cycle, the oxidizing side
Abbreviations:P68o, reaction centre chlorophyll (Chl) of Photosystem II (PS-II); Tyrz, the tyrosineacting as the electrondonor to P680;TyrD, the tyrosine acting as a side path electron donor of PS-ll; QA, primary quinone electron acceptor of PS-II; EPR, electron paramagnetic resonance; EDTA, ethylene diamine tetra acetic acid; EGTA, ethylene glycol bis(/3-aminoethyl ether)N.N,N',N'-tetraacetic acid; Mes, 2-(N-morpholino)ethanesulfonic acid; Hepes, N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid; PPBQ, phenyl-p-benzoquinone;X, symbol for CI or Br ; EXAFS, extended X-ray absorption fine structure; ESEEM, electron spin echo envelope modulation.
of PS-II goes through five different redox states that are denoted S,, n varying from 0 to 4 [3]. The oxygen is released during the S 3 to S o transition in which S 4 is a transient state. Three extrinsic polypeptides, with molecular masses of 17, 23 and 33 kDa, are bound to the PS-II reaction centre on the inside of the thylakoid membrane. The 17 and 23 kDa extrinsic polypeptides can be removed by NaCI washing (reviewed in Ref. [4]). Removal of these polypeptides results in inhibition of oxygen evolution due to an increased requirement for chloride and calcium ions (reviewed in Refs. [1,4-7]). In Ca2+-depleted PS-II, inhibition of the enzyme cycle occurs at the S 3 to S o transition [1,8-10]. The depletion of C I - from PS-II also results in inhibition of oxygen evolution (reviewed in Refs. [1,5,6,11,12]. Two different types of inhibition have been ob-
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A. Boussac / Chemical Physics 194 (1995) 409-418
served, depending on the nature of the counterion used a n d / o r the severity of the C1- depletion. A decrease in oxygen evolution attributed to a slowdown of the S 3 to S O transition was reported when nitrate or acetate were used as the counterion [13]. In contrast, a total block in the S-state cycle was observed when using sulphate as a counterion [13]. The point at which the enzyme cycle is blocked by this kind of C1 depletion procedure has been shown to be also the S 3 to S O transition [10,14]. Ligands exchange experiments generally provide useful information on the roles and location of intrinsic ligands. In PS-II, it has been shown that bromide can replace chloride [15,16]. Two effects have been observed depending the treatment used to exchange chloride with bromide [17]. When chloride-depletion was done by a high pH treatment, no difference was found in the dissociation constant of the bromide and chloride required to reactivate oxygen evolution. The percentage of the activity which is restored is also similar. By contrast, when chloride depletion was done after the 17 and 23 kDa extrinsic polypeptides were removed, the dissociation constant of bromide becomes higher than that of chloride and, under saturating light conditions, all centers are active but the maximum activity restored with bromide is half of that obtained with chloride. This may indicate that the removal of the extrinsic polypeptides induces the depletion of a chloride binding site which is different from or is a modified version.of the CI site which is concerned by the high pH treatment. Until now there is no clear spectroscopic evidence indicating an interaction between chloride and the Mn. EPR performed at X- and S-band were unable to detect modifications in the hyperfine signal after a chloride/bromide exchange [18,19]. Indeed, chloride and bromide have two isotopes and the four nuclei have the same spin ( I = 3 / 2 ) but they possess different Larmor frequencies. The natural abundances and the Larmor frequencies at 3600 gauss are; 35C1, 75.77%, 1.50 MHz; 37C1, 24.23%, 1.26 MHz; 79Br, 50.69%, 3.85 MHz; 8~Br, 49.31%, 4.15 MHz. In this work, pulsed-EPR has been used to look for hyperfine couplings, between the Mn cluster and chloride, which may be too weak to be observed by cw-EPR. Chloride has been exchanged with bromide by 3 different procedures: (1) The removal of the 17 and 23 kDa polypeptides increases the Ca 2+ [1,4]
and chloride (or bromide) [6,20] requirement for oxygen evolution. Therefore, polypeptide-depletion by salt-washing has been done with NaCI or NaBr as the salt. Then the membranes were reconstituted with CaC12 or CaBr 2. (2) An EGTA-treatment has been used for the total depletion of Ca 2+ in the salt-washed PS-II [21] and no Ca2+-reconstitution accompanied halide-reconstitution after the salt-washing. (3) Chloride-depletion was done by a high pH treatment and the presence of sulfate followed by reconstitution with C1- or Br-. In each case, the ESEEM in the bromide and chloride reconstituted samples were compared.
1. Materials and methods
Photosystem-II membranes from spinach chloroplasts were prepared as previously described [9] and were either used immediately or stored in liquid nitrogen. For the C I - / B r - exchange procedure, the PS-II membranes were first resuspended and pelleted (i.e. washed) three times by successive centrifugations (15 min, 30000 g) in 25 mM Mes pH 6.5 and 0.3 M sucrose. Then, after the last centrifugation, the pellets were treated with 3 different protocols. The symbol 'X' replaces CI or Br- in the descriptions that follow.
1.1. The CaX2-reconstituted NaX-washed PS-H The pellet was resuspended and incubated under room light at 4°C in 0.3 M sucrose, 26 mM Mes pH 6.5 and 1.2 M NaBr or 1.2 M NaCI (Chl concentration ---0.5 mg/ml). After 30 min, 50 ixM EGTA was added and the salt-washed PS-II membranes were collected by centrifugation. The pellet was resuspended in 25 mM Mes-5 mM Ca(OH) 2 pH 6.5 (final pH adjustment with NaOH) and 10 Mm NaBr or NaCI respectively, centrifuged as above and resuspended once again in the same media at about 8 mg Chl/ml.
1.2. The EGTA(X)-treated NaX-washed PS-H The pellet was resuspended and incubated under room light at 4°C in 0.3 M sucrose, 25 mM Mes pH 6.5 and 1.2 M NaBr or 1.2 M NaCI (Chl concentra-
A. Boussac / Chemical Physics 194 (1995) 409-418
tion = 0.5 m g / m l ) . After 30 min, 5 mM EGTA was added. Then the membranes were collected by centrifugation, washed once again in 25 mM Mes(NaOH) pH 6.5, 5 mM EGTA and 30 mM NaBr or NaCI respectively and resuspended in the same media at about 8 mg Chl/ml. 1.3. The NaX-reconstituted SO 2 -- and high pHtreated PS-H
The pellet was washed in 0.3 M sucrose, 50 mM Na2SO4, 50 mM Hepes pH 7.5 and 100 IxM EDTA. Then the membranes were collected by centrifugation and resuspended in the same medium at about 8 mg C h l / m l without EDTA. After a 200 K illumination, the samples were thawed and 100 mM NaCI or NaBr (final concentration) were added directly to the samples in the EPR tubes. Then the samples were immediately re-frozen at 200 K then to 77 K. We have observed that freezing and thawing of samples at pH 7.5 can lead to some MnlI release from a fraction of centers in some preparations. This was monitored by the extent of the MnlI hexaquo EPR signal. When MnlI release was observed, the preparation was discarded. This monitoring of MnlI levels was only possible in the absence of EDTA. Therefore, EDTA was avoided in the final resuspending media. A second reason to avoid EDTA is the similarity between the MnlI-EDTA spectrum and the cytb559 signal in field-swept echo experiments (not shown). Cytb559 is associated with PS-II and, after chloride-depletion, is present in its oxidised, low spin (S = 1 / 2 ) form in the dark. Its EPR signal is therefore present in the baseline spectrum. The overlapping of both signals could complicate the interpretation since the MnlI-EDTA complex is a good electron donor to damaged PS-II in the light at room temperature (not shown) resulting in the formation of a MnlII-EDTA complex not detectable by EPR and therefore in an artefactual decrease of the baseline spectrum. In all the treatments, the highest purity NaC1 and NaBr salts commercially available (from Merck and Aldrich) were used. The PS-II membranes were put in quartz EPR tubes and, after dark adaptation for 1 hour at 0°C, 1 mM PPBQ (dissolved in DMSO) was added as an artificial electron acceptor. Then the samples were
411
immediately frozen at 200 K in a CO2-ethanol bath then transferred to 77 K. Illumination of the samples was done with an 800 W projector through water and infrared filters in a non-silvered dewar filled with ethanol cooled to 0°C or 200 K with solid CO 2. CW-EPR spectra were recorded at liquid helium temperatures with a Bruker ER 200D X-band spectrometer equipped with an Oxford Instruments cryostat. Pulsed EPR was done with a Bruker ESP 380 spectrometer already described [22]. The field swept spectra were obtained by measuring the amplitude of the echo as a function of the magnetic field after a two-pulse sequence ( ' r r / 2 - 2 0 0 ns-'rr). The duration of 'rr/2 and 'rr pulses were 8 and 16 ns respectively. The ESEEM data presented in this work result from a three-pulse sequence ( r r / 2 - z - ' r r / 2 - T - r r / 2 ) . The amplitude of the stimulated echo as a function of z + T was measured at 3600 gauss. ~- was chosen equal to 136 ns to suppress most of the weakly coupled proton frequency around 15 MHz at 3600 gauss. The minimum interpulse T was 64 ns and was incremented by steps of 8 ns. The "rr/2 pulse duration was 16 ns. To remove the unwanted echoes in a three-pulse experiment, the phase cycling procedure described in Ref. [23] was used.
2. Results 2.1. The CaX2-reconstituted NaX-washed PS-H
The formation of the S2-state in the CaC12 and CaBr 2 reconstituted salt-washed PS-II was monitored by cw- and field-swept echo EPR (Fig. 1). Spectra a were recorded in dark-adapted samples. The spectra recorded in the dark contains the following features; (1) the intense TyrD ° signal at g = 2.0045 ( = 3350 gauss) which is excised from all spectra shown; (2) the EPR signal from the oxidised Cytb559 which, under the conditions shown, is most evident at its gz line at g = 2.93 ( = 2200 gauss); (3) relatively intense signals, in cw-EPR, at low field arise from contaminating Fe 3+ (S = 5 / 2 ) in different environments. Spectra b were recorded after a 200 K illumination. Both samples exhibited a very similar Mn-multiline signal characteristic of the S 2state (Fig. 1, lower panels). Only a very small g = 4
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A. Boussac / Chemical Physics 194 (1995) 409-418
NaCl-wsshed+ CaCl=~
I
0
0
1000 2000 3000 4000 Magnetic field (gauss)
1000 2000 3000 4000 Magnetic field (gauss)
NaCI-washed+ CaCI
NaBr-washed+ CaBr.
iiii
0
1000 2000 3000 4000 Magnetic field (gauss)
0
iii
ii h
iii
iiiiihll
~1 i t i i h
iiitllll
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1000 2000 3000 4000 Magneticfield (gauss)
Fig. 1. CW-EPR spectra (lower panels) and field swept echo (upper panels) of CaC12-reconstituted NaCl-washed PS-II (left panels) and CaBr2-reconstitutedNaBr-washed PS-II (right panels). Spectra a were recorded in dark-adapted samples i.e. in the S1 state. Spectra b were recorded after a 200 K illumination i.e. in the S2 state. Instrument settings for cw-EPR: temperature, 10 K; modulation amplitude, 22 gauss; microwave power, 20 mW; microwave frequency, 9.4 GHz; modulation frequency, 100 KHz. The central parts of the spectra correspondingto the TyrD° region were deleted. Instrument settings for pulsed-EPR: the amplitude of the echo results from a two-pulse sequence ('rr/2 = 8 ns, r = 200 ns, w = 16 ns); temperature, 4-5 K; microwave frequency, 9.7 GHz. The small distortion in the baseline of spectrum a in the right, lower panel is due to a small amount of oxygen. No normalization procedure was used to plot the field-swept spectra.
signal ( = 1500 gauss) was formed in these samples. The 200 K illumination-induced increase, over the Cytb559 signal, in the field swept echo signal is very similar in C I - and B r - reconstituted samples (Fig. 1, upper panels). Thus it seems that the exchange of chloride with bromide did not affect the yield of the S 1 to S 2 transition under continuous illumination at 200 K in these membranes and no spectral modifica-
tions were observed. After a salt-washing, Cytb559 is almost totally oxidized. Therefore, spectra a, in Fig. 1, correspond to = 1 spin (S = 1 / 2 ) per reaction center. The light-induced signals (spectra b) correspond to approximately the same number of spins. By recording the oxygen evolution under various light intensity, it was found that all the centers are active in the presence of bromide or chloride (not shown). Nevertheless, in bromide reconstituted samples the limiting step in the oxygen evolution process is two times slower than in the chloride-reconstituted sample as already observed [17]. In order to detect the couplings between the Mn cluster in the S2-state and the bromide or chloride nuclei, the ESEEM at 3600 gauss in CaCI 2- and CaBr2-reconstituted salt-washed PS-II was measured. At this magnetic field, the Fe 2+ QA signal formed by the 200 K illumination contributes to the cw-EPR spectra. Nevertheless, the very fast relaxation of the Fe2+ QA species prevents its detection by pulsed-EPR at the temperature used in the present study. The ESEEM recorded in CaC12- and CaBr2-reconstituted salt-washed PS-II (Fig. 2, the two upper panels) seem very similar and in the Fourier transform spectra of both ESEEM, no major differences in frequencies are evident due to the complexity of the S 2 spectra (not shown but see for example Refs. [22,24,25]). Therefore, as already described in the literature (see for example Refs. [23,26]), the quotient and the difference of the ESEEM were used. These procedures should reveal only the differences due to the chloride/bromide exchange. Fig. 2 (the two lower panels) shows both the quotient and the difference of the two ESEEM obtained in CaCI 2and CaBre-reconstituted salt-washed PS-II. Some modulations, which are similar in the quotient and the difference, appear clearly with a maximal amplitude which accounts for about 15% of the total ESEEM. Fig. 3 shows the frequency spectrum which was obtained by taking the real part of the Fourier transform of the time domain of the difference of the ESEEM. The positive and negative peaks may be attributed to the couplings between the Mn cluster in the S 2 state and bromide and chloride respectively. This frequency spectrum presents large positive features around 4 MHz and negative features between 1 and 2 MHz. Smaller positive and negative peaks at around 7.5 and 2.5 MHz respectively could also be
A. Boussac/ Chemical Physics 194 (1995) 409-418
413
significant if they are not due to a b a s e l i n e - r o l l i n g w h i c h occurs w h e n a d e a d - t i m e reconstruction cannot be done as it is the case here. The E S E E M recorded in dark-adapted samples, i.e. mainly on the Cytb55, ~ signal, reveals no difference b e t w e e n the two samples (not shown). W e 1
CaB~
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o
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J l l l l l l l l i L J l i l J ~ l l J J l l L L I l l l l l i l l l l J l l
tau + T (nil)
1
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=.
0
5
10
15
20
Frequency ( M H z )
~0.8 O
~ 0.6
Fig. 3. Real part of the Fourier transform of the difference ESEEM (CaBr 2 minus CaCI 2) shown in the lower panel of Fig. 2. No dead-time reconstruction was done in this particular case. The dashed lines is drawn to visualize the zero amplitude level.
~ 0.4
~ 0.2 W
o
1000
2000 tau + T (nil)
3000
4000
1.2 U.I
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~ 0.9 0.8
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i
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0.06 0.04
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-0.06 1000
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Fig. 2. Three pulse ESEEM in CaClz-reconstituted NaCl-washed PS-II and CaBr2-reconstituted NaBr-washed PS-II recorded in the S z state at a magnetic field of 3600 gauss and a microwave frequency of 9.7 GHz. The offset corresponding to the amplitude of the echo when all the modulations are damped has been subtracted. The amplitude of the echo results from the sequence: '~/2 = 16 ns, 7=136 ns, 'rr/2 = 16 ns, T m i n =64 ns, "~/2 = 16 ns. The two lower panels represent respectively the quotient and the difference of the two ESEEM represented above.
cannot rule out a small c o n t a m i n a t i n g i r o n - s u l f u r signal at about g = 1.9. Nevertheless, CaCI 2- and CaBr2-reconstituted salt-washed m e m b r a n e s arise f r o m the s a m e PS-II preparation and all contaminating species are not e x p e c t e d to contribute in the difference signals.
2.2. The EGTA(X)-treated NaX-washed PS-II T o study the effect of Ca2+-depletion on the c h l o r i d e / b r o m i d e e x c h a n g e , the Ca2+-depletion procedure was a c c o m p a n i e d by an E G T A treatment. This treatment results in m e m b r a n e s in w h i c h the S 2 state is stable in the dark and exhibits a m o d i f i e d M n multiline signal [21]. The E G T A treatment was done so that essentially all the centers w o u l d be inhibited, a situation w h i c h is not necessarily a c h i e v e d in the absence o f the chelator w h e r e typically 20% o f the centers remain active in o x y g e n e v o l u t i o n [9]. Fig. 4 ( l o w e r panels) s h o w s the typical c w - E P R spectra in such a preparation. Spectra a correspond to the m o d i fied multiline signal arising f r o m the stable S 2 state. Spectra b w e r e recorded after a 0°C illumination and exhibit the split S 3 signal. The upper panels correspond to the field-swept spectra recorded on the
414
A. Boussac / Chemical Physics 194 (1995) 409-418
s a m e samples and shows, as p r e v i o u s l y reported, that the S 3 signal appears on top o f the S 2 signal with a similar s t o i c h i o m e t r y to that already o b s e r v e d [22]. N o difference can be o b s e r v e d in the p r e s e n c e o f chloride or b r o m i d e . Fig. 5 s h o w s for both samples, in the S 2 state, the E S E E M recorded at 3600 gauss and their quotient. The quotient only reveals a small m o d u l a t i o n (around 15 M H z ) c o r r e s p o n d i n g to
1
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Magnetic field (gauss) NsCI-washed
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Magnetic field (gauss) NaBr-wa+shed
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,
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i
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i
i
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tau + T (ns) hl . . . . . .
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1000 2000 3000 4000 Magnetic field (gauss)
Fig. 4. CW-EPR spectra (lower panels) and field swept echo (upper panels) of EGTA-Cl-treated NaCl-washed PS-II (left panels) and EGTA-Br-treated NaBr-washed PS-II (right panels). Spectra a were recorded in dark-adapted sample i.e. in the S 2 state. Spectra b were recorded after a 0°C illumination i.e. in the S 3 state. Instrument settings for "cw-EPR: temperature, 10 K; modulation amplitude, 22 gauss; microwave power, 20 mW; microwave frequency, 9.4 GHz; modulation frequency, 100 KHz. The central parts of the spectra corresponding to the TyrD° region were deleted. Instrument settings for pulsed-EPR: the amplitude of the echo results from a two-pulse sequence (-rr/2 = 8 ns, ~"= 200 ns, ir = 16 ns); temperature, 4-5 K; microwave frequency, 9.7 GHz. No normalization procedure was used to plot the field-swept spectra.
Fig. 5. Three pulse ESEEM in EGTA-Br-treated NaBr-washed PS-II (upper panel) and EGTA-Cl-treated NaCl-washed PS-II (middle panel) recorded in the S 2 s t a t e at a magnetic field of 3600 gauss and a microwave frequency of 9.7 GHz. The offset corresponding to the amplitude of the echo when all the modulations are damped has been subtracted. The amplitude of the echo results from the sequence: ~r/2 = 16ns, ~-= 136 ns, "n/2 = 16 ns, Train = 64 ns, I r / 2 = 16 ns. The lower panels represent the quotient of the two ESEEM represented above.
w e a k l y coupled protons. A low a m o u n t o f w e a k l y coupled protons is still o b s e r v e d because (1) the tau v a l u e o f 136 ns does not totally r e m o v e the 15 M H z f r e q u e n c y at 3600 gauss and (2) s o m e additional m o d u l a t i o n s attributed to c o u p l i n g s with the nitro-
A. Boussac / Chemical Physics 194 (1995) 409-418
415
2.3. The NaX-reconstituted S042 - and high pHtreated PS-H
gen(s) of EGTA [22] are present. This increases the difficulty of choosing the correct offset (i.e. the amplitude of the echo when all modulations are damped) before ratioing or subtracting the ESEEM. Therefore, the quotient and difference procedures are less effective in removing identical frequencies in the two ESEEM. Nevertheless, the main conclusion is that no modulations arising from an halide exchange can be observed.
Washing of PS-II membranes at pH 7.5 in the presence of sulfate results in an inhibition of oxygen evolution which can be reversed by addition of chloride. It has been shown that bromide has the same efficiency as chloride to reconstitute oxygen evolution after a high pH treatment [17]. The multi-
i I I [ l l l l l l l l l l l l l l l l l l l l l l l l
2000
3000
4000
Magnetic field (gauss) Br b
0
1000 2000 3000 4000 Magnetic field (gauss)
0
1000 2000 3000 4000 Magnetic field (gauss)
Fig. 6. Upper panel: cw-EPR spectra of sulfate and high pH-treated PS-II after the addition of 100 mM NaCI or NaBr following a 200 K illumination. Instrument settings: temperature, 10 K; modulation amplitude, 22 gauss; microwave power, 20 mW; microwave frequency, 9.4 GHz; modulation frequency, 100 kHz. The central parts of the spectra corresponding to the TyrD ° region were deleted. Lower panels: field swept echo of sulfate and high pH-treated PS-II dark-adapted i.e. in the S l state (spectra a) or after the addition of 100 mM NaC1 (left panel) or NaBr (right panel) following a 200 K illumination, i.e. in the S 2 state (spectra b). Instrument settings for pulsed-EPR: the amplitude of the echo results from a two-pulse sequence (~r/2 = 8ns, r = 200 ns, 7r = 16 ns); temperature, 4 - 5 K; microwave frequency, 9.7 GHz. No normalization procedure was used to plot the field-swept spectra.
A. Boussac / Chemical Physics 194 (1995) 409-418
416
line signal is formed by CI- addition after 200 K illumination [27]. The ability of bromide versus chloride to induce the appearance of the EPR signals characteristic of the S 2 state is shown in Fig. 6. The multiline signals (upper panel) detected by cw-EPR and the field swept spectra (lower panels) show that
ar
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0.6 0.8 0.4 0.2 0 1ooo
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3000
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the S 2 signals were similar when bromide and chloride were added after a 200 K illumination (see also Refs. [18,20,28]). In the conditions used here, the S 2 Mn signal is smaller than that generated by 200 K illumination of a CI- reconstituted sample. This is expected given the time required for mixing and is similar to the situation seen under these conditions earlier [27,29]. Two other effects contribute to the smaller size, when compared to untreated samples, of the light-induced signal in Fig. 6. (1) After chloride-depletion, TyrD ° becomes reduced in 20-30% of the centers (not shown). Illumination at 200 K results in the oxidation of TyrD in at least a fraction of these centers instead of S 2 formation. (2) The protocol used for C1 -depletion results in an irreversible inhibition of a small proportion of centers [10]. Fig. 7 shows the ESEEM measured at 3600 gauss in samples where bromide and chloride were added after a 200 K illumination. The lower panel shows the quotient of the ESEEM. No modulation due to the bromide/chloride exchange is detected.
,-i
~-
0.4
u,I
3. D i s c u s s i o n
uJ 0.2 0 0
1000
2000
3000
4000
tau + T (ns) 1.4
Br/CI
LU tn LU 1.2
0
0.8 0.6
0
1000
2000
tau
3000
4000
+ T (ns)
Fig. 7. Three pulse ESEEM in NaBr-reconstituted (upper panel) and NaCl-reconstituted (middle panel) after a 200 K illumination, i.e. in the S 2 state, of sulfate and high pH-treated PS-II at a magnetic field of 3600 gauss and a microwave frequency of 9.7 GHz. The offset corresponding to the amplitude of the echo when all the modulations are damped has been subtracted. The amplitude of the echo results from the sequence: 9 / 2 = 16 ns, "r = 136 ns, 9 / 2 = 16 ns, Tmin = 64 ns, 'rr/2 = 16 ns. The lower panels represent the quotient of the two ESEEM represented above.
In order to detect small hyperfine couplings between the Mn cluster and chloride, a chloride/bromide exchange has been performed with three different biochemical procedures. Comparison of the ESEEM in CaCl2-reconstituted NaCl-washed PS-II and CaBr2-reconstituted NaBr-washed PS-II reveals differences in the frequency spectra. Since, in Fig. 3, the only difference between the two samples is the halide status, it is reasonable to suggest that these differences result directly from the change in the nature of the halide. Since the larger positive frequencies correspond to those expected for the Larmor frequency from bromide while the larger negative frequencies are in the range expected for the Larmor frequencies of chloride, it is also reasonable to attribute these frequencies to hyperfine coupling between the Mn and the halides. We cannot rule out that the CI / B r - exchange perturbs the Mn cluster resulting in changes in native couplings to e.g. nitrogen which give difference features which are coincidentally identical to those expected from C I - / B r - exchange. Moreover, the
A. Boussac / Chemical Physics 194 (1995) 409-418
small features at around 7.5 and 2.5 MHz in Fig. 3 could be due to perturbations induced by the chloride/bromide exchange. The results would then constitute a spectroscopic demonstration of a bromideinduced perturbation of the Mn cluster. After the depletion of Ca 2+ and the binding of EGTA, the couplings seen above are absent. There are several possible explanations for this: (1) The Ca 2+ depletion prevents the binding of chloride. This fits with the model in which Ca 2÷ acts as a locus for C1- binding close to the Mn [2]. However, Ca 2~ depletion could affect C1- binding through a secondary structural effect. Such an observation was already made in chloride-depleted samples in which Ca 2÷ was found to modulate a proportion of the activity in chloride-reconstituted samples [10,17]; (2) The couplings between the Mn cluster and the halides are sufficiently modified so that they are not detectable; (3) EGTA perturbs the chloride site. Indeed, it has been shown that EGTA binding to PS-II decreased in the absence of C1- which reveals a relationship between the chloride and EGTA binding sites [30]. After the chloride removal by treatment at high pH in the presence of sulfate, the couplings between the Mn and the chloride (or bromide) which is required for the reactivation of oxygen evolution and the appearance of the S 2 EPR signals, cannot be detected by pulsed EPR. This suggests that either the corresponding chloride binding site is too far from the Mn cluster or that chloride is a ligand of the Mn cluster itself. In the latter case, the resulting couplings are predicted to be undetectable by pulsed-EPR from simulation of interactions between a spin S = 1 / 2 and a nucleus with a spin I = 3 / 2 [31]. Direct indications of CI binding to the Mn have not been obtained by chloride/bromide exchange experiments in EXAFS [32]. This may indicate that chloride is not a ligand of the Mn, at least in the redox states of the Mn tested [32]. Nevertheless, the same authors have observed a modification of the EXAFS features after replacement of chloride by fluoride and interpreted this as indicating that fluoride binds to one di-&-oxo bridged Mn dimer. From the differences between the three biochemical procedures used in the present study, one possible explanation is that two types of chloride binding site exist: (1) A chloride site in which the chloride(s)
417
is (are) exchangeable after a high pH treatment and which is (are) not detected by pulsed EPR. This chloride could be a direct ligand of the Mn cluster for the reasons explained above. (2) A chloride site which is exchanged after depletion of the 17 and 23 kDa polypeptides and in which chloride(s) is (are) detected by pulsed EPR. As the value of the coupling of this chloride with the Mn cluster is similar to the Larmor frequency of CI-, it is reasonable to assume that the binding site of this chloride is not in the first coordination shell of the Mn cluster. One (the latter) or both types of chlorides can be exchanged after a salt-washing. Alternatively, only one kind of chloride site might be present but it is modified by the salt-washing procedure.
Acknowledgements A.W. Rutherford and S. Un are gratefully acknowledged for stimulating discussions and careful reading of the manuscript.
References [1] R.C. Debus, Biochim. Biophys. Acta 1102 (1992) 269. [2] A.W. Rutherford, Trends Biochem. Sci. 14 (1989) 227. [3] B. Kok, B. Forbush and M. McGloin, Photochem. Photobiol. 11 (1970) 457. [4] N. Murata and M. Miyao, Trends Biochem. Sci. 10 (1985) 122. [5] P.H. Homann, J. Bioenerg. Biomemb. 19 (1987) 105. [6] P.H. Homann, Biochim. Biophys. Acta 934 (1987) 1. [7] C.F. Yocum, Biochim. Biophys. Acta 1059 (1991) 1. [8] A. Boussac, B. Maison-Peteri, C. Vernone and A.-L. Etienne, Biochim. Biophys. Acta 808 (1985) 231. [9] A. Boussac and A.W. Rutherford, Biochemistry 27 (1988) 3476. [10] A. Boussac, P. S6tif and A.W. Rutherford, Biochemistry 31 (1992) 1224. [11] W.J. Coleman, Photosynth. Res. 23 (1990) 1. [12] A.W. Rutherford, J.-L. Zimmermann and A. Boussac, in: The photosystems: structure, function and molecular biology, ed. J. Barber (Elsevier, Amsterdam, 1992) pp. 179-229. [13] J. Sinclair, Biochim. Biophys. Acta 764 (1984) 247. [14] P.H. Homann, H. Gleiter, T.-A. Ono and Y. Inoue, Biochim. Biophys. Acta 850 (1986) 10. [15] P. Gorham and K.A. Clendenning, Arch. Biochem. Biophys. 37 (1952) 199. [16] S. Isawa, R.L. Heath and G. Hind, Biochim. Biophys. Acta 180 (1969) 388.
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[17] P.H. Homann and Y. Inoue, in: Ion interactions in energy transfer biomembranes, eds. G. Papageorgiou, J. Barber and S. Papa (Plenum Press, New York, 1986) pp. 291-302. [18] V. Yachandra, R.D. Guiles, K. Sauer and M.P. Klein, Biochim. Biophys. Acta 850 (1986) 333. [19] A. Haddy, R. Aasa and L.-E. Andr6asson, Biochemistry 28 (1989) 6954. [20] P.H. Homann, Photosynthes. Res. 15 (1988) 205. [21] A. Boussac, J.-L. Zimmermann and A.W. Rutherford, Biochemistry 28 (1989) 8984. [22] J.-L. Zimmermann, A. Boussac and A.W. Rutherford, Biochemistry 32 (1993) 4831. [23] L. Kevan, in: Modern pulsed and continuous wave electron spin resonance, eds. L. Kevan and M.K. Bowman (Wiley-lnterscience, New York) pp. 231-266. [24] R.D. Britt, J.-L. Zimmermann, K. Sauer and M.P. Klein, J. Am. Chem. Soc. 111 (1989) 3522.
[25] V.J. DeRose, V.K. Yachandra, A.E. McDermott, R.D. Britt, K. Sauer and M.P. Klein, Biochemistry 30 (1991) 1335. [26] J. Peisach, W.B. Mims and J.L. Davis, J. Biol. Chem. 259 (1984) 2704. [27] T.-A. Ono, J.-L. Zimmermann, Y. Inoue and A.W. Rutherford, Biochim. Biophys. Acta 851 (1986) 193. [28] T.-A. Ono, H. Nakayama, H. Gleiter, Y. Inoue and A. Kawamori, Arch. Biochem. Biophys. 256 (1987) 618. [29] A. Boussac and A.W. Rutherford, 1. Biol. Chem. 269 (1994) 12462. [30] P. van Vliet, A. Boussac and A.W. Rutherford, Biochemistry 33 (1994) 12998. [31] E.J. Reijerse and C.P. Keijzers, J. Magn. Reson. 71 (1987) 83. [32] V. Yachandra, V.J. DeRose, M.J. Latimer, I. Mukerji, K. Sauer and M.P. Klein, Science 260 (1993) 675.
Chemical Physics
I'
ELSEVIER
Chemical Physics 194 (1995) 409-418
Exchange of chloride by bromide in the manganese Photosystem-II complex studied by cw- and pulsed-EPR Alain Boussac Sert'ice de Bio~nerg~tique (URA CNRS 1290), DBCM, CEA Saclay, 91191 Gif sur Yt~etteCedex, France Received 16 September 1994
Abstract
C1- is an essential cofactor in Photosystem-II (PS-II) oxygen evolution. By using electron spin echo envelope spectroscopy it is shown that, in some circumstances, magnetic couplings between chloride and the Mn cluster can be observed. In order to extract only the electron nuclear couplings between the Mn cluster, in the S2-state, and those of the halide, a comparison was done between chloride- and bromide-reconstituted samples. In salt-washed, CaC12- or CaBr2-reconstituted Photosystem-II membranes, frequencies similar to the Larmor frequency of bromide and chloride were detected. When CI- and Br- reconstituted PS-I1 membranes are compared to membranes that have also been cae+-depleted, and EGTA-treated, these features in the frequency spectra are absent. After chloride depletion at high pH in the presence of sulfate, no difference could be observed in the frequency spectra of bromide- and chloride-reconstituted samples. These results are discussed in view of possible different C1- binding sites in PS-II.
Photosystem-lI catalyzes light-driven water oxidation resulting in oxygen evolution. A cluster of 4 Mn located in the reaction centre of PS-II probably acts both as the active site and as a charge accumulating device of the water-splitting enzyme ([1,2] for reviews). During the enzyme cycle, the oxidizing side
Abbreviations:P68o, reaction centre chlorophyll (Chl) of Photosystem II (PS-II); Tyrz, the tyrosineacting as the electrondonor to P680;TyrD, the tyrosine acting as a side path electron donor of PS-ll; QA, primary quinone electron acceptor of PS-II; EPR, electron paramagnetic resonance; EDTA, ethylene diamine tetra acetic acid; EGTA, ethylene glycol bis(/3-aminoethyl ether)N.N,N',N'-tetraacetic acid; Mes, 2-(N-morpholino)ethanesulfonic acid; Hepes, N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid; PPBQ, phenyl-p-benzoquinone;X, symbol for CI or Br ; EXAFS, extended X-ray absorption fine structure; ESEEM, electron spin echo envelope modulation.
of PS-II goes through five different redox states that are denoted S,, n varying from 0 to 4 [3]. The oxygen is released during the S 3 to S o transition in which S 4 is a transient state. Three extrinsic polypeptides, with molecular masses of 17, 23 and 33 kDa, are bound to the PS-II reaction centre on the inside of the thylakoid membrane. The 17 and 23 kDa extrinsic polypeptides can be removed by NaCI washing (reviewed in Ref. [4]). Removal of these polypeptides results in inhibition of oxygen evolution due to an increased requirement for chloride and calcium ions (reviewed in Refs. [1,4-7]). In Ca2+-depleted PS-II, inhibition of the enzyme cycle occurs at the S 3 to S o transition [1,8-10]. The depletion of C I - from PS-II also results in inhibition of oxygen evolution (reviewed in Refs. [1,5,6,11,12]. Two different types of inhibition have been ob-
113(11-0104/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0301-0104(94)00419-6
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A. Boussac / Chemical Physics 194 (1995) 409-418
served, depending on the nature of the counterion used a n d / o r the severity of the C1- depletion. A decrease in oxygen evolution attributed to a slowdown of the S 3 to S O transition was reported when nitrate or acetate were used as the counterion [13]. In contrast, a total block in the S-state cycle was observed when using sulphate as a counterion [13]. The point at which the enzyme cycle is blocked by this kind of C1 depletion procedure has been shown to be also the S 3 to S O transition [10,14]. Ligands exchange experiments generally provide useful information on the roles and location of intrinsic ligands. In PS-II, it has been shown that bromide can replace chloride [15,16]. Two effects have been observed depending the treatment used to exchange chloride with bromide [17]. When chloride-depletion was done by a high pH treatment, no difference was found in the dissociation constant of the bromide and chloride required to reactivate oxygen evolution. The percentage of the activity which is restored is also similar. By contrast, when chloride depletion was done after the 17 and 23 kDa extrinsic polypeptides were removed, the dissociation constant of bromide becomes higher than that of chloride and, under saturating light conditions, all centers are active but the maximum activity restored with bromide is half of that obtained with chloride. This may indicate that the removal of the extrinsic polypeptides induces the depletion of a chloride binding site which is different from or is a modified version.of the CI site which is concerned by the high pH treatment. Until now there is no clear spectroscopic evidence indicating an interaction between chloride and the Mn. EPR performed at X- and S-band were unable to detect modifications in the hyperfine signal after a chloride/bromide exchange [18,19]. Indeed, chloride and bromide have two isotopes and the four nuclei have the same spin ( I = 3 / 2 ) but they possess different Larmor frequencies. The natural abundances and the Larmor frequencies at 3600 gauss are; 35C1, 75.77%, 1.50 MHz; 37C1, 24.23%, 1.26 MHz; 79Br, 50.69%, 3.85 MHz; 8~Br, 49.31%, 4.15 MHz. In this work, pulsed-EPR has been used to look for hyperfine couplings, between the Mn cluster and chloride, which may be too weak to be observed by cw-EPR. Chloride has been exchanged with bromide by 3 different procedures: (1) The removal of the 17 and 23 kDa polypeptides increases the Ca 2+ [1,4]
and chloride (or bromide) [6,20] requirement for oxygen evolution. Therefore, polypeptide-depletion by salt-washing has been done with NaCI or NaBr as the salt. Then the membranes were reconstituted with CaC12 or CaBr 2. (2) An EGTA-treatment has been used for the total depletion of Ca 2+ in the salt-washed PS-II [21] and no Ca2+-reconstitution accompanied halide-reconstitution after the salt-washing. (3) Chloride-depletion was done by a high pH treatment and the presence of sulfate followed by reconstitution with C1- or Br-. In each case, the ESEEM in the bromide and chloride reconstituted samples were compared.
1. Materials and methods
Photosystem-II membranes from spinach chloroplasts were prepared as previously described [9] and were either used immediately or stored in liquid nitrogen. For the C I - / B r - exchange procedure, the PS-II membranes were first resuspended and pelleted (i.e. washed) three times by successive centrifugations (15 min, 30000 g) in 25 mM Mes pH 6.5 and 0.3 M sucrose. Then, after the last centrifugation, the pellets were treated with 3 different protocols. The symbol 'X' replaces CI or Br- in the descriptions that follow.
1.1. The CaX2-reconstituted NaX-washed PS-H The pellet was resuspended and incubated under room light at 4°C in 0.3 M sucrose, 26 mM Mes pH 6.5 and 1.2 M NaBr or 1.2 M NaCI (Chl concentration ---0.5 mg/ml). After 30 min, 50 ixM EGTA was added and the salt-washed PS-II membranes were collected by centrifugation. The pellet was resuspended in 25 mM Mes-5 mM Ca(OH) 2 pH 6.5 (final pH adjustment with NaOH) and 10 Mm NaBr or NaCI respectively, centrifuged as above and resuspended once again in the same media at about 8 mg Chl/ml.
1.2. The EGTA(X)-treated NaX-washed PS-H The pellet was resuspended and incubated under room light at 4°C in 0.3 M sucrose, 25 mM Mes pH 6.5 and 1.2 M NaBr or 1.2 M NaCI (Chl concentra-
A. Boussac / Chemical Physics 194 (1995) 409-418
tion = 0.5 m g / m l ) . After 30 min, 5 mM EGTA was added. Then the membranes were collected by centrifugation, washed once again in 25 mM Mes(NaOH) pH 6.5, 5 mM EGTA and 30 mM NaBr or NaCI respectively and resuspended in the same media at about 8 mg Chl/ml. 1.3. The NaX-reconstituted SO 2 -- and high pHtreated PS-H
The pellet was washed in 0.3 M sucrose, 50 mM Na2SO4, 50 mM Hepes pH 7.5 and 100 IxM EDTA. Then the membranes were collected by centrifugation and resuspended in the same medium at about 8 mg C h l / m l without EDTA. After a 200 K illumination, the samples were thawed and 100 mM NaCI or NaBr (final concentration) were added directly to the samples in the EPR tubes. Then the samples were immediately re-frozen at 200 K then to 77 K. We have observed that freezing and thawing of samples at pH 7.5 can lead to some MnlI release from a fraction of centers in some preparations. This was monitored by the extent of the MnlI hexaquo EPR signal. When MnlI release was observed, the preparation was discarded. This monitoring of MnlI levels was only possible in the absence of EDTA. Therefore, EDTA was avoided in the final resuspending media. A second reason to avoid EDTA is the similarity between the MnlI-EDTA spectrum and the cytb559 signal in field-swept echo experiments (not shown). Cytb559 is associated with PS-II and, after chloride-depletion, is present in its oxidised, low spin (S = 1 / 2 ) form in the dark. Its EPR signal is therefore present in the baseline spectrum. The overlapping of both signals could complicate the interpretation since the MnlI-EDTA complex is a good electron donor to damaged PS-II in the light at room temperature (not shown) resulting in the formation of a MnlII-EDTA complex not detectable by EPR and therefore in an artefactual decrease of the baseline spectrum. In all the treatments, the highest purity NaC1 and NaBr salts commercially available (from Merck and Aldrich) were used. The PS-II membranes were put in quartz EPR tubes and, after dark adaptation for 1 hour at 0°C, 1 mM PPBQ (dissolved in DMSO) was added as an artificial electron acceptor. Then the samples were
411
immediately frozen at 200 K in a CO2-ethanol bath then transferred to 77 K. Illumination of the samples was done with an 800 W projector through water and infrared filters in a non-silvered dewar filled with ethanol cooled to 0°C or 200 K with solid CO 2. CW-EPR spectra were recorded at liquid helium temperatures with a Bruker ER 200D X-band spectrometer equipped with an Oxford Instruments cryostat. Pulsed EPR was done with a Bruker ESP 380 spectrometer already described [22]. The field swept spectra were obtained by measuring the amplitude of the echo as a function of the magnetic field after a two-pulse sequence ( ' r r / 2 - 2 0 0 ns-'rr). The duration of 'rr/2 and 'rr pulses were 8 and 16 ns respectively. The ESEEM data presented in this work result from a three-pulse sequence ( r r / 2 - z - ' r r / 2 - T - r r / 2 ) . The amplitude of the stimulated echo as a function of z + T was measured at 3600 gauss. ~- was chosen equal to 136 ns to suppress most of the weakly coupled proton frequency around 15 MHz at 3600 gauss. The minimum interpulse T was 64 ns and was incremented by steps of 8 ns. The "rr/2 pulse duration was 16 ns. To remove the unwanted echoes in a three-pulse experiment, the phase cycling procedure described in Ref. [23] was used.
2. Results 2.1. The CaX2-reconstituted NaX-washed PS-H
The formation of the S2-state in the CaC12 and CaBr 2 reconstituted salt-washed PS-II was monitored by cw- and field-swept echo EPR (Fig. 1). Spectra a were recorded in dark-adapted samples. The spectra recorded in the dark contains the following features; (1) the intense TyrD ° signal at g = 2.0045 ( = 3350 gauss) which is excised from all spectra shown; (2) the EPR signal from the oxidised Cytb559 which, under the conditions shown, is most evident at its gz line at g = 2.93 ( = 2200 gauss); (3) relatively intense signals, in cw-EPR, at low field arise from contaminating Fe 3+ (S = 5 / 2 ) in different environments. Spectra b were recorded after a 200 K illumination. Both samples exhibited a very similar Mn-multiline signal characteristic of the S 2state (Fig. 1, lower panels). Only a very small g = 4
412
A. Boussac / Chemical Physics 194 (1995) 409-418
NaCl-wsshed+ CaCl=~
I
0
0
1000 2000 3000 4000 Magnetic field (gauss)
1000 2000 3000 4000 Magnetic field (gauss)
NaCI-washed+ CaCI
NaBr-washed+ CaBr.
iiii
0
1000 2000 3000 4000 Magnetic field (gauss)
0
iii
ii h
iii
iiiiihll
~1 i t i i h
iiitllll
~1 i i i i
iii
1000 2000 3000 4000 Magneticfield (gauss)
Fig. 1. CW-EPR spectra (lower panels) and field swept echo (upper panels) of CaC12-reconstituted NaCl-washed PS-II (left panels) and CaBr2-reconstitutedNaBr-washed PS-II (right panels). Spectra a were recorded in dark-adapted samples i.e. in the S1 state. Spectra b were recorded after a 200 K illumination i.e. in the S2 state. Instrument settings for cw-EPR: temperature, 10 K; modulation amplitude, 22 gauss; microwave power, 20 mW; microwave frequency, 9.4 GHz; modulation frequency, 100 KHz. The central parts of the spectra correspondingto the TyrD° region were deleted. Instrument settings for pulsed-EPR: the amplitude of the echo results from a two-pulse sequence ('rr/2 = 8 ns, r = 200 ns, w = 16 ns); temperature, 4-5 K; microwave frequency, 9.7 GHz. The small distortion in the baseline of spectrum a in the right, lower panel is due to a small amount of oxygen. No normalization procedure was used to plot the field-swept spectra.
signal ( = 1500 gauss) was formed in these samples. The 200 K illumination-induced increase, over the Cytb559 signal, in the field swept echo signal is very similar in C I - and B r - reconstituted samples (Fig. 1, upper panels). Thus it seems that the exchange of chloride with bromide did not affect the yield of the S 1 to S 2 transition under continuous illumination at 200 K in these membranes and no spectral modifica-
tions were observed. After a salt-washing, Cytb559 is almost totally oxidized. Therefore, spectra a, in Fig. 1, correspond to = 1 spin (S = 1 / 2 ) per reaction center. The light-induced signals (spectra b) correspond to approximately the same number of spins. By recording the oxygen evolution under various light intensity, it was found that all the centers are active in the presence of bromide or chloride (not shown). Nevertheless, in bromide reconstituted samples the limiting step in the oxygen evolution process is two times slower than in the chloride-reconstituted sample as already observed [17]. In order to detect the couplings between the Mn cluster in the S2-state and the bromide or chloride nuclei, the ESEEM at 3600 gauss in CaCI 2- and CaBr2-reconstituted salt-washed PS-II was measured. At this magnetic field, the Fe 2+ QA signal formed by the 200 K illumination contributes to the cw-EPR spectra. Nevertheless, the very fast relaxation of the Fe2+ QA species prevents its detection by pulsed-EPR at the temperature used in the present study. The ESEEM recorded in CaC12- and CaBr2-reconstituted salt-washed PS-II (Fig. 2, the two upper panels) seem very similar and in the Fourier transform spectra of both ESEEM, no major differences in frequencies are evident due to the complexity of the S 2 spectra (not shown but see for example Refs. [22,24,25]). Therefore, as already described in the literature (see for example Refs. [23,26]), the quotient and the difference of the ESEEM were used. These procedures should reveal only the differences due to the chloride/bromide exchange. Fig. 2 (the two lower panels) shows both the quotient and the difference of the two ESEEM obtained in CaCI 2and CaBre-reconstituted salt-washed PS-II. Some modulations, which are similar in the quotient and the difference, appear clearly with a maximal amplitude which accounts for about 15% of the total ESEEM. Fig. 3 shows the frequency spectrum which was obtained by taking the real part of the Fourier transform of the time domain of the difference of the ESEEM. The positive and negative peaks may be attributed to the couplings between the Mn cluster in the S 2 state and bromide and chloride respectively. This frequency spectrum presents large positive features around 4 MHz and negative features between 1 and 2 MHz. Smaller positive and negative peaks at around 7.5 and 2.5 MHz respectively could also be
A. Boussac/ Chemical Physics 194 (1995) 409-418
413
significant if they are not due to a b a s e l i n e - r o l l i n g w h i c h occurs w h e n a d e a d - t i m e reconstruction cannot be done as it is the case here. The E S E E M recorded in dark-adapted samples, i.e. mainly on the Cytb55, ~ signal, reveals no difference b e t w e e n the two samples (not shown). W e 1
CaB~
~oe @
~0.6
aE 0.4 ~ 0.2 W
o
3ooo
,ooo
J l l l l l l l l i L J l i l J ~ l l J J l l L L I l l l l l i l l l l J l l
tau + T (nil)
1
CaCI 2
=.
0
5
10
15
20
Frequency ( M H z )
~0.8 O
~ 0.6
Fig. 3. Real part of the Fourier transform of the difference ESEEM (CaBr 2 minus CaCI 2) shown in the lower panel of Fig. 2. No dead-time reconstruction was done in this particular case. The dashed lines is drawn to visualize the zero amplitude level.
~ 0.4
~ 0.2 W
o
1000
2000 tau + T (nil)
3000
4000
1.2 U.I
~ 1.1
~ 0.9 0.8
14J
i
t
1000
i
c.rc.c,2II I
i
2000 tau + T (ns)
I
3000
4000
0.06 0.04
~6 0.02
~
o
-0.02 • -0.04
-0.06 1000
2000 tau + T (ns)
3000
4000
Fig. 2. Three pulse ESEEM in CaClz-reconstituted NaCl-washed PS-II and CaBr2-reconstituted NaBr-washed PS-II recorded in the S z state at a magnetic field of 3600 gauss and a microwave frequency of 9.7 GHz. The offset corresponding to the amplitude of the echo when all the modulations are damped has been subtracted. The amplitude of the echo results from the sequence: '~/2 = 16 ns, 7=136 ns, 'rr/2 = 16 ns, T m i n =64 ns, "~/2 = 16 ns. The two lower panels represent respectively the quotient and the difference of the two ESEEM represented above.
cannot rule out a small c o n t a m i n a t i n g i r o n - s u l f u r signal at about g = 1.9. Nevertheless, CaCI 2- and CaBr2-reconstituted salt-washed m e m b r a n e s arise f r o m the s a m e PS-II preparation and all contaminating species are not e x p e c t e d to contribute in the difference signals.
2.2. The EGTA(X)-treated NaX-washed PS-II T o study the effect of Ca2+-depletion on the c h l o r i d e / b r o m i d e e x c h a n g e , the Ca2+-depletion procedure was a c c o m p a n i e d by an E G T A treatment. This treatment results in m e m b r a n e s in w h i c h the S 2 state is stable in the dark and exhibits a m o d i f i e d M n multiline signal [21]. The E G T A treatment was done so that essentially all the centers w o u l d be inhibited, a situation w h i c h is not necessarily a c h i e v e d in the absence o f the chelator w h e r e typically 20% o f the centers remain active in o x y g e n e v o l u t i o n [9]. Fig. 4 ( l o w e r panels) s h o w s the typical c w - E P R spectra in such a preparation. Spectra a correspond to the m o d i fied multiline signal arising f r o m the stable S 2 state. Spectra b w e r e recorded after a 0°C illumination and exhibit the split S 3 signal. The upper panels correspond to the field-swept spectra recorded on the
414
A. Boussac / Chemical Physics 194 (1995) 409-418
s a m e samples and shows, as p r e v i o u s l y reported, that the S 3 signal appears on top o f the S 2 signal with a similar s t o i c h i o m e t r y to that already o b s e r v e d [22]. N o difference can be o b s e r v e d in the p r e s e n c e o f chloride or b r o m i d e . Fig. 5 s h o w s for both samples, in the S 2 state, the E S E E M recorded at 3600 gauss and their quotient. The quotient only reveals a small m o d u l a t i o n (around 15 M H z ) c o r r e s p o n d i n g to
1
Br
=, 0.8 I
0.6
0.4 ¢~ 0.2 LU
0 1000
0
[NaCl-+~lshed
~
2000 3000 tau + T (ns)
c1
[ NaBr-~+sh.~ "
4000
0.8
~ 0.6 E
0.4
t~
LU ¢n 0.2 i.u 1000
2OOO
3OOO
4O00
tau + T (ns) 0
1000 2000 3000 4000
0
Magnetic field (gauss) NsCI-washed
2.5
1000 2000 3000 4000
Magnetic field (gauss) NaBr-wa+shed
Ib
uJ 2 (/) LU "6 "E 1.5
1
0" 0.5
,
i
i
1000
2000
i
i
3000
4000
tau + T (ns) hl . . . . . .
0
in iii iii i thllllll
i i h i i i II I I I h l l l l l
1000 2000 3000 4000 Magnetic field (gauss)
tl
hi .......
0
I'''"l'
' t h 11 ' l l l l l h l l l l
I Illhl
Illlll
1000 2000 3000 4000 Magnetic field (gauss)
Fig. 4. CW-EPR spectra (lower panels) and field swept echo (upper panels) of EGTA-Cl-treated NaCl-washed PS-II (left panels) and EGTA-Br-treated NaBr-washed PS-II (right panels). Spectra a were recorded in dark-adapted sample i.e. in the S 2 state. Spectra b were recorded after a 0°C illumination i.e. in the S 3 state. Instrument settings for "cw-EPR: temperature, 10 K; modulation amplitude, 22 gauss; microwave power, 20 mW; microwave frequency, 9.4 GHz; modulation frequency, 100 KHz. The central parts of the spectra corresponding to the TyrD° region were deleted. Instrument settings for pulsed-EPR: the amplitude of the echo results from a two-pulse sequence (-rr/2 = 8 ns, ~"= 200 ns, ir = 16 ns); temperature, 4-5 K; microwave frequency, 9.7 GHz. No normalization procedure was used to plot the field-swept spectra.
Fig. 5. Three pulse ESEEM in EGTA-Br-treated NaBr-washed PS-II (upper panel) and EGTA-Cl-treated NaCl-washed PS-II (middle panel) recorded in the S 2 s t a t e at a magnetic field of 3600 gauss and a microwave frequency of 9.7 GHz. The offset corresponding to the amplitude of the echo when all the modulations are damped has been subtracted. The amplitude of the echo results from the sequence: ~r/2 = 16ns, ~-= 136 ns, "n/2 = 16 ns, Train = 64 ns, I r / 2 = 16 ns. The lower panels represent the quotient of the two ESEEM represented above.
w e a k l y coupled protons. A low a m o u n t o f w e a k l y coupled protons is still o b s e r v e d because (1) the tau v a l u e o f 136 ns does not totally r e m o v e the 15 M H z f r e q u e n c y at 3600 gauss and (2) s o m e additional m o d u l a t i o n s attributed to c o u p l i n g s with the nitro-
A. Boussac / Chemical Physics 194 (1995) 409-418
415
2.3. The NaX-reconstituted S042 - and high pHtreated PS-H
gen(s) of EGTA [22] are present. This increases the difficulty of choosing the correct offset (i.e. the amplitude of the echo when all modulations are damped) before ratioing or subtracting the ESEEM. Therefore, the quotient and difference procedures are less effective in removing identical frequencies in the two ESEEM. Nevertheless, the main conclusion is that no modulations arising from an halide exchange can be observed.
Washing of PS-II membranes at pH 7.5 in the presence of sulfate results in an inhibition of oxygen evolution which can be reversed by addition of chloride. It has been shown that bromide has the same efficiency as chloride to reconstitute oxygen evolution after a high pH treatment [17]. The multi-
i I I [ l l l l l l l l l l l l l l l l l l l l l l l l
2000
3000
4000
Magnetic field (gauss) Br b
0
1000 2000 3000 4000 Magnetic field (gauss)
0
1000 2000 3000 4000 Magnetic field (gauss)
Fig. 6. Upper panel: cw-EPR spectra of sulfate and high pH-treated PS-II after the addition of 100 mM NaCI or NaBr following a 200 K illumination. Instrument settings: temperature, 10 K; modulation amplitude, 22 gauss; microwave power, 20 mW; microwave frequency, 9.4 GHz; modulation frequency, 100 kHz. The central parts of the spectra corresponding to the TyrD ° region were deleted. Lower panels: field swept echo of sulfate and high pH-treated PS-II dark-adapted i.e. in the S l state (spectra a) or after the addition of 100 mM NaC1 (left panel) or NaBr (right panel) following a 200 K illumination, i.e. in the S 2 state (spectra b). Instrument settings for pulsed-EPR: the amplitude of the echo results from a two-pulse sequence (~r/2 = 8ns, r = 200 ns, 7r = 16 ns); temperature, 4 - 5 K; microwave frequency, 9.7 GHz. No normalization procedure was used to plot the field-swept spectra.
A. Boussac / Chemical Physics 194 (1995) 409-418
416
line signal is formed by CI- addition after 200 K illumination [27]. The ability of bromide versus chloride to induce the appearance of the EPR signals characteristic of the S 2 state is shown in Fig. 6. The multiline signals (upper panel) detected by cw-EPR and the field swept spectra (lower panels) show that
ar
i
0.6 0.8 0.4 0.2 0 1ooo
2000
3000
4000
tau + T (ns) CI
~' 0.8 08
the S 2 signals were similar when bromide and chloride were added after a 200 K illumination (see also Refs. [18,20,28]). In the conditions used here, the S 2 Mn signal is smaller than that generated by 200 K illumination of a CI- reconstituted sample. This is expected given the time required for mixing and is similar to the situation seen under these conditions earlier [27,29]. Two other effects contribute to the smaller size, when compared to untreated samples, of the light-induced signal in Fig. 6. (1) After chloride-depletion, TyrD ° becomes reduced in 20-30% of the centers (not shown). Illumination at 200 K results in the oxidation of TyrD in at least a fraction of these centers instead of S 2 formation. (2) The protocol used for C1 -depletion results in an irreversible inhibition of a small proportion of centers [10]. Fig. 7 shows the ESEEM measured at 3600 gauss in samples where bromide and chloride were added after a 200 K illumination. The lower panel shows the quotient of the ESEEM. No modulation due to the bromide/chloride exchange is detected.
,-i
~-
0.4
u,I
3. D i s c u s s i o n
uJ 0.2 0 0
1000
2000
3000
4000
tau + T (ns) 1.4
Br/CI
LU tn LU 1.2
0
0.8 0.6
0
1000
2000
tau
3000
4000
+ T (ns)
Fig. 7. Three pulse ESEEM in NaBr-reconstituted (upper panel) and NaCl-reconstituted (middle panel) after a 200 K illumination, i.e. in the S 2 state, of sulfate and high pH-treated PS-II at a magnetic field of 3600 gauss and a microwave frequency of 9.7 GHz. The offset corresponding to the amplitude of the echo when all the modulations are damped has been subtracted. The amplitude of the echo results from the sequence: 9 / 2 = 16 ns, "r = 136 ns, 9 / 2 = 16 ns, Tmin = 64 ns, 'rr/2 = 16 ns. The lower panels represent the quotient of the two ESEEM represented above.
In order to detect small hyperfine couplings between the Mn cluster and chloride, a chloride/bromide exchange has been performed with three different biochemical procedures. Comparison of the ESEEM in CaCl2-reconstituted NaCl-washed PS-II and CaBr2-reconstituted NaBr-washed PS-II reveals differences in the frequency spectra. Since, in Fig. 3, the only difference between the two samples is the halide status, it is reasonable to suggest that these differences result directly from the change in the nature of the halide. Since the larger positive frequencies correspond to those expected for the Larmor frequency from bromide while the larger negative frequencies are in the range expected for the Larmor frequencies of chloride, it is also reasonable to attribute these frequencies to hyperfine coupling between the Mn and the halides. We cannot rule out that the CI / B r - exchange perturbs the Mn cluster resulting in changes in native couplings to e.g. nitrogen which give difference features which are coincidentally identical to those expected from C I - / B r - exchange. Moreover, the
A. Boussac / Chemical Physics 194 (1995) 409-418
small features at around 7.5 and 2.5 MHz in Fig. 3 could be due to perturbations induced by the chloride/bromide exchange. The results would then constitute a spectroscopic demonstration of a bromideinduced perturbation of the Mn cluster. After the depletion of Ca 2+ and the binding of EGTA, the couplings seen above are absent. There are several possible explanations for this: (1) The Ca 2+ depletion prevents the binding of chloride. This fits with the model in which Ca 2÷ acts as a locus for C1- binding close to the Mn [2]. However, Ca 2~ depletion could affect C1- binding through a secondary structural effect. Such an observation was already made in chloride-depleted samples in which Ca 2÷ was found to modulate a proportion of the activity in chloride-reconstituted samples [10,17]; (2) The couplings between the Mn cluster and the halides are sufficiently modified so that they are not detectable; (3) EGTA perturbs the chloride site. Indeed, it has been shown that EGTA binding to PS-II decreased in the absence of C1- which reveals a relationship between the chloride and EGTA binding sites [30]. After the chloride removal by treatment at high pH in the presence of sulfate, the couplings between the Mn and the chloride (or bromide) which is required for the reactivation of oxygen evolution and the appearance of the S 2 EPR signals, cannot be detected by pulsed EPR. This suggests that either the corresponding chloride binding site is too far from the Mn cluster or that chloride is a ligand of the Mn cluster itself. In the latter case, the resulting couplings are predicted to be undetectable by pulsed-EPR from simulation of interactions between a spin S = 1 / 2 and a nucleus with a spin I = 3 / 2 [31]. Direct indications of CI binding to the Mn have not been obtained by chloride/bromide exchange experiments in EXAFS [32]. This may indicate that chloride is not a ligand of the Mn, at least in the redox states of the Mn tested [32]. Nevertheless, the same authors have observed a modification of the EXAFS features after replacement of chloride by fluoride and interpreted this as indicating that fluoride binds to one di-&-oxo bridged Mn dimer. From the differences between the three biochemical procedures used in the present study, one possible explanation is that two types of chloride binding site exist: (1) A chloride site in which the chloride(s)
417
is (are) exchangeable after a high pH treatment and which is (are) not detected by pulsed EPR. This chloride could be a direct ligand of the Mn cluster for the reasons explained above. (2) A chloride site which is exchanged after depletion of the 17 and 23 kDa polypeptides and in which chloride(s) is (are) detected by pulsed EPR. As the value of the coupling of this chloride with the Mn cluster is similar to the Larmor frequency of CI-, it is reasonable to assume that the binding site of this chloride is not in the first coordination shell of the Mn cluster. One (the latter) or both types of chlorides can be exchanged after a salt-washing. Alternatively, only one kind of chloride site might be present but it is modified by the salt-washing procedure.
Acknowledgements A.W. Rutherford and S. Un are gratefully acknowledged for stimulating discussions and careful reading of the manuscript.
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