Skip to main content
NCBI home page
Philosophical Transactions of the Royal Society B: Biological Sciences logoLink to Philosophical Transactions of the Royal Society B: Biological Sciences
. 2007 Oct 25;363(1494):1203–1210. doi: 10.1098/rstb.2007.2216

Low-temperature photochemistry in photosystem II from Thermosynechococcus elongatus induced by visible and near-infrared light

Alain Boussac 1,*, Miwa Sugiura 2, Thanh-Lan Lai 1, A William Rutherford 1
PMCID: PMC2614093  PMID: 17965006

Abstract

The active site for water oxidation in photosystem II (PSII) consists of a Mn4Ca cluster close to a redox-active tyrosine residue (TyrZ). The enzyme cycles through five sequential oxidation states (S0 to S4) in the water oxidation process. Earlier electron paramagnetic resonance (EPR) work showed that metalloradical states, probably arising from the Mn4 cluster interacting with TyrZ·, can be trapped by illumination of the S0, S1 and S2 states at cryogenic temperatures. The EPR signals reported were attributed to S0TyrZ·, S1TyrZ· and S2TyrZ·, respectively. The equivalent states were examined here by EPR in PSII isolated from Thermosynechococcus elongatus with either Sr or Ca associated with the Mn4 cluster. In order to avoid spectral contributions from the second tyrosyl radical, TyrD·, PSII was used in which Tyr160 of D2 was replaced by phenylalanine. We report that the metalloradical signals attributed to TyrZ· interacting with the Mn cluster in S0, S1, S2 and also probably the S3 states are all affected by the presence of Sr. Ca/Sr exchange also affects the non-haem iron which is situated approximately 44 Å units away from the Ca site. This could relate to the earlier reported modulation of the potential of QA by the occupancy of the Ca site. It is also shown that in the S3 state both visible and near-infrared light are able to induce a similar Mn photochemistry.

Keywords: photosystem II, oxygen evolution, electron paramagnetic resonance, Mn photochemistry

1. Introduction

Photosystem II (PSII), the enzyme responsible for oxygen evolution, catalyses light-driven water oxidation using a Mn4Ca cluster that acts as both a device for accumulating oxidizing equivalents and the active site. The structure and ligand environment of the Mn4Ca cluster is still unclear despite recent advances in X-ray crystallography (Ferreira et al. 2004; Loll et al. 2005) and in Mn-X-ray absorption fine structure (EXAFS) (Yano et al. 2006; Pushkar et al. 2007). Absorption of a photon by PSII induces a charge separation that results in the oxidation of a chlorophyll molecule (P680) and the reduction of a pheophytin molecule. The pheophytin anion transfers the electron to a quinone, QA, and P680+ oxidizes a tyrosine residue of the D1 polypeptide, tyrosine residue (TyrZ), which in turn oxidizes the Mn4Ca cluster and the associated substrate water. During the enzyme cycle, the oxidizing side of PSII goes through five sequential redox states, denoted Sn where n varies from 0 to 4. Oxygen is released during the S3 to S0 transition in which S4 is a transient state (Debus 2001; Diner 2001; Goussias et al. 2002; Britt et al. 2004; McEvoy & Brudvig 2004; Rutherford & Boussac 2004; Sauer & Yachandra 2004).

When either of the essential ions Ca2+ or Cl− is removed from its site, manganese oxidation can still take place, allowing the formation of the S2 state, but in the following step, the normal S3 state is not formed. Instead, an alternative abnormally stable form appears to be induced (Boussac et al. 1989). This state is thought to be the one in which the metal cluster is in the same redox state as it was in S2 state but in addition a nearby radical is also present (Boussac et al. 1989). This radical is proposed to be the tyrosine residue TyrZ (e.g. Tang et al. 1996; Un et al. 2007; see also Mino & Itoh 2005). In this state, the Mn4Ca cluster (S=1/2) and TyrZ· (S=1/2) magnetically interact, giving rise to a characteristic electron paramagnetic resonance (EPR) signal that is known as the split signal. More recently, several other ‘split’ signals were also observed in functional PSII. They were generated by illuminating PSII in the S0, S1 or S2 states with visible light or in the S3 state with near-infrared (NIR) light either at liquid helium temperatures or at 200 K. The species that were formed under these conditions were proposed to correspond to ‘non-relaxed’ S0TyrZ· S1TyrZ· and S2TyrZ· states (Ioannidis et al. 2002; Koulougliotis et al. 2003, 2004; Zhang & Styring 2003; Zhang et al. 2004; Petrouleas et al. 2005; Havelius et al. 2006; Ioannidis et al. 2006) that could correspond to intermediates in the oxygen-evolving process.

It has been shown that the biosynthetic replacement of Ca2+ by Sr2+ in the thermophilic cyanobacterium Thermosynechococcus elongatus had multiple effects on kinetics properties of the enzyme probably arising from Sr2+-induced S-state-dependent shifts in the redox properties of both the manganese complex and the TyrZ (Boussac et al. 2004). In this work we report fully resolved spectra from the split signals arising in oxygen-evolving Ca- and Sr-containing PSII isolated from T. elongatus. In order to obtain spectra without spectroscopic overlap from TyrD·, we used the D2-Y160F mutant of PSII which was engineered to remove TyrD· (Sugiura et al. 2004). In addition we report a study of the different effects of NIR and visible light in the S3 state. Lastly and somewhat surprisingly, the replacement of Ca2+ by Sr2+ appears to influence the non-haem iron which is located on the other side of the reaction centre protein.

2. Material and methods

His-tagged wild-type (WT) (Sugiura & Inoue 1999) and His-tagged D2-Y160F (Sugiura et al. 2004) T. elongatus cells were grown in the presence of either Ca or Sr salts (Boussac et al. 2004) and PSII were purified as previously described (Boussac et al. 2004) with the following modifications. The presence of glycerol prevents the detection of the split signals induced by visible light (Ioannidis et al. 2002; Zhang & Styring 2003; Havelius et al. 2006), therefore, glycerol was omitted from the elution of the PSII cores from the Ni column and in the final re-suspension medium which contained 1 M betaine, 40 mM Mes, 15 mM MgCl2 and 15 mM CaCl2 (the pH was adjusted with NaOH to 6.5). The PSII samples were stored in liquid nitrogen at a concentration of approximately 2 mg of Chl ml−1 until they were used.

Continuous wave-EPR (cw-EPR) spectra were recorded using a standard ER 4102 (Bruker) X-band resonator with a Bruker Elexsys 500 X-band spectrometer equipped with an Oxford Instruments cryostat (ESR 900). Flash illumination at room temperature was provided by an Nd : YAG laser (532 nm, 550 mJ, 8 ns Spectra Physics GCR-230-10). PSII samples at 1.2 mg of Chl ml−1 were loaded under dim light into quartz EPR tubes and further dark adapted for 1 hour at room temperature. Then, the samples were synchronized in the S1 state with one pre-flash (Styring & Rutherford 1987). After another dark period for 1 hour at room temperature, 0.5 mM PPBQ dissolved in Me2SO was added (the final concentration of Me2SO was approx. 2%). After illumination by 0, 1, 2 or 3 flashes to form the S1, S2, S3 and S0 states, respectively, the samples were frozen in the dark at 198 K in a dry ice ethanol bath, degassed at 198 K and then transferred to 77 K. NIR illumination of the samples was done directly in the EPR cavity and was provided with a laser diode emitting at 820 nm (Coherent, diode S-81-1000C) with a power of 600–700 mW at the level of the sample. Illumination of the samples with visible light was done with a 250 W projection lamp filtered with water, five calflex filters and a Corion LS-650 filter resulting in a broadband illumination from 400 to 650 nm (designated visible light hereafter). Illumination with visible light at 180–200 K of the samples was done in a nitrogen gas flow system (Bruker, B-VT-3000).

3. Results and discussion

The Sn states were generated by laser flashes illumination at room temperature in D2-Y160F PSII containing either Ca (black spectra) or Sr (red spectra). In figure 1a, PSII in the S0 state were illuminated at 4.2 K for 1–2 s by visible light. EPR spectra were recorded immediately after this short illumination and then after dark adaptation for 2–4 min at 4.2 K. The signal that decayed during this dark period is shown in figure 1a. On the time scale required for the recording of these spectra, the small amount of Car·+/Chl·+ radicals generated in a fraction of centres by the low-temperature illumination is virtually stable (not shown), so that the difference spectra shown in figure 1a and attributed to S0TyrZ· (Zhang & Styring 2003) are essentially without any contribution from these free radical species. The spectra in Ca- and Sr-PSII are similar but not identical. The width (low-field peak to high-field trough) varies from 130 gauss in Sr-PSII to 146 gauss in Ca-PSII. This latter value found in the cyanobacterium studied here is slightly smaller than that found in plant Ca-PSII (approx. 165 gauss; Havelius et al. 2006).

Figure 1.

Figure 1

Open in a new tab

Split EPR spectra in D2-Y160F-PSII with either Ca (black spectra) or Sr (red spectra) in the Mn4 cluster. For (a–c), the spectra were first recorded immediately after the 400–350 nm illumination (light spectra). Then, the black spectra were recorded after 2–4 min at 4.2 K. The light-minus-dark difference spectra are shown in (a) S0, (b) S1, (c) S2. For (d) S3, the dark spectra were recorded prior to the NIR illumination. Instrument settings: modulation amplitude, 10 G; microwave power, 20 mW; microwave frequency, 9.4 GHz; modulation frequency, 100 kHz and temperature, 4.2 K. The chlorophyll concentration was 1.2 mg ml−1. In each panel, spectra were roughly scaled to the same spin concentration as estimated by a double integration.

Figure 1b shows the spectra induced by an illumination with visible light at 4.2 K in the S1 state. The protocol was identical to that described in figure 1a. Spectra in Ca- and Sr-PSII attributed to S1TyrZ· (Zhang & Styring 2003) are similar but not identical. This split signal is slightly broader in Sr-PSII (87 gauss, low-field peak to high-field trough) than in Ca-PSII (73 gauss). In plant Ca-PSII, the width was found to be slightly narrower (approx. 68 gauss; Havelius et al. 2006).

In figure 1c, the Ca-PSII sample trapped in the S2 state was illuminated in the visible range at 200 K (note the different magnetic field scale in this figure). In plant PSII, this protocol was shown to be able to induce a split signal attributed to S2TyrZ· and that decays in the minutes time scale by charge recombination at 4.2 K with the electron from QA− (Ioannidis et al. 2006). Similar behaviour occurs in Ca-PSII from T. elongatus (not shown). The Car·+/Chl·+ radicals generated in a fraction of centres by the 200 K illumination were stable at 4.2 K in the time scale where most of the split signal decayed (not shown), so that the difference spectrum shown in figure 1c (black spectrum) is not contaminated by these free radical species. In Sr-PSII, we found that the 200 K illumination induced only a very weak signal (not shown). Therefore, several other temperatures were tested and we found that a signal was induced with its highest amplitude (although still weaker than that in Ca-PSII) upon illumination at 180 K (N.B. in each panel of figure 1 the spectra are normalized, see the legend). A minor contribution from another free radical signal may be present in the weaker S2TyrZ· signal in Sr-PSII (red spectrum, figure 1c). Clearly, the presence of Sr in the place of Ca changes the enzyme in such a way that its temperature dependence is affected. In plant Ca-PSII, the low-field peak to high-field trough spacing was 116 gauss (Ioannidis et al. 2006) and is found here in T. elongatus to be 110 gauss in Ca-PSII and 100 gauss in Sr-PSII.

The split signals shown in figure 1a–c in T. elongatus were generated by a charge separation triggered by visible light at low temperature and are interpreted as originating from the S0TyrZ· (figure 1a), S1TyrZ· (figure 1b) and S2TyrZ· (figure 1c) states in accordance with the literature reports in plant PSII (Ioannidis et al. 2002; Koulougliotis et al. 2003; Zhang & Styring 2003; Zhang et al. 2004; Petrouleas et al. 2005; Havelius et al. 2006; Ioannidis et al. 2006). The use of the D2-Y160F mutant allowed us to detect the central part of the spectra without any additional treatment like those used in plant PSII to reduce TyrD· (Havelius et al. 2006).

An S3TyrZ· state has yet to be reported. In S3, NIR illumination at 4.2 K does result in the formation of a split signal (Petrouleas et al. 2005). However, this signal was attributed to S2TyrZ· formed by NIR-induced conversion of the Mn cluster into a state so oxidizing that the back reaction, S3TyrZ to S2TyrZ·, occurs (Petrouleas et al. 2005). The split signals generated in the S3 state by NIR illumination in Ca-PSII (218 gauss width) and Sr-PSII (295 gauss width) are shown in figure 1d. The use of the D2-Y160F mutant makes the detection of these spectra possible without any contributions from any free radicals generated by the reaction centre photochemistry. The two spectra present some similarities in their structure, but clearly the signal is broader in Sr-PSII than in Ca-PSII.

The miss parameter and the S-state composition in dark adapted samples upon a pre-flash illumination have been shown to be very similar in Ca- and Sr-containing PSII (Boussac et al. 2004), so that a different S-state population in these two PSII samples after 0, 1, 2 and 3 flashes cannot explain the spectral differences detected in figure 1. There are several further arguments in support of the conclusion that the spectral differences between Ca- and Sr-PSII shown for each S state are real effects due to the metal exchange rather than mixing S states. (i) The S2 split signal is light induced at a temperature much higher than that at which the S1 split signal can be generated, so that we can exclude contributions from the S1 split signal in the spectra of figure 1c. (ii) The S3 split signal is induced by NIR illumination at 4.2 K in the sample in the S3 state. Thus, in figure 1d, contributions from both S1 and S2 split signals, which are induced only by visible light, are unlikely. (iii) In the S0 state, the effect of Sr exchange is a slight narrowing of the split signal (figure 1a). In contrast, in the S3 state, the split signal generated (in figure 1d by NIR light but a similar effect is seen with visible light, see figure 5) in Sr-containing PSII is markedly broader than that in the Ca-containing PSII. Thus, contributions from the centres starting in the S3 state (after two flashes) to the spectra shown in figure 1a (after three flashes) are not detectable.

Figure 5.

Figure 5

Open in a new tab

Effect of illumination with visible and NIR light in S3, in WT PSII, in samples pre-illuminated at 180 K. In (a), the S3 sample illuminated at 180 K with visible light was further illuminated with visible light at 4.2 K (red spectrum). Then blue spectrum was recorded after a dark period of 4 hours at 4.2 K. Green spectrum was then recorded after a further NIR illumination for 2 min at 4.2 K. In (b), the S3 sample illuminated at 180 K with visible light was illuminated with visible NIR light at 4.2 K (red spectrum). Then blue spectrum was recorded after a dark period of 2 hours at 4.2 K. Green spectrum was then recorded after another NIR illumination for 2 min at 4.2 K and the black spectrum after a further illumination with visible light. Instrument settings are same as in figure 2. The central part of the spectra corresponding to the TyrD· signal was deleted. The insets show an expanded view of the split signal magnetic field region.

We can conclude that in addition to the well-known effect of the Ca/Sr exchange on the EPR properties of the S2 state (Boussac & Rutherford 1988; Boussac et al. 2004), figure 1 shows that Ca/Sr exchange also affects the magnetic properties of the Mn4 cluster in all the Sn states, and/or the magnetic coupling between the cluster and TyrZ·. All of these split signals are different from those formed in PSII inhibited by either acetate treatment or Ca depletion and also slightly differ from those in PSII from plants. The fully resolved split signals presented here should be used in future work to characterize structural changes in the TyrZ–Mn4 environment occurring in the different states.

NIR illumination in the S3 state (black spectrum) produces a complex signal (red spectrum; figure 2). In addition to the split signal with a peak to trough width of 176 gauss, signals at lower field and higher field were also induced. The NIR-induced spectrum (blue spectrum) exhibits a feature which peaks at g≈5.20 (turning point at g≈4.35) and a smaller peak at g≈1.51 (Boussac et al. 2000). The signal at g≈5.20 is thought to correspond to a spin 7/2 state of the Mn cluster (Sanakis et al. 2001).

Figure 2.

Figure 2

Open in a new tab

Effect of near-infrared light (820 nm) in S3 in D2-Y160F-PSII. The black spectrum was recorded in S3 and the red spectrum was recorded after an additional illumination at 820 nm at 4.2 K in the EPR cavity for 2 min. The blue spectrum is the difference spectrum after-minus-before the NIR illumination. Instrument settings: modulation amplitude, 25 G; microwave power, 20 mW; microwave frequency, 9.4 GHz; modulation frequency, 100 kHz; and temperature, 4.2 K. The chlorophyll concentration was 1.2 mg ml−1.

In our conditions the NIR-induced split signal was stable for at least 4 hours at 4.2 K after the NIR illumination (not shown). This result contrasts somewhat with previous reports in which a decay of this split signal was observed (Petrouleas et al. 2005). This discrepancy could originate from a difference in the quality of light, i.e. visible light filtered by an RG 715 Schott filter (Petrouleas et al. 2005) versus 820 nm laser light used here. Alternatively, the different protocol used to generate the S3 state could explain the different stabilities of the signal, i.e. the continuous illumination at 240 K resulting in a reduced electron acceptor side (Petrouleas et al. 2005) versus flash illumination at room temperature resulting in an oxidized electron acceptor side as used in this work. In figure 3 the effects of light in the NIR and visible range were therefore tested separately in the S3 state. The black trace shows the formation of the split signal (at the magnetic field position indicated by the arrow in figure 2) under continuous illumination at 820 nm at 4.2 K. Then, following the NIR illumination, the sample was further illuminated with visible light (400–650 nm; red trace). This visible illumination induced an increase of the signal, the spectrum of which was similar to that induced by NIR light (not shown). Unlike the signal induced by NIR light, this signal was not stable at 4.2 K and decayed to the original level reached prior to the visible illumination. Each further illumination in the visible range regenerated a signal with similar amplitude and a similar decay (not shown). In a second experiment, the sample in S3 was illuminated only with visible light (blue trace). The EPR spectrum of this signal was similar to that induced by NIR light (not shown) and as shown by the blue trace, a fraction of which decayed with kinetics comparable to that of the red trace and the rest of the signal was stable.

Figure 3.

Figure 3

Open in a new tab

Formation and decay of the split signal in S3. Amplitude of the split signal at the field position indicated by the arrow in figure 2 was followed versus time at 4.2 K in different conditions. Black trace; the sample in S3 was illuminated at 820 nm (upward arrow) until the light was switched off (downward arrow). Red trace; at the end of the recording of the black trace, the sample was further illuminated (upward arrow) with visible light (400–650 nm) until the light was switched off (downward arrow). The blue trace was recorded on a different S3 sample which was illuminated with visible light (400–650 nm; upward arrow) until the light was switched off (downward arrow). Instrument settings are the same as in figure 2.

The experiment reported in figure 3 indicates that the Mn4 clusters that exhibit an absorption band in the NIR range (Boussac et al. 2005) also exhibit an absorption band in the visible range (blue trace). In addition, some of the centres in which Mn photochemistry was not triggered by NIR light could be nevertheless triggered by visible light (red trace). Upon illumination with visible light (blue and red traces), part of the split signal decayed at 4.2 K. Two explanations can be put forward for this decay: (i) the visible light induces a charge separation in the S3 state resulting in a S3X·+QA·− state (where S3X·+ is responsible for the split signal) that would recombine into the S3XQA state at 4.2 K. In this case, the S3X·+ EPR signal would have to be coincidentally very similar to that of the S2TyrZ·. This weighs somewhat against this hypothesis. (ii) Alternatively, the visible light induces two photochemical events in the same reaction centre: first, the conversion of S3TyrZ to S2TyrZ· by Mn photochemistry, where S2TyrZ· is responsible for the split signal, and second, the formation of S2TyrZ·-(Car·+/Chl·+/cytb559ox)-QA− by chlorophyll photochemistry, which then would decay to S2TyrZ-(Car·+/Chl·+/Cytb559ox)-QA at 4.2 K by a charge recombination between TyrZ· and QA−.

These two hypotheses were addressed in the following experiment. Samples in S3 were pre-illuminated at 180 K with visible light to form QA− and reduce the oxidized non-haem iron thus minimizing further charge separation upon additional visible light illumination at 4.2 K. In order to better quantify the effect of these low-temperature illuminations, a normal WT PSII was used in order to benefit from the TyrD· signal as a spin-counting reference.

The effects of the 180 K pre-illumination in S3 are depicted in figure 4. The black and red spectra were recorded prior to and after the 180 K illumination, respectively. The difference spectra (after-minus-before the 180 K illumination) are in blue. Spectra were recorded at 4.2 K in figure 4a and at 15 K in figure 4b,c. Several comments can be made: (i) the non-haem iron, which was oxidized in a fraction of the centres, was reduced by the 180 K illumination (negative signals at 870 and 1182 gauss, g values=7.7 and 5.65, respectively), (ii) the 180 K illumination generated the QA−FeII state (signal at 3400–3800 gauss, g value approx. 1.9) presumably in centres in which the non-haem iron was not oxidized, (iii) cytb559 was oxidized in a fraction (less than or equal to 40%) of centres (signals at 2213 gauss (gz=3.04), 3074 gauss (gy=2.19) and 4700 gauss (gx=1.43)), (iv) the 180 K illumination did not affect the amplitude of the S3 signal observed at 300–1000 gauss and centred at g=9.4, and (v) in figure 4c, it can be seen that the 180 K illumination induced a Car·+/Chl·+ signal in approximately 5% of the centres (assuming TyrD was oxidized in all centres).

Figure 4.

Figure 4

Open in a new tab

Effect of illumination with visible light at 180 K in S3 in WT-PSII. Spectra in (a) were recorded at 4.2 K and spectra in (b,c) were recorded at 15 K. The black spectra were recorded in S3 and the red spectra were recorded after an additional illumination with visible light at 180 K. The blue spectra are the difference spectra after-minus-before the 180 K illumination. Microwave frequency, 9.4 GHz; modulation frequency, 100 kHz. Other instrument settings: (a), modulation amplitude, 25 G; microwave power, 20 mW; (b), modulation amplitude, 25 G; microwave power, 5 mW; (c), modulation amplitude, 2.8 G; microwave power, 20 μW. The central part of the spectra corresponding to the TyrD· signal was deleted.

Based on the estimate of the proportion of oxidized electron donors, the number of centres containing QA− after the 180 K illumination may not be 100%. However, it is possible that other species, not detected under the experimental conditions (i.e. the buffer composition) used here, may also act as electron donors. In any case, the effect of 180 K pre-illumination should significantly decrease the proportion of centres that are able to undergo a charge separation upon a subsequent illumination at 4.2 K.

Figure 5a shows an experiment in which the S3 sample was illuminated by visible light at 4.2 K immediately after the 180 K illumination (red spectrum). The amount of the split signal generated under these conditions is comparable to (although systematically slightly larger than, not shown) that in the absence of the 180 K pre-illumination procedure (figures 2 and 3). This confirms that formation of the split signal under illumination with visible light occurred independently of the charge separation process. Upon dark adaptation for 4 hours at 4.2 K (blue spectrum), approximately 50% of the split signal decayed. This decay was accompanied by a decrease in the QA−FeII signal and this is consistent with a charge recombination process, i.e. the hypothesis (ii) stated above.

Unfortunately while hypothesis (ii) may occur, an additional experiment showed that the situation is not so simple. When an additional NIR illumination at 820 nm was given after the recording of the blue spectrum, i.e. when approximately 50% of the S2TyrZ· state had disappeared, the split signal was fully regenerated (green spectrum). From hypothesis (ii) the centres were expected to be in the S2TyrZ-(Car·+/Chl·+/cytb559ox)-QA state after the 4 hours incubation at 4.2 K, therefore it is difficult to understand how the NIR illumination could reform the split signal (i.e. S2TyrZ·) from the S2TyrZ state. We can tentatively put forward the following hypothesis. It is well established that the spin state of the Mn4 cluster in S2 is strongly affected by NIR illumination (Boussac et al. 2005 and references therein). Moreover, it was reported that NIR illumination at 5 K of the S2 state could probably induce an S2TyrZ to S1TyrZ· back reaction (Koulougliotis et al. 2003) in a small proportion of centres. Therefore, we cannot exclude that upon NIR illumination, S1TyrZ· could be formed in a fraction of the centres that decayed into the S2TyrZ state during the dark period at 4.2 K (figure 5a). If so, the split signal formed in these conditions would have to be identical to that originating from S2TyrZ· upon NIR illumination of the S3TyrZ state, and this seems rather unlikely.

In the experiment reported in figure 5b, the S3 sample was illuminated by NIR light (red spectrum) at 4.2 K immediately after the 180 K pre-illumination procedure. Again a large split signal was observed (this NIR-induced split signal is also slightly larger here than that produced in the conditions of figure 2). More than half of the NIR-induced split signal disappeared in 2 hours at 4.2 K (blue spectrum) together with a decay of the QA−FeII signal. A second NIR illumination at 4.2 K almost completely regenerated the split signal (green spectrum). Then, an additional visible illumination increased the split signal (black spectrum).

From the results in figure 5 it appears that a pre-illumination with visible light of an S3 sample at 180 K closed a high proportion of centres by reducing the electron acceptors, but does not prevent the formation of the split signal generated at 4.2 K by both visible and NIR illumination. Moreover, the amplitude of the split signals generated at 4.2 K is systematically larger than that induced without the 180 K pre-illumination. The origin of this effect remains to be explained. It is probably not an influence of the positive charges (Car·+/Chl·+) generated by the 180 K illumination because the proportion of centres in which these radicals were formed is small. Quantification with a field-swept electron spin-echo experiment of the split signal induced by NIR in the S3 state done as previously described for Ca2+-depleted PSII (Boussac 1996) indicates that it is formed in 20–30% of the centres (not shown). Visible light at 180 K seems to increase slightly this proportion by triggering a photochemical event in/or around the Mn4 cluster. Further experiments will be required to test these hypotheses and it seems probable that some of the structural heterogeneities at the origin of the different photosynthetic electron transfer process at low temperature could also affect the Mn photochemistry triggered by NIR and/or visible lights.

It has been proposed that the photoinhibition process of PSII is triggered in part by the absorption of blue light by the Mn4 cluster (Vass et al. 2002; Ohnishi et al. 2005; Sarvikas et al. 2006). It is possible that this phenomenon and the effects of visible light described in this work reflect the same Mn photochemistry.

During the course of this work, we observed that the non-haem iron signal slightly differed in Ca- and Sr-containing D2-Y160F PSII, which is shown in figure 6. The red spectrum (a, Ca sample) and blue spectrum (c, Sr sample) were recorded in the S2 state formed by one flash at room temperature in the presence of PPBQ. The PPBQ semiquinone formed on the flash oxidizes the non-haem iron in a large fraction of the centres (Zimmermann & Rutherford 1986). Then the Ca and Sr samples were further illuminated at 200 K (black spectra b and d). This resulted in the reduction of FeIII to FeII by the QA− formed under the illumination. The difference spectra show that the non-haem iron environment differs slightly in these two samples (e.g. a shift from 7.7 to 7.5 for the g value of one of the resonances). It was recently reported that the electron transfer from QA− to QB was slowed down in Sr-PSII (Kargul et al. 2007). Such a slowdown could be the consequence of the structural change around the non-haem iron observed here. Since this Sr-PSII preparation contains only one Sr per PSII (Boussac et al. 2004), this observation appears to reflect a long-distant effect between the specific Ca site in the oxygen-evolving complex and the PSII electron acceptor side that may be related to the change of the midpoint potential of QA observed when Ca2+ is removed from PSII (Krieger & Rutherford 1997). Such a change on the acceptor side is the second long-distant effect upon Ca/Sr exchange which has been already observed. The first one was a lower affinity of the extrinsic proteins that could be countered by the addition of betaine (Boussac et al. 2004).

Figure 6.

Figure 6

Open in a new tab

EPR spectra of the non-haem iron. Spectra a and c were recorded in the S2 state of Ca and Sr containing PSII, respectively. Spectra b and d were recorded after a further 200 K illumination in Ca and Sr containing PSII, respectively. The difference spectra after-minus-before the 200 K illumination are shown in the centre part of the panel. Instrument settings: modulation amplitude, 25 G; microwave power, 20 mW; microwave frequency, 9.4 GHz; modulation frequency, 100 kHz; temperature, 8.5 K. The chlorophyll concentration was 1.2 mg ml−1.

Discussion

Boussac Q & A edited by J. Barber

F. Ho (Uppsala University, Uppsala, Sweden). How definitive is your assignment of Mn1 as the Mn(III) ion in the S2 state? To which extent would it affect spectral simulations and exchange coupling schemes?

A. Boussac. In order to reproduce the isotopic hyperfine coupling obtained from X-band EPR simulations, we placed the Mn(III) in different positions compared to J. Messinger in his talk. The location shown is favoured on the basis of the J-values and the possibility to explain other EPR signals (such as g=4.1).

Acknowledgments

Vasili Petrouleas and Sun Un are acknowledged for discussions. This work was supported in part by EU NEST STRP programme SolarH and the JSPS-CNRS Japan–France Cooperative Research Programme.

Footnotes

One contribution of 20 to a Discussion Meeting Issue ‘Revealing how nature uses sunlight to split water’.

References

  1. Boussac A. Quantification of the number of spins in the S2- and S3-states of Ca2+-depleted photosystem II by pulsed-EPR spectroscopy. Biochim. Biophys. Acta. 1996;1277:253–266. doi:10.1016/S0005-2728(96)00107-7 [Google Scholar]
  2. Boussac A, Rutherford A.W. Nature of the inhibition of the oxygen-evolving enzyme of photosystem II induced by NaCl-washing and reversed by the addition of Ca2+ or Sr2+ Biochemistry. 1988;27:3476–3483. doi:10.1021/bi00409a052 [Google Scholar]
  3. Boussac A, Zimmermann J.L, Rutherford A.W. EPR Signals from modified charge accumulation states of the oxygen evolving enzyme in Ca2+-deficient photosystem-II. Biochemistry. 1989;28:8984–8989. doi: 10.1021/bi00449a005. doi:10.1021/bi00449a005 [DOI] [PubMed] [Google Scholar]
  4. Boussac A, Sugiura M, Inoue Y, Rutherford A.W. EPR study of the oxygen evolving complex in His-tagged photosystem II from the cyanobacterium Synechococcus elongatus. Biochemistry. 2000;39:13 788–13 799. doi: 10.1021/bi001159r. doi:10.1021/bi001159r [DOI] [PubMed] [Google Scholar]
  5. Boussac A, Rappaport F, Carrier P, Verbavatz J.-M, Gobin R, Kirilovsky D, Rutherford A.W, Sugiura M. Biosynthetic Ca2+/Sr2+ exchange in the photosystem II oxygen evolving enzyme of Thermosynechococcus elongatus. J. Biol. Chem. 2004;279:22 809–22 819. doi: 10.1074/jbc.M401677200. doi:10.1074/jbc.M401677200 [DOI] [PubMed] [Google Scholar]
  6. Boussac A, Sugiura M, Kirilovsky D, Rutherford A.W. Near-infrared-induced transitions in the manganese cluster of photosystem II: action spectra for the S2 and S3 redox states. Plant Cell Physiol. 2005;46:837–842. doi: 10.1093/pcp/pci088. doi:10.1093/pcp/pci088 [DOI] [PubMed] [Google Scholar]
  7. Britt R.D, Campbell K.A, Peloquin J.M, Gilchrist M.L, Aznar C.P, Dicus M.M, Robblee J, Messinger J. Recent pulsed EPR studies of the photosystem II oxygen-evolving complex: implications as to water oxidation mechanisms. Biochim. Biophys. Acta. 2004;1655:158–171. doi: 10.1016/j.bbabio.2003.11.009. doi:10.1016/j.bbabio.2003.11.009 [DOI] [PubMed] [Google Scholar]
  8. Debus R.J. Amino acid residues that modulate the properties of tyrosine Y–Z and the manganese cluster in the water oxidizing complex of photosystem II. Biochim. Biophys. Acta. 2001;1503:164–186. doi: 10.1016/s0005-2728(00)00221-8. doi:10.1016/S0005-2728(00)00221-8 [DOI] [PubMed] [Google Scholar]
  9. Diner B.A. Amino acid residues involved in the coordination and assembly of the manganese cluster of photosystem II. Proton-coupled electron transport of the redox-active tyrosines and its relationship to water oxidation. Biochim. Biophys. Acta. 2001;1503:147–163. doi: 10.1016/s0005-2728(00)00220-6. doi:10.1016/S0005-2728(00)00220-6 [DOI] [PubMed] [Google Scholar]
  10. Ferreira K.N, Iverson T.M, Maghlaoui K, Barber J, Iwata S. Architecture of the photosynthetic oxygen evolving center. Science. 2004;303:1831–1838. doi: 10.1126/science.1093087. doi:10.1126/science.1093087 [DOI] [PubMed] [Google Scholar]
  11. Goussias C, Boussac A, Rutherford A.W. Photosystem II and photosynthetic oxidation of water: an overview. Phil. Trans. R. Soc. B. 2002;357:1369–1381. doi: 10.1098/rstb.2002.1134. doi:10.1098/rstb.2002.1134 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Havelius K.G.W, Su J.H, Feyziyev Y, Mamedov F, Styring S. Spectral resolution of the split EPR signals induced by illumination at 5 K from the S1, S3, and S0 states in photosystem II. Biochemistry. 2006;45:9279–9290. doi: 10.1021/bi060698e. doi:10.1021/bi060698e [DOI] [PubMed] [Google Scholar]
  13. Ioannidis N, Nugent J.H.A, Petrouleas V. Intermediates of the S3 state of the oxygen-evolving complex of photosystem II. Biochemistry. 2002;41:9589–9600. doi: 10.1021/bi0159940. doi:10.1021/bi0159940 [DOI] [PubMed] [Google Scholar]
  14. Ioannidis N, Zahariou G, Petrouleas V. Trapping of the S2 to S3 state intermediate of the oxygen-evolving complex of photosystem II. Biochemistry. 2006;45:6252–6259. doi: 10.1021/bi060520s. doi:10.1021/bi060520s [DOI] [PubMed] [Google Scholar]
  15. Kargul J, Maghlaoui K, Murray J.W, Deak Z, Boussac A, Rutherford A.W, Vass I, Barber J. Purification, crystallization and X-ray diffraction analyses of the T. elongatus PSII core dimer with strontium replacing calcium in the oxygen-evolving complex. Biochim. Biophys. Acta. 2007;1767:404–413. doi: 10.1016/j.bbabio.2007.01.007. doi:10.1016/j.bbabio.2007.01.007 [DOI] [PubMed] [Google Scholar]
  16. Koulougliotis D, Shen J.R, Ioannidis N, Petrouleas V. Near-IR irradiation of the S2 state of the water oxidizing complex of photosystem II at liquid helium temperatures produces the metalloradical intermediate attributed to S1YZ·. Biochemistry. 2003;42:3045–3053. doi: 10.1021/bi027051o. doi:10.1021/bi027051o [DOI] [PubMed] [Google Scholar]
  17. Koulougliotis D, Teutlolf C, Sanakis Y, Lubitz W, Petrouleas V. The S1YZ· metalloradical intermediate in photosystem II: an X- and W-band EPR study. Phys. Chem. Chem. Phys. 2004;6:4859–4863. doi:10.1039/b407355j [Google Scholar]
  18. Krieger A, Rutherford A.W. Comparison of chloride-depleted and calcium-depleted PSII: the midpoint potential of Q(A) and susceptibility to photodamage. Biochim. Biophys. Acta. 1997;1319:91–98. doi:10.1016/S0005-2728(96)00117-X [Google Scholar]
  19. Loll B, Kern J, Saenger W, Zouni A, Biesiadka J. Towards complete cofactor arrangement in the 3.0 angstrom resolution structure of photosystem II. Nature. 2005;438:1040–1044. doi: 10.1038/nature04224. doi:10.1038/nature04224 [DOI] [PubMed] [Google Scholar]
  20. McEvoy J.P, Brudvig G.W. Structure-based mechanism of photosynthetic water oxidation. Phys. Chem. Chem. Phys. 2004;6:4754–4763. doi:10.1039/b407500e [Google Scholar]
  21. Mino H, Itoh S. The origin of split EPR signals in the Ca2+-depleted photosystem II. Photosynth. Res. 2005;84:333–337. doi: 10.1007/s11120-005-0476-9. doi:10.1007/s11120-005-0476-9 [DOI] [PubMed] [Google Scholar]
  22. Ohnishi N, Allakhverdiev S.I, Takahashi S, Higashi S, Watanabe M, Nishiyama Y, Murata N. Two-step mechanism of photodamage to photosystem II: step 1 occurs at the oxygen-evolving complex and step 2 occurs at the photochemical reaction center. Biochemistry. 2005;44:8494–8499. doi: 10.1021/bi047518q. doi:10.1021/bi047518q [DOI] [PubMed] [Google Scholar]
  23. Petrouleas V, Koulougliotis D, Ioannidis N. Trapping of metalloradical intermediates of the S-states at liquid helium temperatures. Overview of the phenomenology and mechanistic implications. Biochemistry. 2005;44:6723–6728. doi: 10.1021/bi0503201. doi:10.1021/bi0503201 [DOI] [PubMed] [Google Scholar]
  24. Pushkar Y, Yano J, Glatzel P, Messinger J, Lewis A, Sauer K, Bergmann U, Yachandra V. Structure and orientation of the Mn4Ca Cluster in plant photosystem II membranes studied by polarized range-extended X-ray absorption spectroscopy. J. Biol. Chem. 2007;282:7198–7208. doi: 10.1074/jbc.M610505200. doi:10.1074/jbc.M610505200 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rutherford A.W, Boussac A. Water photolysis in biology. Science. 2004;303:1782–1784. doi: 10.1126/science.1096767. doi:10.1126/science.1096767 [DOI] [PubMed] [Google Scholar]
  26. Sanakis Y, Ioannidis N, Sioros G, Petrouleas V. A novel S=7/2 configuration of the Mn cluster of photosystem II. J. Am. Chem. Soc. 2001;123:10 766–10 767. doi: 10.1021/ja016418u. doi:10.1021/ja016418u [DOI] [PubMed] [Google Scholar]
  27. Sarvikas P, Hakala M, Patsikka E, Tyystjarvi T, Tyystjarvi E. Action spectrum of photoinhibition in leaves of wild type and npq1-2 and npq4-1 mutants of Arabidopsis thaliana. Plant Cell Physiol. 2006;47:391–400. doi: 10.1093/pcp/pcj006. doi:10.1093/pcp/pcj006 [DOI] [PubMed] [Google Scholar]
  28. Sauer K, Yachandra V.K. The water-oxidation complex in photosynthesis. Biochim. Biophys. Acta. 2004;1655:140–148. doi: 10.1016/j.bbabio.2003.07.004. doi:10.1016/j.bbabio.2003.07.004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Styring S, Rutherford A.W. In the oxygen-evolving complex of photosystem II the S0 state is oxidized to the S1 state by D+ (signal II slow) Biochemistry. 1987;26:2401–2405. doi:10.1021/bi00383a001 [Google Scholar]
  30. Sugiura M, Inoue Y. Highly purified thermo-stable oxygen-evolving photosystem II core complex from the thermophilic cyanobacterium Synechococcus elongatus having His-tagged CP43. Plant Cell Physiol. 1999;40:1219–1231. doi: 10.1093/oxfordjournals.pcp.a029510. [DOI] [PubMed] [Google Scholar]
  31. Sugiura M, Rappaport F, Brettel K, Noguchi T, Rutherford A.W, Boussac A. Site-directed mutagenesis of Thermosynechococcus elongatus photosystem II: the O2 evolving enzyme lacking the redox active tyrosine D. Biochemistry. 2004;43:13 549–13 563. doi: 10.1021/bi048732h. doi:10.1021/bi048732h [DOI] [PubMed] [Google Scholar]
  32. Tang X.S, Randall D.W, Force D.A, Diner B.A, Britt R.D. Manganese–tyrosine interaction in the photosystem II oxygen-evolving complex. J. Am. Chem. Soc. 1996;118:7638–7639. doi:10.1021/ja9610592 [Google Scholar]
  33. Un S, Boussac A, Sugiura M. Characterization of the tyrosine-Z radical and its environment in the spin-coupled S2TyrZ· state of photosystem II from Thermosynechococcus elongatus. Biochemistry. 2007;46:3138–3150. doi: 10.1021/bi062084f. doi:10.1021/bi062084f [DOI] [PubMed] [Google Scholar]
  34. Vass I, Turcsányi E, Touloupakis E, Ghanotakis D, Petrouleas V. The mechanism of UV-A radiation-induced inhibition of photosystem II electron transport studied by EPR and chlorophyll fluorescence. Biochemistry. 2002;41:10 200–10 208. doi: 10.1021/bi020272+. doi:10.1021/bi020272+ [DOI] [PubMed] [Google Scholar]
  35. Yano J, et al. Where water is oxidized to dioxygen: structure of the photosynthetic Mn4Ca cluster. Science. 2006;314:821–825. doi: 10.1126/science.1128186. doi:10.1126/science.1128186 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Zhang C, Styring S. Formation of split electron paramagnetic resonance signals in photosystem II suggests that tyrosineZ can be photooxidized at 5 K in the S0 and S1 states of the oxygen-evolving complex. Biochemistry. 2003;42:8066–8076. doi: 10.1021/bi0269299. doi:10.1021/bi0269299 [DOI] [PubMed] [Google Scholar]
  37. Zhang C, Boussac A, Rutherford A.W. Low temperature electron transfer in photosystem II: a tyrosyl radical and semiquinone charge pair. Biochemistry. 2004;43:13 787–13 795. doi: 10.1021/bi048631j. doi:10.1021/bi048631j [DOI] [PubMed] [Google Scholar]
  38. Zimmermann J.-L, Rutherford A.W. Photoreductant-induced oxidation of Fe2+ in the electron-acceptor complex of photosystem-II. Biochim. Biophys. Acta. 1986;851:416–423. doi:10.1016/0005-2728(86)90078-2 [Google Scholar]

Articles from Philosophical Transactions of the Royal Society B: Biological Sciences are provided here courtesy of The Royal Society

RESOURCES