Phowsynthesis Research 48: 197-203, 1996. ¢~) 1996 Kluwer Academic Publishers. Printed in the Netherlands.
Regular paper
Effect of the redox state of QB on electric field-induced charge r e c o m b i n a t i o n in Photosystem II Petra W. Hemelrijk & Hans J. van Gorkom Department of Biophysics, Huygens Laboratory of the State University, Leiden, P.O. Box 9504, 2300 RA Leiden, The Netherlands Received 20 September 1995; accepted in revised form 9 January 1996
Key words: blebs, charge recombination, electroluminescence, Photosystem II
Abstract
Electric field-induced charge recombination in Photosystem II (PS II) was studied in osmotically swollen spinach chloroplasts ('blebs') by measurement of the concomitant chlorophyll luminescence emission (electroluminescence). A pronounced dependence on the redox state of the two-electron gate QB was observed and the earlier failure to detect it is explained. The influence of the QB/Qff oscillation on electroluminescence was dependent on the redox state of the oxygen evolving complex; at times around one millisecond after flash illumination a large effect was observed in the states $2 and $3, but not in the state '$4' (actually Z+S3). The presence of the oxidized secondary electron donor, tyrosine Z +, appeared to prevent expression of the QB/QB effect on electroluminescence, possibly because this effect is primarily due to a shift of the redox equilibrium between Z/Z + and the oxygen evolving complex.
Abbreviations: B S A - b o v i n e serum albumin; EDTA-ethylene-diaminetetraacetic acid; EL-electroluminescence; FCCP-carbonylcyanide p-trifluoromethyloxyphenyl-hydrazone; HEPESI-4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; I-primary electron acceptor; M O P S - 3-(N-morpholino) propane sulfonic acid; P 6 8 0 primary electron donor of Photosystem II; P 7 0 0 - primary electron donor of Photosystem I; QA and QB - secondary and tertiary electron acceptors of Photosystem II; Z-secondary electron donor (D 1 Tyr 161) Introduction
Photosynthetic reaction centers convert the very shortlived excited states produced by light absorption in the antenna into a stable separation of charges across the photosynthetic membrane, stable enough to provide the driving force for the biochemistry of photosynthesis. The stabilization is achieved by a sequence of electron transfer reactions, (de)protonations and possibly protein conformational changes. In priciple, each of those steps is expected to decrease the (quasi-) equilibrium concentration of the primary radical pair and hence the rate of charge recombination. Electron transport in Photosystem II (PS II) (van Gorkom 1985) starts with the formation of the primary radical pair that consists of an oxidized chlorophyll,
P680 +, and a reduced pheophytin, I - . The main stabilization step is electron transfer in about 300 ps from I - to a permanently bound plastoquinone molecule, QA, which normally does not become protonated and can accept only one electron. P680 + oxidizes a tyrosine residue (D1 Tyr 161) called Z in the sub-ps time range, the exact time being dependent on the 'S-state', the redox state of the oxygen evolving complex (Debus 1992). This complex can reduce Z + four times, on four successive photoreactions, and then oxidizes two water molecules to oxygen (Joliot and Kok 1975). On the acceptor side, QA can be oxidized twice, on two successive photoreactions, by a transiently bound plastoquinone molecule QB, which then is protonated, released as a plastoquinol and replaced by a new plastoquinone molecule (Velthuys 1981). The reduction
198 of Z + and oxidation of QA and the associated proton release and uptake take place largely in the 0.1-1 ms time range, with kinetics and equilibrium constants which depend on the number of charges stored already. See Refs. (Rappaport et al. 1994; Haumann and Junge 1994) for the most recent data on the kinetics of these reactions. Vos et al. (1991) have studied the stabilization processes in PSII by means of electroluminescence (EL) (Arnold and Azzi 1971; Ellenson and Sauer 1976; van Gorkom, 1996). This is chlorophyll fluorescence due to reversal of the photosynthetic charge separation by an electric field. Strong electric field pulses were obtained by making use of the large enhancement of the field strength in the membrane of osmotically swollen chloroplasts ('blebs') in an externally applied electrical field (Ellenson and Sauer 1976; de Grooth et al. 1980). In general, EL has contributions from both PS I and PS II (Symons et al. 1985). In a previous study of PS II by Vos et al. (1991) P S I contribution was eliminated by using ferricyanide, which leaves P700 in the oxidized, inactive state after the first photoreaction. Vos et al. (1991) have carried out a comprehensive survey of PS II-EL as a function of flash number, time between flash and electrical pulse, and time during the pulse. Many kinetic components were detected. Some of those were not dependent on flash number (after the first) and could be assigned to a minor fraction of non-oscillating centers, known also from UV absorbance difference spectroscopy (Dekker et al. 1984; Lavergne 1987) and fluorescence induction (Lavergne and Leci 1993), in which electron transport beyond QA is blocked. Most components, however, were clearly S-state dependent. Surprisingly, in the above work ofVos et al. (1991) no dependence on the redox state of QB was found, although control measurements of the 325 nm absorption band of Q~ (van Gorkom et al. 1982) indicated a normal binary oscillation ofQB/Q~ with flash number. One would expect the redox state of QB to be reflected in recombination luminescence because it influences the rate and extent of QA oxidation and proton uptake (Robinson and Crofts 1983), recently shown to be electrogenic (Mamedov et al. 1994). A pronounced influence was in fact observed in thermoluminescence (Rutherford et al. 1982). We have therefore reinvestigated this paradox using the same approach as Vos et al. (1991), but using a different method to eliminate PS I.
Materials and methods
Chloroplasts from laboratory-grown spinach were isolated by grinding in a cooled blender in 50 mM HEPES (pH 7.5)/0.4 M NaC1/1 mM EDTA/0.2% w/v BSA, filtration through a 25 #m mesh nylon cloth and centrifugation for 10 min at 10.000 x g. The pellet was resuspended in a buffer containing 50 mM HEPES (pH 7.5)/0.15 MNa C1/5 mM MgC12 to a chlorophyll concentration of 2 mg/ml and stored at 77 K until use. After thawing, about 10 min before measurement the sample was diluted 400-fold in a buffer containing 1 mM CaCI2/1 mM MOPS (pH 6.6) at 18 °C, the temperature of measurement. A fresh sample was used for each measurement. The experimental set-up was described previously (Vos and van Gorkom 1988). In short, a 1 × 1 × 0.2 cm cuvette was used of which two opposite sides (0.2 cm distance) consisted of platinum electrodes; one side was used for inlet and outlet of a stoppedflow system and the remaining sides were used for flash-illumination and detection of luminescence. The cuvette was placed in the focus of an ellipsoidal mirror for efficient light collection. The emitted light was detected with a gated photomultiplier through a 680 nm interference filter and a Schott RG 665 cut-off filter. Saturating laser flashes (532 nm, 20 ns half-width) were provided at 10 Hz and filtered through a Balzers Calflex C and a Corning CS 4-96 broad band blue filter. Electric pulses with a field strength of 1650 V/cm and duration of 140 ~uswere used. For all measurements the signal observed without electric pulse was subtracted after digitalization.
Results
In order to investigate the effect of the redox state of QB on EL we followed the same procedure as Vos et al. (1991): EL, induced by an electric field pulse 0.5 ms after illumination and integrated from 25 to 125/~s after the onset of the pulse, was measured as a function of the number of flashes fired on a sample initially set predominantly to the state SIQB or to the state S1Q~. The initial condition was prepared by firing 0, 1, or 2 'QB-setting flashes' on a dark-adapted sample, producing predominantly S1QB, S2Q~, or S3QB, respectively. Then the sample was kept in darkness long enough to allow deactivation of the high S-states back to $1, which was accelerated by the presence of FCCP.
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Figure 1. (A) Kinetics of the deactivation of the S-states in ~he
presence of 5 nM (open circles), 10 nM (open squares) and 20 ~M FCCP (solid circles). The ratio of the EL of the 7th flash and the ~th flash is plotted as a function of the time between the 2nd and 3rd flashes. The other flashes were spaced at 100 ms. (B) The EL a~a function of flash number (flashes spaced at 100 ms intervals) in the absence (solid circles) and presence (open squares) of 20 nM FCCP.
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Figure 2. The influence of ferricyanide on EL as a function of flash
number. (A) 50 #M ferricyanide and (B) no ferricyanide. After preillumination by 0 (solid circles, only in A), 1 (open circles) or 2 (solid squares) QB-setting flashes spaced at 100 ms and 7 s dark time for S-state deactivation, a series of 1 to 10 flashes was fired at 10 Hz and EL was measured 0.5 ms after the last flash.
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The deactivation kinetics of the higher S-states ~y F C C P was checked by monitoring the phase shif! of the oscillation pattern as a function o f the dark time between QB-Setting preillumination and flash series. ~n our case 20 nM F C C P was sufficient to deactivate the S-states in about 7 s, as illustrated in Figure 1A. Figure 1B shows that for up to 8 flashes, at a flash frequency of 10 Hz, this deactivation does not cause a substantlal difference between E L oscillation patterns measurpd with (open squares) and without (solid circles) FCCP. In all experiments described below 20 nM F C C P x~as present, i In the presence o f 20 nM F C C P and 50 # M fermicyanide, as used by Vos et al. (1991) to inactivate Photosystem I, preillumination by 1 or 2 QB-setting flashes followed by 7 s dark adaptation had little effect on EL amplitudes, as shown in Figure 2A. Only the amplitude after the first flash is much smaller without preillumination and some o f this effect is still seen on the second flash. This effect is due to the 'non-oscillating' centers mentioned in the Introduction and does not concern ~s
here. After 1 or 2 QB-setting flashes the EL amplitude oscillation with flash number is identical. This result confirms the findings of Vos et al. (1991) and shows not only that 100% SI was present initially, irrespective of the number of QB-setting flashes, but also that QB either has no influence on EL or was in the same initial redox state, independent of preillumination. Figure 2B shows the same experiment with one (open circles) and two (solid squares) QB-setting flashes, carried out in the absence of ferricyanide. Clearly, the number of Q~-setting flashes now does have an effect and in addition the direction o f the effect appears to be alternating with flash number, disregarding flash numbers 4 and 8 for reasons explained below. These results strongly suggest that the redox state of QB influences the amplitude o f E L and that this was not observed earlier because 50 # M ferricyanide oxidized all Q~ within the dark time between QB-setting preillumination and flash series. In the absence of ferricyanide EL emission by P S I cannot be avoided, but it decreases much more rapid-
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ly during the electric field pulse than EL from PS II (Symons et al. 1985; Vos and van Gorkom 1988). As shown in Figure 3, EL appears on the pulseinduced luminescence as an initial spike which does not decrease with increasing delay time between flash and pulse (that takes 0.1 s (Vos and van Gorkom 1988))• It also does not depend on flash number and could thereby be distinguished from PS II EL. Discarding EL emitted during the first 25 #s of the pulse effectively removed the P S I contribution. When the emission at later times was plotted as a function of the delay time between flash and pulse it was found to decrease largely in two exponential phases, with time constants of about 0.2 and 1.4 ms, and a small much longer-lived component (Figure 4). The relative amplitude of the fast component was smaller for luminescence emitted at later times during the pulse (solid
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Figure 5. Decay of integrated (25 to 50 /zs) EL as a function of the time between the last flash and the electrical pulse after 1 (solid circles), 2 (open circles), 3 (solid squares) and 4 (open squares) flashes. Preillumination in (A) none, (B) one and (C) two QB-setting flashes.
lines: integrated from 25 to 50 #s; open circles: from 70 to 85 #s). Apparently the fast component also shows a faster decay during the pulse• The decay of EL, integrated from 25 to 50/zs after the onset of the pulse, as a function of time between the last of I to 4 flashes and the electrical pulse is shown in Figure 5A, B, and C, for 0, 1, and 2 QB-setting flashes, respectively. The curves are fits obtained with a sum of two exponentials, and an offset• Their amplitudes and time constants are listed in Table 1. The offset was independent of QB-setting preillumination and of the number of flashes in the series, in agreement with the conclusion by Vos et al. (1991) that EL measured at 5 ms after the flash is dominated by emission from 'non-oscillating centers'•
201 Table 1. Amplitudes (betweenparentheses)and time constants, in milliseconds, of two exponential phases and a long-lived component, of the decayof integrated EL contributions (Figure 5A, B and C) for 0, 1 and 2 QB-settingpreflashes(integration from 25 to 50/~s)
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The 0.2 and 1.4 ms components can be attributed to stabilization of the charge separation in active, oxygen evolving reaction centers, because a clear Sstate dependence is seen in the time constant of the fast phase and in the amplitude of the slow phase, both being larger for higher S-states. Qualitatively, we associate the former with the S-state dependence of the reduction time of Z + by the oxygen evolving complex (Rappaport et al. 1994; Dekker et al. 1984) and the latter with a different extent of stabilization achieved by each S-state transition, presumably expressed in a different equilibrium concentration of Z +, and hence of P680 +. The reoxidation of QA presumably contributed to the fast phase. The total amplitude at short delay time oscillates with flash number, indicating that the energy of the state Z+QA is S-state dependent. A quantitative interpretation requires additional assumptions, but does not invalidate these global assignments, as discussed already (Vos et al. 1991). The amplitude of the 1.4 ms phase in the decay of EL with increasing time between the last flash and the electric pulse was clearly dependent on the number of QB-setting flashes that had been used. The ratio of this amplitude for the experiment with 2 QB-setting flashes over that with 1 QB-setting flash shows the same pattern during the flash series, 1.9, 0.8, 1.1, 1.1, as already indicated by the data in Figure 2B. Figure 2B indicates that the pattern seen on the first 4 flashes was
repeated on the next cycle of the S-states, suggesting that the effect is specific for the $1 --4 $2 and $2 --4 $3 transitions. For both transitions the electroluminescence signal inducible during the slow stabilization phase was larger when QB was expected to be in the oxidized state before the flash and reduced to Q~ after the flash. The effect of the QB/QB oscillation should be most clearly observed by comparing the results obtained with 1 and 2 Qa-setting flashes, respectively. At any rate the experiment with one flash should be disregarded because of the different behaviour of the nonoscillating centers. Quantitative simulations to determine the actual QB/QB and S-state stoichiometries will require a more extensive data set, including independent information on the QA reoxidation kinetics in these conditions.
Discussion
The results reported here indicate that the earlier failure to detect an influence of the redox state of QB on electroluminescence (Vos et al. 1991 ) was due to reoxidation Of QB by ferricyanide during the 5 seconds dark time after the QB-setting flashes and cannot be ascribed to non-B acceptors (Lavergne and Etienne 1980) as suggested by Lavergne and Leci (1993). In the control experiments showing a binary oscillation with flash number in 325 nm absorbance at low flash frequency (Vos et al. 1991) the addition of 1 mM CaCI2 may have been omitted. The role of Ca 2+ here is not specific, because it can be replaced by Mg 2+ (not shown), and most likely is to screen the negative surface charge at the acceptor side of PS II so that it becomes accessible to ferricyanide (Itoh 1978). The assignments of the various decay phases of the EL precursor as discussed by Vos et al. (1991) are not much affected by the present findings. An 0.4 s phase observed in the presence of 50 #M ferricyanide may now be assigned to Q~ oxidation by ferricyanide, a possibility already recognized by Vos et al. (1991), in agreement with our observation that 50 #M ferricyanide completely abolished the effect of a QB-setting flash followed by 7 s dark adaptation. The assignment of a stabilization phase of about 1 ms to non-oscillating centers must be reconsidered. This was postulated because such a phase was seen on all flashes, including the first two flashes, where no $3 --~ So transition can occur. The amplitude of the 1.4 ms component is now found to oscillate with the
202 redox state of QB, at least in $2 and 83. Therefore, we attribute this component to a stabilization process in active, oscillating centers. In the absence of ferricyanide we do find a specific effect of the redox state of QB on the amplitude of the EL signal at times around a ms after the flash. The amplitude appears to be larger in the presence of Qff than in the presence of QB. One might expect that this is due to a smaller equilibrium constant being associated with electron transfer from QA to QB than with that from QA to Q~ assuming that these reactions take less than a ms. However, the difference is seen only on flash numbers 1 and 2, 5 and 6, etc., corresponding to the S1 --~ $2 and $2 --~ $3 transitions (Figure 2B). It seems reasonable to assume that these transitions contribute to the fast stabilization phase and that the electroluminescence inducible during the slow phase originates from the state reached after equilibration of electron transfer between Tyr Z and the subsequent electron donor. On the Z+S3 --4 ZSo transition, however, Z + probably remains present during the slow phase. This difference between the $3 --~ So transition and the $1 ~ $2 and $2 ---r $3 transitions seems the only likely basis to explain the absence of a Qff/QB effect on the 3rd flash. The slow EL component after the 4th flash may have the same origin as that after the 3rd and be attributed to misses; centers in the Sl-State may not yield detectable EL after Z + reduction (Vos et al. 1991). If the QB/QB effect is due to an influence on the concentration or potential of QA, we have to assume that this influence disappears in the presence of Z +. Alternatively, the QB/QB effect may not be expressed in EL via the acceptor side, but via an influence of QB/QB on the (quasi steady state) concentration of Z + during the electric field induced charge recombination in the states S2QA and S3QA. Both options are somewhat hard to envisage mechanistically. On the other hand, it is also hard to imagine that the observed differences between one and two QB-setting flashes in our experiments are due to anything else than the redox state of QB, and their S-state dependence is undeniable. Our findings may be related to the observation by Bouges-Bocquet (1973) that the reopening of PS II centers on the $3 to So transition is a first order process and does not show the sigmoidal kinetics expected if QA reoxidation and Z + rereduction were independent.
Acknowledgements We are indebted to an anonymous reviewer for great help with the manuscript. This work was supported by the Netherlands Foundation for Chemical Research (SON) of the Netherlands Organization for Scientific Research (NWO) and a EU DGXII Human Capital & Mobility network grant (CHRX-CT94-0524).
References Arnold WAand Azzi R (1971) The mechanismof delayed light production by photosyntheticorganismsand a new effect of electric fields on chloroplasts. Photochem Photobiol 14:233-240 Bouges-Bocquet B (1973) Limiting steps in Photosystem II and water decomposition in Chlorella and spinach chloroplasts. Biochim Biophys Acta 292:772-785 Debus RJ (1992) The manganeseand calciumions of photosynthetic oxygen evolution.BiochimBiophys Acta 1102:269-352 De Grooth BG, van Gorkom HJ and Meiburg RF (1980) Electrochromic absorbancechanges in spinachchloroplasts induced by an external electrical field. Biochim Biophys Acta 589: 299314 Dekker JP, van Gorkom HJ, Wensink J and Ouwehand L (1984) Absorbancedifference spectra of the successiveredox states of the oxygen-volvingapparatus of photosynthesis. Biochim Biophys Acta 767:1-9 EllensonJL and Saner K (1976) The electrophotoluminescenceof chloroplasts. Photochem Photobiol23:113-123 Haumann M and Junge W (1994) The rates of proton uptake and electron transfer at the reducing side of Photosystem II in thylakiods. FEBS Lett 347:45-50 ltoh S (1978) Membranesurface potentialand the reactivity of the system II primary electron acceptor to charged electroncarders in the medium.Biochim Biophys Acta 504:324-340 Joliot P and Kok B (1975) Oxygen evolution in photosynthesis. In: Govindjee(ed) Bioenergeticsof Photosynthesis,pp 387-412. Academic Press, New York Lavergne J (1987) Optical-differencespectra of the S-state transitions in the photosynthetic oxygen-evolvingcomplex. Biochim Biophys Acta 894:91-107 LavergneJ and EtienneAL (1980) Promptand delayed fluorescence of chloroplasts upon mixing with dichlorophenyldimethylurea. Biochim Biophys Acta 593:136--148 LavergneJ and Leci E (1993) Properties of inactivePhotosystemII centers. PhotosynthRes 35:323-343 Mamedov MD, Beshta OE, Samuilov VD and Semenov AY (1994) Electrogenicity at the secondary quinone acceptor site of cyanobacterialPhotosystemII. FEBS Lett 35 96-98 Rappaport F, Blanchard-Desce M and Lavergne J (1994) Kinetics of electron transfer and electrochromic changes during the redox transitions of the photosynthetic oxygen-evolvingcomplex. BiochimBiophys Acta 1184:178-192 Robinson HH and Crofts AR (1983) Kinetics of the oxidationreductionreactions of the PhotosystemII quinoneacceptor complex, and the pathway for deactivation.FEBS Lett 153:221-226 RutherfordAW,CroftsAR and InoueY (1982) Thermoluminescence as a probe of Photosystem II photochemistry the origin of the flash-inducedglow peaks. Biophys BiochimActa 682:457-465
203 Symons M, Korenstein R and Malkin S (1985) External electric-field effects on photosynthetic vesicles. The relationship of the rapid and slow phase of electrophotohiminescence in hypotonically swollen chloroplasts to PSI and PS II activity. Biochim Biophys Acta 806:305-310 Van Gorkom HJ (1985) Electron transfer in Photosystem II. Photosynth Res 6:97-112 Van Gorkom HJ (1996) Electroluminescence. Photosynth Res 48: 107-116 (this issue) Van Gorkom HI, Thielen APGM and Gorren ACF (1982) The secondary electron acceptor of Photosystem II. In: Trumpower BL (ed) Function of Quinones in Energy Conserving Systems, pp 13-225. Academic Press, New York.
Velthuys BR (1981) Electron-dependent competition between plastoquinone and inhibitors for binding to Photosystem II. FEBS Lett 126:277-281 Vos MH and van Gorkom HJ (1988) Thermodynamics of electron transport in Photosystem I studied by electric field-stimulated charge recombination. Biochim Biophys Acta 934:293-302 Vos MH, van Gorkom HJ and van Leeuwen PJ (1991) An electroluminescence study of stabilization reactions in the oxygen evolving complex of Photosystem II. Biochim Biophys Acta 1056: 27-39
Regular paper
Effect of the redox state of QB on electric field-induced charge r e c o m b i n a t i o n in Photosystem II Petra W. Hemelrijk & Hans J. van Gorkom Department of Biophysics, Huygens Laboratory of the State University, Leiden, P.O. Box 9504, 2300 RA Leiden, The Netherlands Received 20 September 1995; accepted in revised form 9 January 1996
Key words: blebs, charge recombination, electroluminescence, Photosystem II
Abstract
Electric field-induced charge recombination in Photosystem II (PS II) was studied in osmotically swollen spinach chloroplasts ('blebs') by measurement of the concomitant chlorophyll luminescence emission (electroluminescence). A pronounced dependence on the redox state of the two-electron gate QB was observed and the earlier failure to detect it is explained. The influence of the QB/Qff oscillation on electroluminescence was dependent on the redox state of the oxygen evolving complex; at times around one millisecond after flash illumination a large effect was observed in the states $2 and $3, but not in the state '$4' (actually Z+S3). The presence of the oxidized secondary electron donor, tyrosine Z +, appeared to prevent expression of the QB/QB effect on electroluminescence, possibly because this effect is primarily due to a shift of the redox equilibrium between Z/Z + and the oxygen evolving complex.
Abbreviations: B S A - b o v i n e serum albumin; EDTA-ethylene-diaminetetraacetic acid; EL-electroluminescence; FCCP-carbonylcyanide p-trifluoromethyloxyphenyl-hydrazone; HEPESI-4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; I-primary electron acceptor; M O P S - 3-(N-morpholino) propane sulfonic acid; P 6 8 0 primary electron donor of Photosystem II; P 7 0 0 - primary electron donor of Photosystem I; QA and QB - secondary and tertiary electron acceptors of Photosystem II; Z-secondary electron donor (D 1 Tyr 161) Introduction
Photosynthetic reaction centers convert the very shortlived excited states produced by light absorption in the antenna into a stable separation of charges across the photosynthetic membrane, stable enough to provide the driving force for the biochemistry of photosynthesis. The stabilization is achieved by a sequence of electron transfer reactions, (de)protonations and possibly protein conformational changes. In priciple, each of those steps is expected to decrease the (quasi-) equilibrium concentration of the primary radical pair and hence the rate of charge recombination. Electron transport in Photosystem II (PS II) (van Gorkom 1985) starts with the formation of the primary radical pair that consists of an oxidized chlorophyll,
P680 +, and a reduced pheophytin, I - . The main stabilization step is electron transfer in about 300 ps from I - to a permanently bound plastoquinone molecule, QA, which normally does not become protonated and can accept only one electron. P680 + oxidizes a tyrosine residue (D1 Tyr 161) called Z in the sub-ps time range, the exact time being dependent on the 'S-state', the redox state of the oxygen evolving complex (Debus 1992). This complex can reduce Z + four times, on four successive photoreactions, and then oxidizes two water molecules to oxygen (Joliot and Kok 1975). On the acceptor side, QA can be oxidized twice, on two successive photoreactions, by a transiently bound plastoquinone molecule QB, which then is protonated, released as a plastoquinol and replaced by a new plastoquinone molecule (Velthuys 1981). The reduction
198 of Z + and oxidation of QA and the associated proton release and uptake take place largely in the 0.1-1 ms time range, with kinetics and equilibrium constants which depend on the number of charges stored already. See Refs. (Rappaport et al. 1994; Haumann and Junge 1994) for the most recent data on the kinetics of these reactions. Vos et al. (1991) have studied the stabilization processes in PSII by means of electroluminescence (EL) (Arnold and Azzi 1971; Ellenson and Sauer 1976; van Gorkom, 1996). This is chlorophyll fluorescence due to reversal of the photosynthetic charge separation by an electric field. Strong electric field pulses were obtained by making use of the large enhancement of the field strength in the membrane of osmotically swollen chloroplasts ('blebs') in an externally applied electrical field (Ellenson and Sauer 1976; de Grooth et al. 1980). In general, EL has contributions from both PS I and PS II (Symons et al. 1985). In a previous study of PS II by Vos et al. (1991) P S I contribution was eliminated by using ferricyanide, which leaves P700 in the oxidized, inactive state after the first photoreaction. Vos et al. (1991) have carried out a comprehensive survey of PS II-EL as a function of flash number, time between flash and electrical pulse, and time during the pulse. Many kinetic components were detected. Some of those were not dependent on flash number (after the first) and could be assigned to a minor fraction of non-oscillating centers, known also from UV absorbance difference spectroscopy (Dekker et al. 1984; Lavergne 1987) and fluorescence induction (Lavergne and Leci 1993), in which electron transport beyond QA is blocked. Most components, however, were clearly S-state dependent. Surprisingly, in the above work ofVos et al. (1991) no dependence on the redox state of QB was found, although control measurements of the 325 nm absorption band of Q~ (van Gorkom et al. 1982) indicated a normal binary oscillation ofQB/Q~ with flash number. One would expect the redox state of QB to be reflected in recombination luminescence because it influences the rate and extent of QA oxidation and proton uptake (Robinson and Crofts 1983), recently shown to be electrogenic (Mamedov et al. 1994). A pronounced influence was in fact observed in thermoluminescence (Rutherford et al. 1982). We have therefore reinvestigated this paradox using the same approach as Vos et al. (1991), but using a different method to eliminate PS I.
Materials and methods
Chloroplasts from laboratory-grown spinach were isolated by grinding in a cooled blender in 50 mM HEPES (pH 7.5)/0.4 M NaC1/1 mM EDTA/0.2% w/v BSA, filtration through a 25 #m mesh nylon cloth and centrifugation for 10 min at 10.000 x g. The pellet was resuspended in a buffer containing 50 mM HEPES (pH 7.5)/0.15 MNa C1/5 mM MgC12 to a chlorophyll concentration of 2 mg/ml and stored at 77 K until use. After thawing, about 10 min before measurement the sample was diluted 400-fold in a buffer containing 1 mM CaCI2/1 mM MOPS (pH 6.6) at 18 °C, the temperature of measurement. A fresh sample was used for each measurement. The experimental set-up was described previously (Vos and van Gorkom 1988). In short, a 1 × 1 × 0.2 cm cuvette was used of which two opposite sides (0.2 cm distance) consisted of platinum electrodes; one side was used for inlet and outlet of a stoppedflow system and the remaining sides were used for flash-illumination and detection of luminescence. The cuvette was placed in the focus of an ellipsoidal mirror for efficient light collection. The emitted light was detected with a gated photomultiplier through a 680 nm interference filter and a Schott RG 665 cut-off filter. Saturating laser flashes (532 nm, 20 ns half-width) were provided at 10 Hz and filtered through a Balzers Calflex C and a Corning CS 4-96 broad band blue filter. Electric pulses with a field strength of 1650 V/cm and duration of 140 ~uswere used. For all measurements the signal observed without electric pulse was subtracted after digitalization.
Results
In order to investigate the effect of the redox state of QB on EL we followed the same procedure as Vos et al. (1991): EL, induced by an electric field pulse 0.5 ms after illumination and integrated from 25 to 125/~s after the onset of the pulse, was measured as a function of the number of flashes fired on a sample initially set predominantly to the state SIQB or to the state S1Q~. The initial condition was prepared by firing 0, 1, or 2 'QB-setting flashes' on a dark-adapted sample, producing predominantly S1QB, S2Q~, or S3QB, respectively. Then the sample was kept in darkness long enough to allow deactivation of the high S-states back to $1, which was accelerated by the presence of FCCP.
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Figure 1. (A) Kinetics of the deactivation of the S-states in ~he
presence of 5 nM (open circles), 10 nM (open squares) and 20 ~M FCCP (solid circles). The ratio of the EL of the 7th flash and the ~th flash is plotted as a function of the time between the 2nd and 3rd flashes. The other flashes were spaced at 100 ms. (B) The EL a~a function of flash number (flashes spaced at 100 ms intervals) in the absence (solid circles) and presence (open squares) of 20 nM FCCP.
Number of flashes
Figure 2. The influence of ferricyanide on EL as a function of flash
number. (A) 50 #M ferricyanide and (B) no ferricyanide. After preillumination by 0 (solid circles, only in A), 1 (open circles) or 2 (solid squares) QB-setting flashes spaced at 100 ms and 7 s dark time for S-state deactivation, a series of 1 to 10 flashes was fired at 10 Hz and EL was measured 0.5 ms after the last flash.
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The deactivation kinetics of the higher S-states ~y F C C P was checked by monitoring the phase shif! of the oscillation pattern as a function o f the dark time between QB-Setting preillumination and flash series. ~n our case 20 nM F C C P was sufficient to deactivate the S-states in about 7 s, as illustrated in Figure 1A. Figure 1B shows that for up to 8 flashes, at a flash frequency of 10 Hz, this deactivation does not cause a substantlal difference between E L oscillation patterns measurpd with (open squares) and without (solid circles) FCCP. In all experiments described below 20 nM F C C P x~as present, i In the presence o f 20 nM F C C P and 50 # M fermicyanide, as used by Vos et al. (1991) to inactivate Photosystem I, preillumination by 1 or 2 QB-setting flashes followed by 7 s dark adaptation had little effect on EL amplitudes, as shown in Figure 2A. Only the amplitude after the first flash is much smaller without preillumination and some o f this effect is still seen on the second flash. This effect is due to the 'non-oscillating' centers mentioned in the Introduction and does not concern ~s
here. After 1 or 2 QB-setting flashes the EL amplitude oscillation with flash number is identical. This result confirms the findings of Vos et al. (1991) and shows not only that 100% SI was present initially, irrespective of the number of QB-setting flashes, but also that QB either has no influence on EL or was in the same initial redox state, independent of preillumination. Figure 2B shows the same experiment with one (open circles) and two (solid squares) QB-setting flashes, carried out in the absence of ferricyanide. Clearly, the number of Q~-setting flashes now does have an effect and in addition the direction o f the effect appears to be alternating with flash number, disregarding flash numbers 4 and 8 for reasons explained below. These results strongly suggest that the redox state of QB influences the amplitude o f E L and that this was not observed earlier because 50 # M ferricyanide oxidized all Q~ within the dark time between QB-setting preillumination and flash series. In the absence of ferricyanide EL emission by P S I cannot be avoided, but it decreases much more rapid-
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ly during the electric field pulse than EL from PS II (Symons et al. 1985; Vos and van Gorkom 1988). As shown in Figure 3, EL appears on the pulseinduced luminescence as an initial spike which does not decrease with increasing delay time between flash and pulse (that takes 0.1 s (Vos and van Gorkom 1988))• It also does not depend on flash number and could thereby be distinguished from PS II EL. Discarding EL emitted during the first 25 #s of the pulse effectively removed the P S I contribution. When the emission at later times was plotted as a function of the delay time between flash and pulse it was found to decrease largely in two exponential phases, with time constants of about 0.2 and 1.4 ms, and a small much longer-lived component (Figure 4). The relative amplitude of the fast component was smaller for luminescence emitted at later times during the pulse (solid
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Figure 5. Decay of integrated (25 to 50 /zs) EL as a function of the time between the last flash and the electrical pulse after 1 (solid circles), 2 (open circles), 3 (solid squares) and 4 (open squares) flashes. Preillumination in (A) none, (B) one and (C) two QB-setting flashes.
lines: integrated from 25 to 50 #s; open circles: from 70 to 85 #s). Apparently the fast component also shows a faster decay during the pulse• The decay of EL, integrated from 25 to 50/zs after the onset of the pulse, as a function of time between the last of I to 4 flashes and the electrical pulse is shown in Figure 5A, B, and C, for 0, 1, and 2 QB-setting flashes, respectively. The curves are fits obtained with a sum of two exponentials, and an offset• Their amplitudes and time constants are listed in Table 1. The offset was independent of QB-setting preillumination and of the number of flashes in the series, in agreement with the conclusion by Vos et al. (1991) that EL measured at 5 ms after the flash is dominated by emission from 'non-oscillating centers'•
201 Table 1. Amplitudes (betweenparentheses)and time constants, in milliseconds, of two exponential phases and a long-lived component, of the decayof integrated EL contributions (Figure 5A, B and C) for 0, 1 and 2 QB-settingpreflashes(integration from 25 to 50/~s)
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The 0.2 and 1.4 ms components can be attributed to stabilization of the charge separation in active, oxygen evolving reaction centers, because a clear Sstate dependence is seen in the time constant of the fast phase and in the amplitude of the slow phase, both being larger for higher S-states. Qualitatively, we associate the former with the S-state dependence of the reduction time of Z + by the oxygen evolving complex (Rappaport et al. 1994; Dekker et al. 1984) and the latter with a different extent of stabilization achieved by each S-state transition, presumably expressed in a different equilibrium concentration of Z +, and hence of P680 +. The reoxidation of QA presumably contributed to the fast phase. The total amplitude at short delay time oscillates with flash number, indicating that the energy of the state Z+QA is S-state dependent. A quantitative interpretation requires additional assumptions, but does not invalidate these global assignments, as discussed already (Vos et al. 1991). The amplitude of the 1.4 ms phase in the decay of EL with increasing time between the last flash and the electric pulse was clearly dependent on the number of QB-setting flashes that had been used. The ratio of this amplitude for the experiment with 2 QB-setting flashes over that with 1 QB-setting flash shows the same pattern during the flash series, 1.9, 0.8, 1.1, 1.1, as already indicated by the data in Figure 2B. Figure 2B indicates that the pattern seen on the first 4 flashes was
repeated on the next cycle of the S-states, suggesting that the effect is specific for the $1 --4 $2 and $2 --4 $3 transitions. For both transitions the electroluminescence signal inducible during the slow stabilization phase was larger when QB was expected to be in the oxidized state before the flash and reduced to Q~ after the flash. The effect of the QB/QB oscillation should be most clearly observed by comparing the results obtained with 1 and 2 Qa-setting flashes, respectively. At any rate the experiment with one flash should be disregarded because of the different behaviour of the nonoscillating centers. Quantitative simulations to determine the actual QB/QB and S-state stoichiometries will require a more extensive data set, including independent information on the QA reoxidation kinetics in these conditions.
Discussion
The results reported here indicate that the earlier failure to detect an influence of the redox state of QB on electroluminescence (Vos et al. 1991 ) was due to reoxidation Of QB by ferricyanide during the 5 seconds dark time after the QB-setting flashes and cannot be ascribed to non-B acceptors (Lavergne and Etienne 1980) as suggested by Lavergne and Leci (1993). In the control experiments showing a binary oscillation with flash number in 325 nm absorbance at low flash frequency (Vos et al. 1991) the addition of 1 mM CaCI2 may have been omitted. The role of Ca 2+ here is not specific, because it can be replaced by Mg 2+ (not shown), and most likely is to screen the negative surface charge at the acceptor side of PS II so that it becomes accessible to ferricyanide (Itoh 1978). The assignments of the various decay phases of the EL precursor as discussed by Vos et al. (1991) are not much affected by the present findings. An 0.4 s phase observed in the presence of 50 #M ferricyanide may now be assigned to Q~ oxidation by ferricyanide, a possibility already recognized by Vos et al. (1991), in agreement with our observation that 50 #M ferricyanide completely abolished the effect of a QB-setting flash followed by 7 s dark adaptation. The assignment of a stabilization phase of about 1 ms to non-oscillating centers must be reconsidered. This was postulated because such a phase was seen on all flashes, including the first two flashes, where no $3 --~ So transition can occur. The amplitude of the 1.4 ms component is now found to oscillate with the
202 redox state of QB, at least in $2 and 83. Therefore, we attribute this component to a stabilization process in active, oscillating centers. In the absence of ferricyanide we do find a specific effect of the redox state of QB on the amplitude of the EL signal at times around a ms after the flash. The amplitude appears to be larger in the presence of Qff than in the presence of QB. One might expect that this is due to a smaller equilibrium constant being associated with electron transfer from QA to QB than with that from QA to Q~ assuming that these reactions take less than a ms. However, the difference is seen only on flash numbers 1 and 2, 5 and 6, etc., corresponding to the S1 --~ $2 and $2 --~ $3 transitions (Figure 2B). It seems reasonable to assume that these transitions contribute to the fast stabilization phase and that the electroluminescence inducible during the slow phase originates from the state reached after equilibration of electron transfer between Tyr Z and the subsequent electron donor. On the Z+S3 --4 ZSo transition, however, Z + probably remains present during the slow phase. This difference between the $3 --~ So transition and the $1 ~ $2 and $2 ---r $3 transitions seems the only likely basis to explain the absence of a Qff/QB effect on the 3rd flash. The slow EL component after the 4th flash may have the same origin as that after the 3rd and be attributed to misses; centers in the Sl-State may not yield detectable EL after Z + reduction (Vos et al. 1991). If the QB/QB effect is due to an influence on the concentration or potential of QA, we have to assume that this influence disappears in the presence of Z +. Alternatively, the QB/QB effect may not be expressed in EL via the acceptor side, but via an influence of QB/QB on the (quasi steady state) concentration of Z + during the electric field induced charge recombination in the states S2QA and S3QA. Both options are somewhat hard to envisage mechanistically. On the other hand, it is also hard to imagine that the observed differences between one and two QB-setting flashes in our experiments are due to anything else than the redox state of QB, and their S-state dependence is undeniable. Our findings may be related to the observation by Bouges-Bocquet (1973) that the reopening of PS II centers on the $3 to So transition is a first order process and does not show the sigmoidal kinetics expected if QA reoxidation and Z + rereduction were independent.
Acknowledgements We are indebted to an anonymous reviewer for great help with the manuscript. This work was supported by the Netherlands Foundation for Chemical Research (SON) of the Netherlands Organization for Scientific Research (NWO) and a EU DGXII Human Capital & Mobility network grant (CHRX-CT94-0524).
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