The present study describes the formation of different forms of peroxide in Photosystem II (PS II) by using a chemiluminescence detection technique.
Photosynthesis Research 38: 409-416, 1993. © 1993 Kluwer Acadeliic Publishers. Printed in the Netherlands.
Regular paper
Photoproduction of hydrogen peroxide in Photosystem II membrane fragments: A comparison of four signals Vyacheslav Klimov 1,*, Gennady Ananyev 1, Olga Zastryzhnaya 1, Tom Wydrzynski 2 & Gemot Renger 3,*
IInstitute of Soil Science and Photosynthesis, Russian Academy of Science, Pushchino, Moscow Region, 142292, Russia; 2Research School of Biological Science, The Australian National University, GPO Box 475, ACT2601, Australia; 3Max-Volmer-Institute, Technical University Berlin, Strafle des 17. Juni 135, D-10632 Berlin, Germany; *Authors for correspondence Received 19 April 1993; acceptedin revised form 29 August 1993
Key words." peroxide formation, oxygen evolution, water oxidation Abstract
The present study describes the formation of different forms of peroxide in Photosystem II (PS II) by using a chemiluminescence detection technique. Four chemiluminescence signals (A, B, C and D) of the luminolperoxidase (Lu-Per) system, which detects peroxide, are found in illuminated O,-evolving Photosystem II (PS II) membrane fragments isolated from spinach. Signal A ('free peroxide') peafdng around 0.2-0.3 s after mixing PS II membrane fragments with Lu-Per is eliminated by catalase or removal of oxygen from the suspension and ascribed to 02 interaction with reduced PS II electron acceptors. In contrast, signal B peaking around 1.5 min remains largely unaffected under anaerobic conditions, as well as in the presence of catalase (20 pg/ml). Under flash illumination the extent of this signal exhibits a weak period four oscillation (maximum at third and 7th flash). Its yield increases up to the third flash, but is close to zero in the fourth flash. An analogous behaviour is observed in flashes 5 to 8. Signal B is ascribed to Lu-Per interaction with the water-oxidizing system being in S2 and/or S3-state. Signal C ('bound peroxide') detected as free peroxide after acid decomposition of illuminated PS II particles is observed on the 1st flash and oscillates with period 2 with superposition of period 4. It is evidently related to peroxide either released from S2or formed at S2 upon acid shock treatment. Signal D ('slowly released peroxide') peaking around 2-3 s after mixing is observed in samples after various treatments (LCC-incubation, washing with 1 M NaC1 at pH 8 or with 1 M CaC12, C1-depletion) that lead to at least partial removal of the extrinsic proteins of 18, 24 and 33 kDa without Mn extraction. The average amplitude of this signal corresponds with a yield of about 0.2 H202 molecules per RC and flash. In a flash train, the extent of signal D exhibits an oscillation pattern with a minimum at the 3rd flash. We assume that these treatments increase the release of 'bound' peroxide (upon injection into the Lu-Per assay) either formed in the normal oxidative pathway of the water oxidase in the S2 or the S3-state or give rise to peroxide formation due to higher accessibility of the Mn-cluster to water molecules.
Abbreviations." DCPIP - 2,6-dichlorophenolindophenol; DPC - diphenylcarbazide; LCC - lauroylcholine chloride; Lu-Per - luminol-peroxidase; PS II - Photosystem II; RC - reaction center; $2, S3 - redox states of the water oxidizing system; TEMED-N,N,N',N' - tetramethylethylenediamine
Introduction
Photosynthetic water oxidation to molecular oxygen occurs in a multimeric pigment-protein complex
referred to as Photosystem II (PS II) which is anisotropically incorporated into the thylakoid membrane. The PS II complex can be considered as consisting of two main functional blocks: 1) the
410 photochemical reaction center (RC) which converts the energy of Chl singlet state excitation into the free energy of separated charges and produces the strong biological oxidant P680 (for a review see Renger 1992), and 2) a Mn-containing enzymatic system (designated as water oxidase) where formation of molecular oxygen takes place (for reviews see Rutherford et al. 1992, Debus 1992, Renger 1993, Govindjee and Coleman 1993). Based on the fundamental work of Pierre Joliot and Bessel Kok and their coworkers (for a review see Joliot and Kok 1975) the latter process was shown to take place via a sequence of four univalent redox steps. This highly endergonic pathway referred to as the Kok-cycle (Kok et al. 1970) is energetically driven by P:a0 with a redox active tyrosine (Yz) as an intermediate electron carrier (Debus 1992). From a comparison of the redox potential of pheophytin (Pheo) as the primary (intermediary) electron acceptor of PS II @610 mV), the energy barrier between the states [P:80 Pheo-] and [P680Pheo] (6080 meV) and the energy of singlet excitation of P6,0 (1.8-1.82 eV), the redox potential of the pair P680/ P680was evaluated to be +1.12 + 0.05 V (Klimov et al. 1979) [this value is consistent with an earlier evaluation leading to 1.0-1.3 V (Jursinic and Govindjee 1977)]. Several attempts have been made to use this value in order to decide whether or not water oxidation to molecular oxygen requires a simultaneous ('concerted') extraction of four electrons from two substrate water molecules without formation of long-lived intermediary products of water oxidation, but a satisfying answer is still lacking (for a thorough discussion see Krishtalik 1990). It is now widely assumed that the key step of oxygen-oxygen bond formation takes place only at the highest (transient) oxidation state S4 of the water oxidase (Rutherford et al. 1992, Debus 1992). Alternative mechanisms have been proposed where a peroxidic state is formed below the redox level S4 (Andreasson et al. 1983, Renger 1978, 1987). In order to analyze this point of central mechanistic relevance, studies on the possible formation of a peroxidic state within the water oxidase are required. Different lines of evidence indicate that H202 can be formed at the PS II donor side (see Wydrzynski et al. 1989, Ananyev et al. 1992, Fine and Frasch 1992 and references therein). It was found that the rates of the electron flow from water
to an exogenous acceptor and the concomitant O2-evolution are not always consistent: In PS II membrane fragments lacking Mn, up to 80% of the electron transport can be reactivated by the addition of catalytic (0.1-0.2 #M) concentrations of Mn > without an essential restoration of O2-evolution (Klimov et al. 1982, 1990). PS II membrane fragments treated with lauroylcholine chloride (LCC) completely lose O2-evolution, while up to 50% of the electron flow still remains (Wydrzynski et al. 1985). Possibly such results suggest an oxidation of water without O2-evolution; however, alternative explanations cannot be excluded. In our recent work (Ananyev and Klimov 1988, 1989a,b, Klimov et al. 1990, 1992, Ananyev et al. 1992), the photoproduction ofH202 in PS II was demonstrated using peroxide-induced chemiluminescence of the luminol-peroxidase (Lu-Per) system. At least four different luminescence signals (A, B, C and D) related to H202 formation were found in illuminated PS II preparations. The present paper is devoted to a comparison of these signals and of their characteristic features (especially their oscillation patterns in a sequence of brief flashes of light). Based on the experimental results, possible routes of H202 formation in PS II and implications for the mechanism of photosynthetic water oxidation will be discussed.
Materials and methods
Oxygen-evolving PS II membrane fragments [activity 200-300 #moles 0 2 • (mg Chl. h)-1] were isolated form spinach chloroplasts as described earlier (Berthold et al. 1981). Glycerol was added as cryoprotectant for storage in liquid N 2. To remove loosely bound divalent cations, the suspension was washed twice with a buffer medium containing 20 mM MES-NaOH, pH 6.5, 35 mM NaC1, 300 mM sucrose, and 0.5 mM EDTA before starting the experiments. To study H202-production, a sample (50#1, 0.2-1 mg Chl ml 1) in a plastic pipette tip was illuminated with either continuous red light (200 W • m -2) for 10-20 s or a sequence of brief (5 #s) saturating light flashes given at 0.5 Hz repetition rate, followed by rapid injection into 1 ml of the assay medium consisting of 50 mM phosphate
411 buffer pH 8.0, horseradish peroxidase (0.5-5#M) and luminol (20-200/_tM). The chemiluminescence emitted from this assay was measured as described previously (Ananyev and Klimov 1988). To release bound peroxide from PS II membrane fragments, the 'acid decomposition' method was used as outlined in Ananyev and Klimov (1988). For this purpose, 50 #1 ofPS II membrane fragments were illuminated with 1-10 flashes in the presence of catalase and subsequently mixed with 50/A of H2SO4 (final pH was adjusted to 1.4), incubated for 30 s and neutralized with 50/.tl of Tris to a final pH of 8.0. Anaerobic conditions were established by purging of the sample suspension with argon. For lauroylcholine chloride (LCC) treatment, samples were suspended to 200 ]~g Chl/ml in a buffer medium containing 10 mM MES-NaOH, pH 6.5 and 10 mM NaC1 to which LCC was added as a small aliquot to obtain a ratio of 3:1 (w/w) to Chl. Samples were incubated in the dark at room temperature for 5 min under gentle shaking before being centrifuged. The pellet was then resuspended in the same buffer medium to an appropriate Chl concentration (Ananyev et al. 1992). CaC12 treatment of PS II membrane fragments with 1 M CaC12 was performed as described previously (Ono and Inoue 1983). Mn was removed (> 90%) from PS II membrane fragments by incubating samples at 0.25 mg Chl/ml in a medium consisting of 20 mM MES-NaOH (pH 6.5) and 20 mM N,N,N',N'-tetramethylethylenediamine (TEMED) for 5 min on ice in the dark, as outlined by Ananyev et al. (1992).
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Fig. 1. Chemiluminescence of the mixture containing 60-200 /IM luminol, 0.5 mM peroxidase form horse-radish and 50/tim phosphate buffer, pH 8.0 upon the addition of: (1) 0.1/tM H202 in 20 mM MES-NaOH, pH 6.5; (2) PS II membrane fragments after 10 saturating microsecond light flashes (aerobic conditions); (3) same as 2 under anaerobic conditions; (4) same as 2 plus catalase (20/lg/ml); (5) same as 2, after 'the acid decomposition'; (6) same as 5 under anaerobic conditions; (7) same as 5 plus catalase (5 /~g/ml) added after 'the acid decomposition' of illuminated particles; (8) LCC-treated PS II membrane (LCC: Chl = 3:1) after 10 s continuous illumination; (9) same as 8 plus catalase (5/~g/ml); (10) same as 8 plus catalase (50/~g/ml); (11) CaC12-treated PS II membrane fragments particles after 20 s continuous illuminations; (12) same as 11 plus DPC (10 HIM); (13) same as 1 l after removal of Mn (> 90%) form CaC12treated samples by TEMED; (14) same as 11 plus the extract obtained as a result of CaC12-treatment and subsequent dialysis; (15) same as 11 plus DCMU (20 ktM). The arrow denotes the moment &injection of the sample into the Lu-Per assay medium.
The response of the luminol-peroxidase (Lu-Per) system to different additions is summarized in Fig. 1.
membrane fragments did not reveal detectable chemiluminescence of Lu-Per (evidently due to a slowly reacting catalase-like activity ofPS II). After illumination of the sample with 10 short flashes of light in the absence of added electron acceptors, a chemiluminescence signal appears (Fig. l, trace 2) that consists of two kinetic components denoted as signals A and B.
Signals A and B
Signal A
A test experiment revealed that injection of 50 yl of 0.1 #M H202 into the (Lu-Per) assay system results in the luminescence emission from luminol which reaches the maximum after 0.2-0.3 s and disappears 3-5 s after mixing, as is shown in Fig. 1, trace 1. Control measurements with dark adapted PS II
Signal A is characterized by fast kinetics closely resembling those due to the addition of HzO2 in solution (compare traces 1 and 2). It disappears completely in the presence of catalase and is suppressed down to levels of 20-30% (trace 3) under anaerobic conditions (Fig. l). Based on the
Results and discussion
412 kinetic features and the sensitivity to catalase, signal A is assumed to be due to 'free peroxide'. Illumination with a sequence of/.ts flashes gives rise to 'free peroxide' formation starting from the second flash. It gradually accumulates in the medium with increasing total number of flashes (data not shown). The yield is very low (about 0.01 H202 molecules per RC and saturating flash) and decreases by a factor of 4-5 times upon the addition of 20/.tM DCMU. All these data show that 'free peroxide' results from the interaction between O 2 and the reduced PS II electron acceptor side yielding superoxide followed by its dismutation to H202 (see Fig. 3). This conclusion is in line with the previous findings reported in the literature (Schr6der and Akerlund 1990)
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flush N ~° Fig. 2. Signal A, B, C and D as a function of the number of saturating 5 #s flashes. The difference in the amplitude of the signals from samples illuminated with n and n - 1 flashes, respectively, is used as the yield of the n th flash.
Signal B Signal B attains its maximum around 1-1.5 min after mixing the illuminated sample with Lu-Per, followed by a slow decay down to zero within less than 5 min (Fig. 1, trace 2). The dark decay time of signal B in the absence of Lu-Per is 5-7 min and 11-12 rain for the 50% and 90% decline, respectively (data not shown). In contrast to signal A, anaerobic conditions or addition of catalase do not prevent signal B (see traces 3 and 4 of Fig. 1). After rapid centrifugation of illuminated PS II membrane fragments, signal B was found to be associated with the pellet (data not shown). If samples are illuminated (either with 10 flashes or 20 s continuous light) in the presence of DCMU, signal B is vanishingly small (not shown). Accordingly, this signal is intimately related to the photoinduced electron transfer in PS II. Furthermore, signal B is inferred to be due to photoaccumulation of an oxidized species at the donor side of PS II because it is activated 2.5-3-fold by addition of the electron acceptor DCPIP (10 #M), while upon the addition of DCPIPH 2 or ascorbate it is completely inhibited. This assignment is supported by the finding that signal B is not observed in PS II membrane fragments lacking Mn, and becomes restored by readdition of Mn z+ while the replacement of Mn 2+ by ascorbate only increases signal A without signal B restoration (Ananyev and Klimov 1989b). Interestingly, Fig. 2 shows that in samples illuminated with a train of flashes, signal B increases up to the 3rd flash and then returns back to the dark
level in the 4th flash. A similar pattern arises in flashes 5 to 8. This period four pattern markedly differs from that of O2-evolution (also measured in the absence of artificial electron acceptors), i.e. signal B is observed already at the first (and second) flash. This finding suggests that signal B is evidently related to an interaction of the Lu-Per system with the Mn-containing water oxidizing complex in the oxidation state S2 and Sr Measurements of the S2 life time under our assay conditions (values of 8-10 min were obtained, data not shown) support this idea. The absence of signal B in the presence of DCMU can be explained by a rapid decay of S2 ($3) with Q~ as reductant. Two alternative mechanisms could be responsible for the generation of signal B. It might originate from the direct interaction of LuPer with oxidized forms of Mn acting at the donor side ofPS II (see Fig. 1; trace 4). In this respect it is interesting to mention that a similar signal can be induced by MnC12 mixed with phosphate buffer in the presence of oxygen. However, it should be emphasized that the manganese of the water oxidase in S2 ($3) attain redox states higher than Mn(II) (see e.g. Kusunoki 1992 and references therein). As an alternative interpretation, signal B could be caused by 'bound' H202 complexed at the catalytic site which is slowly released from the manganese cluster (see Fig. 3). In order to account for the lack of a catalase effect on signal B it would have to be assumed that the interaction of 'bound' H202 with the Lu-Per assay cannot be prevented by catalase due to steric circumstances.
413
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