Photosynthesis Research 31: 113-126, 1992. © 1992 Kluwer Academic Publishers. Printed in the Netherlands. Regular paper

Effects of photoinhibition on the PS II acceptor side including the endogenous high spin Fe 2+ in thylakoids, PS II-membrane fragments and PS II core complexes E. Haag, H.M. Gleiter & G. Renger* Max-Volmer-Institut fiir Biophysikalische und Physikalische Chemie, Technische Universitiit Berlin, Strafle des 17. Juni 135, D-IO00 Berlin 12, Germany; *Author for correspondence

Received 5 June 1991; accepted in revised form 27 October 1991

Key words:

Photosystem II, Photoinhibition, non-heme Fe 2÷, PS II core complexes

Abstract

Effects of photoinhibition at 0 °C on the PS II acceptor side have been analyzed by comparative studies in isolated thylakoids, PS II membrane fragments and PS II core complexes from spinach under conditions where degradation of polypeptide(s) DI(D2) is highly retarded. The following results were obtained by measurements of the transient fluorescence quantum and oxygen yield, respectively, induced by a train of short flashes in dark-adapted samples: (a) in the control the decay of the fluorescence quantum yield is very rapid after the first flash, if the dark incubation was performed in the presence of 300/xM K3[Fe(CN)6]; whereas, a characteristic binary oscillation was observed in the presence of 100/xM phenyl-p-benzoquinone with a very fast relaxation after the even flashes (2nd, 4 t h . . . ) of the sequence; (b) illumination of the samples in the presence of K3[Fe(CN)6 ] for only 5 min with white light (180 W m -2) largely eliminates the very fast fluorescence decay after the first flash due to Q~ reoxidation by preoxidized endogenous non-heme Fe 3÷, while a smaller effect arises on the relaxation kinetics of the fluorescence transients induced by the subsequent flashes; (c) the extent of the normalized variable fluorescence due to the second (and subsequent) flash(es) declines in all sample types with a biphasic time dependence at longer illumination. The decay times of the fast (6-9 min) and the slow degradation component (60-75 min) are practically independent of the absence or presence of K3[Fe(CN)6 ] and of anaerobic and aerobic conditions during the photo-inhibitory treatment, while the relative extent of the fast decay component is higher under anaerobic conditions. (d) The relaxation kinetics of the variable fluorescence induced by the second (and subsequent) flash(es) become retarded due to photoinhibition, and (e) the oscillation pattern of the oxygen yield caused by a flash train is not drastically changed due to photoinhibition. Based on these findings, it is concluded that photoinhibition modifies the reaction pattern of the PS II acceptor side prior to protein degradation. The endogenous high spin Fe 2÷ located between QA and QB is shown to become highly susceptible to modification by photoinhibition in the presence of K3[Fe(CN)6 ] (and other exogenous acceptors), while the rate constant of QA reoxidation by QB(QB) and other acceptors (except the special reaction via Fe 3÷) is markedly less affected by a short photoinhibition. The equilibrium constant between Q~, and QB(QB) is not drastically changed as reflected by the damping parameters of the oscillation pattern of oxygen evolution.

114 Introduction

Solar radiation exerts multiple effects of functional relevance to photosynthetic organisms. Apart from its indispensable role as driving force of photosynthesis, many regulatory functions of light are known to play a key role in the development of the photosynthetic apparatus (Senger 1982, Smith 1982, Shopshire and Mohr 1983). Cyanobacteria, red algae and green plants have developed a variety of adaptation mechanisms in order to cope with different environmental conditions (for a recent review, see Renger 1992). At high light intensities, however, prolonged illumination initially leads to a diminished photosynthetic activity, referred to as photoinhibition, which can cause the eventual death of the organism due to further processes of photodynamic destruction (for a review, see e.g., Powles 1984, Critchley 1988). Numerous studies in the past two decades unequivocally revealed that photoinhibition is primarily due to a deterioration of Photosystem II activity. The process comprises a sequence of events which can be generalized in the following way: light-induced modification of primary target(s) ~ triggering of endogenous proteolysis ~ degradation of the apoprotein of PS II, especially of polypeptide D1. The detailed mechanisms of these events are not yet fully resolved, but it seems clear now that photoinhibition is not induced by only a single, unique target site. Depending on the functional integrity of the PS II complex and on the experimental conditions, at least three different types of reactions were found to be susceptible to photoinhibition: a) the formation of the 'stabilized' radical pair, P680+Q~ (Krause et al. 1985, Cleland et al. 1986, Arntz and Trebst 1986, Demeter et al. 1987, Allakhverdiev et al. 1987, Vass et al. 1988, Eckert et al. 1991), b) the PS II donor side (including the water oxidase) (Zhao and Brand 1988, Eckert et al. 1991), and c) the PS II acceptor side, especially the QB-site (Kyle et al. 1984, Reisman and Ohad 1986, Kirilovsky et al. 1989, Ohad et al. 1990). The triggering of an endoproteolytic attack on the PS II reaction center apoprotein by these functional modifications probably relies on mod-

ifications of the tertiary structure, possibly comprising a PEST-type sequence of D1 near the QB-binding site (Greenberg et al. 1989). The degradation might involve an autocatalytic process (Virgin et al. 1990). Different lines of evidence support the contribution of oxygen radicals in the D1 destruction (Kuhn and B6ger 1990, Sopory et al. 1990, Casado et al. 1990, Hundall et al. 1990). In a recent report, photo-inhibitory effects on the capacity for stable charge separation and the electron flow at the donor side of PS II were analyzed predominantly by laser flash-induced absorption changes at 830nm, reflecting the P680 turnover (Eckert et al. 1991). In the present study, PSII acceptor side photoinhibition was investigated by comparative measurements of the transient flash-induced fluorescence quantum yield in thylakoids, PSII membrane fragments and PS II-core complexes from spinach. In addition, the oscillation pattern of flash-induced oxygen yield was detected in control and photoinhibited samples.

Materials and methods

Thylakoids, PS II-membrane fragments and PS II-core complexes with high oxygen-evolution capacity were prepared from spinach according to the procedures described by Winget et al. (1965), Berthold et al. (1981) (with modifications by V61ker et al. 1985) and Haag et al. (1990), respectively. The photo-inhibitory treatment was performed by exposing 1.5 ml of a sample suspension in a 2cm-wide circular Petri dish, kept on ice, to white light (500 W tungsten lamp, heat filter K3, Schott) at an incident light intensity of 180W -2 m For control measurements, the samples were kept under the same incubation conditions in complete darkness or under dim light. This storage of the control samples did not cause any deleterious effect on the functional activity. The suspensions contained: the sample preparation (100 t~M Chl), 10mM NaCI, 10mM CaCI2, 20mM M E S / N a O H , p H = 6 . 5 , addition of 2 m M K3[Fe(CN)6 ] as indicated in the figure legends. In order to achieve unaerobic conditions, glucose (50mM), glucose oxidase

115 The oxygen-yield pattern induced by a train of single turnover flashes was detected with a Joliot-type electrode (Joliot 1972) as outlined in Messinger and Renger (1990). Suitable signal averaging procedures were used in order to achieve the required high signal/noise ratios (see figure legends).

(0.001% w/v), and catalase (0.001% w/v) were added to the suspensions• The flash-induced transient fluorescence quantum yield was measured with a home-built equipment (Gleiter 1988), as described in Renger et al. (1988), which resembles in its basic principles that described in Schreiber et al. (1986)• In order to achieve a high time resolution, the samples were excited with saturating pulses from a Nd :YAG laser (E = 15 mJ/pulse, FMHW: 3 ns). The fluorescence changes were detected via a modulation technique using high-frequency trains of weak LED-pulses (duration 1/zs) of variable frequency as non-disturbing monitoring system. The fluorescence emission caused by these very weak LED-pulses was detected by an avalanche photodiode (RCA C 30872)• The time resolution of the equipment is of the order of 5/xs. The data were transiently stored in a Nicolet 1147 and further numerical processing was performed•

Results

The major part of this study comprises the measurement and analysis of transient fluorescence quantum yield changes which are induced by laser flashes in dark-adapted samples• Therefore, at first, check experiments were performed in control samples: (a) to define the parameters which are used as indicators of the functional activities affected by photoinhibition, and (b) to show how the overall feature of these fluorescence transients depends on the sample type

thylakoids i

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time Fig. 1. Transient changes of fluorescence yield induced by a train of four laser flashes in the absence of exogenous electron acceptors in dark-adapted thylakoids (top traces), PS II-membrane fragments (middle traces) and PS II-core complexes (bottom traces). Experimental details as described in Materials and methods.

116 (i.e., thylakoids, PS II-membrane fragments and PS II core complexes) and on the experimental conditions (i.e., presence of different electron acceptors). Figure 1 depicts typical traces of transient fluorescence quantum yield changes induced by a train of four laser flashes in darkadapted thylakoids (top), PS II-membrane fragments (middle) and PS II core complexes (bottom). Figure 2 reveals the feature of these transients in PSII-membrane fragments in the absence (top) and presence of either 300/~M K3[Fe(CN)6 ] (middle) or 100/xM phenyl-pbenzoquinone (Ph-p-BQ) (bottom). The flashinduced rise of the fluorescence quantum yield from the F 0 level is not kinetically resolved at our time resolution of about 5/.ts. It is caused by

the QA formation taking place with a 300ps kinetics (Eckert et al. 1988) and the multiphasic P680 ÷ reduction in the ns- and/~s-time domain (Renger et al. 1983, Brettel and Witt 1983, Eckert et al. 1984)• The subsequent decay, dominated by kinetics of a few hundred microseconds, mainly reflects the QA reoxidation (Robinson and Crofts 1983). These kinetics markedly depend on the sample type (Fig. t). A striking phenomenon caused by Ka[Fe(CN)6 ] are the markedly faster relaxation kinetics of the fluorescence quantum yield change induced by the first flash, while the decay after the subsequent flashes appears much less affected [compare the relaxation kinetics after the second flash (middle trace of Fig. 2) with those of the fluorescence

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time Fig. 2. T r a n s i e n t changes of fluorescence yield induced by a train of four laser flashes in dark-adapted PS I I - m e m b r a n e f r a g m e n t s at p H = 6.5. T o p traces: control; middle traces: 5 min dark incubation with 300 p.M K3[Fe(CN)6 ] before the m e a s u r e m e n t s ; b o t t o m traces: 5 min dark incubation with 100/~M P h - p - B Q before the m e a s u r e m e n t s . In order to improve the signal/noise ratio, the f r e q u e n c y of the m e a s u r i n g L E D - p u l s e sequence was reduced a n d the intensity of each pulse was increased. F u r t h e r m o r e , signal averaging was used. 39 signals were averaged for the top traces and 49 signals for the b o t t o m traces, no signal averaging in the middle traces. In the case of P h - p - B Q , the variable fluorescence is rather small due to quenching effects. For the sake of clarity the F 0 level of this m e a s u r e m e n t was suppressed (break in the ordinate). T h e following extents and life times of the fast relaxation were obtained from the traces of the control: first flash (a I = 0.43; r 1 = 1.6 ms), second flash (a 1 = 0.66; r~ = 1.0 ms), third flash (a 1 = 0.56; r 1 = 1.6) and fourth flash (a] = 0.62, r 1 = 1.0 ms), and of samples preincubated with 300 p.M K3[Fe(CN)6]: second flash (al = 0.45, r~ = 1.8 ms), third flash (a~ = 0 . 4 6 , rl = 1.5 ms) and fourth flash (a~ = 0.45, r 1 = 1.5 ms). For o t h e r experimental details see Materials and methods.

117 exhibit a striking binary oscillation of both, the extent of the maximum fluorescence and the relaxation kinetics. This phenomenon is in perfect agreement with the idea of reductantinduced oxidation of the endogenous Fe 2÷, giving rise to a population of redox state Fe 3+, which exhibits a characteristic period-two oscillation, if dark-adapted samples are excited with a flash train (Zimmermann and Rutherford 1986, Diner and Petrouleas 1988). The effect of photoinhibition on the very fast QA reoxidation by Fe 3+ is illustrated in Fig. 3. It shows that illumination of thylakoids with 180 W m -2 at 0 °C and pH = 6.5 for only 5 min under aerobic conditions in the presence of an electron acceptor like K3[Fe(CN)6 ] or D C B Q (2,6-dichloro-p-benzoquinone) (data not shown) largely eliminates the very fast QA reoxidation after the first flash (compare top traces of control with bottom traces of photo-inhibited samples). This effect evolved markedly slower, if K3[Fe(CN)6 ] is omitted during the photo-inhibitory treatment and only added before the fluorescence measure-

yield decay after the first flash in the control (top trace of Fig. 2)]. The very fast relaxation is also responsible for the significantly reduced extent of the maximum fluorescence caused by the first flash, because a large part of the signal escapes detection due to the incomplete rise and the limited time resolution of our equipment. The very fast decay kinetics are assumed to reflect the rapid electron transfer from QA to the endogenous Fe 3÷ which has been slowly oxidized before the first flash by K3[Fe(CN)6 ] during the dark incubation period (Diner and Petrouleas 1989) but not during the comparatively short time of 1 s between the first and second flash (Renger et al. 1987). Accordingly, for kinetic reasons the ratio of the variable fluorescence, measured 100/xs after the second and first flash, respectively, Fvar(2)/Fvar(1) (see Fig. 3), can be used as a qualitative measure of the extent of rapid Q~ reoxidation with the endogenous Fe 3+ as acceptor, formed by K3[Fe(CN)6 ] before the first flash. This idea is highly supported by the data obtained in the presence of Ph-p-BQ which

control

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time Fig. 3. Transient changes of fluorescence yield induced by a train of four laser flashes in dark-adapted PS I I - m e m b r a n e fragments

preincubated with 300/zM K3[Fe(CN)6] for 5 min under aerobic conditionsbefore the measurements. Top traces: control sample; bottom traces: samples photo-inhibited by 5 min white light illumination at 180 W m -2 at 0 °C and 2 mM K3[Fe(CN)6] before subsequent dark incubation and measurement. Other experimental details as in Materials and methods.

118 ments (see Fig. 4). In order to test whether the strong photoinhibition of the Ka[Fe(CN)6 ]induced, very fast QA reoxidation after the first flash is a general phenomenon of the PS II acceptor side, comparative experiments were performed with thylakoids, PS II-membrane fragments and PS II-core particles. These sample types exhibit marked differences near the QB site as reflected by modified QA reoxidation kinetics (see Fig. 1) and atrazine-binding properties (Renger et al. 1986). Figure 4 depicts the ratio Fvar(2)/Fvar(1 ) (see Fig. 3) as a function of photoinhibition time in thylakoids, PS IImembrane fragments and PS II-core complexes. The data reveal that the K3[Fe(CN)6]-induced, very fast QA reoxidation exhibits similarly high susceptibilities to photoinhibition performed in the presence of this electron acceptor, regardless of the detergent-induced changes at or near the QB-binding site in PS II-membrane fragments and PS II-core complexes. These results provide the first evidence that under certain conditions the microenvironment of the endogenous high spin Fe 2+ between QA and QB exhibits a very

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the second laser flash and after the first flash, respectively, [Fvar(2) / Fva~(1)],pp as a function of photo-inhibitoryillumination time tp] in thylakoids, PS II-membrane fragments and PS II-core complexes. The data were obtained from types of measurements described in Fig. 3. The photoinhibitiontreatment was performed in suspensions containing 2mM K3[Fe(CN)6] (open symbols). For a comparison, closed symbols show the effect of photoinhibition performed in the absence of K3[Fe(CN)6] in PSII-membrane fragments. Other experimental details as described in Materials and methods.

high sensitivity to modifications by light stress (see Discussion). In previous studies photoinhibition was shown to affect the properties of the QB-binding site (Ohad et al. 1990, Kirilovsky et al. 1990). As the endogenous high spin Fe 2÷ is in close proximity to this site, both phenomena might be interrelated. In order to test this idea, the extent of the flash-induced maximum variable fluorescence and its dark relaxation time were measured as a function of illumination with white light (180 W m -2) under different conditions. Typical traces are depicted in Fig. 5 for the control (top) and thylakoids illuminated for 15 min (middle) and 60min (bottom) on ice in the absence of K3[Fe(CN)6 ]. Three striking phenomena emerge from Fig. 5: i) the maximum of the flash-induced variable fluorescence drastically declines, ii) the relaxation kinetics become markedly retarded, especially in the time domain up to 10 ms, and iii) the F 0 level increases. The drastically diminished extent of the flashinduced increase of the fluorescence-quantum yield in photo-inhibited samples is consistent with the decline of the maximum fluorescence reported in numerous studies in the literature for the conventional fluorescence induction curve (see Setlik et al. 1990, Kirilovsky et al. 1990, and references therein). In order to analyze the time course of the photo-inhibitory treatment on different samples and various conditions, the normalized maximum of the variable fluorescence induced by the second flash, FvmaX(2)/F0(1), was used as an appropriate indicator. In this way, possible effects on the K3[Fe(CN)6]-induced fast QA reoxidation are eliminated, because this reaction is restricted to the relaxation after the first flash (vide supra). Figure 6 depicts in a semilogarithmic plot the extent of FvmaX(2)/F0(1) as a function of photoinhibition time in thylakoids illuminated either under anaerobic or aerobic conditions. The time course can be described by the expression: [Fvm~X(2)/Fo(1)Lpl

[F~m2x(2)/Fo( 1)],,i=o = a exp(- t.i/r~ ) + (1 - a) exp(-tpi/P2 )

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time Fig. 5. Transient changes of fluorescence yield induced by a train of four laser flashes in dark-adapted thylakoids without addition of exogenous acceptors. Top traces: control; middle traces: samples photo-inhibited by illumination with white light of 180 W m-2 for 15 min at 0 °C before subsequent dark incubation and measurement; bottom traces: 60 min photoinhibition. The following life times of the fast phase of Q~-reoxidation after the first flash were obtained: control (z I = 0.4 ms), sample illuminated for 15 min (~'1 = 2.1 ms), sample illuminated for 60 min (rl = 3.1 ms). Other experimental details as described in Materials and methods.

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Fig. 6. Semilogarithmic plot of the normalized maximum variable fluorescence due to the second flash, [Fm~,~(2)/ Fo(1)],p/[F~,x(2)/Fo(1)],p~=o, as a function of photo-inhibitory illumination time tpt of thylakoids (triangles), PS IImembrane fragments (squares) and PS II-core complexes (circles) under aerobic conditions in the absence of K3[Fe(CN)6 ]. Other experimental details as described in Materials and methods.

This behavior of a biphasic decay was also observed in PS II-membrane fragments and PS IIcore complexes, regardless of the absence or presence of K3[Fe(CN)6 ] and aerobic or unaerobic conditions, as shown by a compilation of the results in Table 1. In general, the following conclusions can be drawn from these data: a) the lifetimes of the fast (r I ~ 7 min) and the slow (r 2 ~ 60-70 min) phases are almost independent of the presence of 0 2 (aerobic vs. anaerobic conditions) and/or K3[Fe(CN)6 ] in the assay medium for photoinhibitory treatment; b) in the absence of K3[Fe(CN)6], the extent of the fast decline is dominating under anaerobic conditions, whereas it is almost equal to that of the slow phase under aerobic conditions; c) the presence of K3[Fe(CN)6 ] during photoinhibition under anaerobic conditions causes an enhanced extent of the slow decline, while it has only marginal effects under aerobic conditions. If one accepts that the variable fluorescence mainly reflects the capacity of the stable charge

120 Table I. Normalized variable fluorescence at 100/xs after the second laser flash, Fvar(2)/F0(1), of a train given to dark-adapted samples as a function of illumination time of photo-inhibitory pretreatment, tpr (see Material and methods):

[F~=~(2)/Fo(1)l,p, FmaX = a exp(- tp]rl) + (1 - a) exp(-tm/r2)

(1)

[ v., ( 2 ) / F 0 ( 1 ) ] , , , = 0

Sample

Aerobic conditions PI: minus M: minus

PI: plus M: plus

a

~'Jmin

~-2/min

a

rl/min

~'2/min

Thylakoids PS II-membrane fragments PS II-core complexes

0.40 0.45 small

9 7 -

60 60 65

0.38 0.36 0.34

7 6 6

70 70 70

Sample

Anaerobic conditions PI: minus M: minus

Thy|akoids PS II-membrane fragments PS II-core complexes

PI: plus M: plus

a

r I / min

r: / m i n

a

r 1/ m i n

r 2/

0.77 0.71 0.53

7 7 7

70 70 75

0.55 0.53 0.50

7 7 9

60 70 70

min

PI: absence or presence of 2 mM K3[Fe(CN)6] during the photo-inhibitory treatment. M: omission or addition of 300/xM K3[Fe(CN)6] in the assay for fluorescence measurements. separation leading to the radical-pair state, P680+PheoQA, the present data would imply that the elimination of this activity exhibits a two-phase degradation. A n analogous biphasic b e h a v i o r has not been observed for the photoinhibition of the P680 turnover in O2-evolving PS I I - m e m b r a n e fragments (Eckert et al. 1991). T h e r e f o r e , the m o r e complex time course of the photoinhibitory [Fv~a~X(2)/F0(1)]-decline probably reflects the contribution of additional effects, as will be outlined in the Discussion. F u r t h e r m o r e , photoinhibition p e r f o r m e d in the presence of K3[Fe(CN)6 ] leads to a faster d r o p of FvmaaX(2)/Fvar(1) than of FvmaaX(2)/F0(1) ( c o m p a r e Figs. 4 and 6). This indicates that the loss of the capacity of the very fast QA reoxidation after the first flash, ascribed to the presence of e n d o g e n o u s Fe 3+, is kinetically not intimately related to the deterioration of the PS II activity as reflected by the decrease of FvmaX(2)/F0(1). T h e traces in Fig. 5 also revealed a retardation of the relaxation kinetics of the flash-induced transient fluorescence q u a n t u m yield. As the kinetics in the 0.1-10 ms time domain reflect the QA reoxidation by Q B ( Q ~ ) (Robinson and Crofts 1983, Weiss and R e n g e r 1984), the data of Fig. 5 are interpreted as a slowing down of the electron transfer f r o m Q~ to Q s ( Q B ) due to

photoinhibition. It has to be emphasized that the relaxation kinetics of the untreated control exhibit m a r k e d differences between the different sample types (see Fig. 1). A numerical evaluation of the data in Fig. 1 leads to the following results: In thylakoids, about 8 0 - 9 0 % of the fluorescence decay takes place with a rate constant of about kfast = 4000s -1, while in PS I I - m e m b r a n e fragments and PS II-core complexes about 50% decays rapidly with rate constants of about 1500 s -1 and 300-400 s -1, respectively. In order to analyze a possible correlation between light stress-induced decrease of [Fv~X(2)/ F0(1)] and the kinetics of the fast relaxation c o m p o n e n t (see Fig. 5), the rate constant, kraft, of the fluorescence decay after the second flash was m e a s u r e d as a function of the photoinhibition time. A semilogarithmic plot ln[kfa~t(tpi)/ kf,~t(tpi = 0)] = f ( t ) revealed that a biphasic dep e n d e n c e prevails under aerobic conditions in thylakoids and PS I I - m e m b r a n e fragments, while under anaerobic conditions and in PS II-core complexes, the results are better described by a m o n o - e x p o n e n t i a l time course of the kfast ret a r d a t i o n due to photoinhibition (data not shown). A s u m m a r y of these results is given in Table 2. A comparison of the data in Tables 1 and 2 readily shows that the flash-induced, nor-

121 Table 2. Normalized rate constant kraft of the fast decay of flash-induced variable fluorescence as a function of illumination time of photo-inhibitory pretreatment: [kf..], m

[kfast]q,[=o a exp(-tvl/rl) + (1 - a) exp(-tp,/~2) -

Sample

Aerobic conditions PI: minus M: minus

PI: plus M: plus

a

'7"1 / min

r 2/ min

a

r 1/ min

%/ min

Thylakoids PS II-membrane fragments PS II-core complexes

0.27 -

4 -

50 50 40

0.32 no change

9 -

80 50

Sample

Anaerobic conditions r 2/ min

a

r 1/

n.r. -

-

35

-

10

PI: minus M: minus Thylakoids PS II-membrane fragments PS II-core complexes

min

PI: plus M: plus

a

r 1/

n.r. -

-

35

-

10

min

r 2/ min

n.r. - not resolvable. m a l i z e d m a x i m u m v a r i a b l e f l u o r e s c e n c e a n d its relaxation kinetics exhibit different dependences on the time of photoinhibition and the assay c o n d i t i o n s . L i k e w i s e , t h e m o d e o f kfast r e t a r d a t i o n e x h i b i t s a significant d e p e n d e n c e o n t h e sample type, which can be explained by a det e r g e n t - i n d u c e d m o d i f i c a t i o n o f t h e a c c e p t o r side (see D i s c u s s i o n ) . T h e a p p a r e n t r a t e c o n s t a n t o f QA r e o x i d a t i o n by QB(QB) actually represents the sum of the r a t e c o n s t a n t s for t h e f o r w a r d a n d b a c k reactions, respectively, because of the equilibrium between QA/QA and QB/QB (QBH2/QB) (for a r e v i e w s e e C r o f t s a n d W r i g h t 1983). A c c o r d i n g ly, t h e m e a s u r e d r e t a r d a t i o n o f kfast b y p h o t o i n h i b i t i o n c o u l d i m p l y a shift in t h e e q u i l i b r i u m c o n s t a n t t o w a r d s l o w e r v a l u e s , as o b s e r v e d in C h l a m y d o m o n a s reinhardtii ( K i r i l o v s k y et al. 1990). I n o r d e r to test this p o s s i b i l i t y , t h e oscillation pattern of the oxygen yield induced by a flash t r a i n w a s m e a s u r e d in d a r k - a d a p t e d s a m pies. F i g u r e 7 s h o w s t h e r e s u l t s o b t a i n e d in t h e c o n t r o l a n d in s a m p l e s i l l u m i n a t e d f o r 60 m i n w i t h w h i t e light ( 1 8 0 W m - 2 ) , under anaerobic conditions, before performing the measurem e n t s . I n o r d e r to i l l u s t r a t e m o r e c l e a r l y p o s sible effects o n t h e o s c i l l a t i o n p a t t e r n , t h e d a t a were normalized on the oxygen yield of the third flash. I n t h y l a k o i d s ( t o p ) , t h e c h a r a c t e r i s t i c

ol.

T.YLAKo,os

/i ~ ' d -o

o'A

/,

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/'-'o

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PSII-CORE

o 2

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6 8 flashnumber

, 10

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Fig. 7. Oxygen yield as a function of flash number in darkadapted thylakoids (top traces), PS II-membrane fragments (middle traces) and PS II-core complexes (bottom traces) without and with photoinhibition treatment for 60 min on ice at 180 W m 2 Control samples: open symbols; photo-inhibited samples: closed symbols. p e r i o d - f o u r o s c i l l a t i o n p a t t e r n is o b s e r v e d . This p a t t e r n e x h i b i t s a slightly e n h a n c e d d a m p i n g in s a m p l e s e x p o s e d to a 6 0 m i n - p h o t o i n h i b i t i o n

122 treatment before the measurement. These data are in line with the finding of Ohad et al. (1988) that cells of Clamydomonas reinhardtii show no changes in the normalized flash induced oscillation pattern of 0 2 evolution after photoinhibitory treatment. The traces for the control PS IImembrane fragments (middle) exhibit a pronounced period-four oscillation, which rapidly fades due to the limited electron acceptor capacity. An interesting phenomenon emerges in the photo-inhibited PS II-membrane fragments. In this case, the normalized oscillation pattern prevails for more than only two quaternary periods. This finding can be explained by the blockage of PS II due to photoinhibition and the assumption that the total pool capacity is available for the remaining active centers. An analogous effect has been observed recently in PS II-membrane fragments that were partially inhibited by D C M U (Renger et al. 1989). In PS II-core complexes, the oscillation pattern is highly damped due to the very limited electron acceptor capacity. The data of this study closely resemble those published recently (L/ibbers and Junge 1990). This shows that the pattern at the bottom of Fig. 7 is typical for PS IIcore complexes. In samples, after 60 min photoinhibition, the oscillation pattern is not markedly improved in terms of a further period-four oscillation. This phenomenon indicates that the highly damped oscillation is due to a complex pattern of acceptor side limitations, as will be outlined in a forthcoming paper (Gleiter, Haag, Inoue and Renger, submitted for publication). An evaluation of the experimental data of the thylakoids and PSII-membrane fragments in terms of the classical Si-state model of Kok (Kok et al. 1970) does not reveal a significant increase of the probability of misses (data not shown). Based on these findings, we conclude that photoinhibition under our conditions does not markedly change the redox equilibrium between QA and the secondary plastoquinone at the QB site. The photoinhibition treatment in the present study was performed at pH 6.5 on an ice bath, because under these conditions the degradation of polypeptides was reported to be very slow (Kuhn 1989, Aro et al. 1990, Virgin et al. 1990) and, therefore, a primary attack on the functional integrity is expected to be clearly separable

from secondary effects due to protein degradation. Silver-staining of SDS-poly-acrylamide gels confirmed that under our experimental conditions no marked D1 degradation was detected in all samples exposed to photoinhibition in the absence of K3[Fe(CN)6 ]. Preliminary experiments seem to indicate a diminution of D1, if the illumination was performed in the presence of K3[Fe(CN)6 ] (data not shown). Interestingly enough, under these conditions, also the very fast QA reoxidation after the first flash was found to be highly sensitive to photoinhibition (see Figs. 2 and 4). Therefore, it appears attractive to assume that a modification in the microenvironment of the endogenous non-heme iron leads to a high susceptibility for polypeptide D1 degradation even at 0 °C and pH = 6.5. The underlying mechanism for such an effect is unknown.

Discussion

The aim of this study was the analysis of photoinhibition effects on the functional pattern of PS II with special emphasis on the acceptor side reactions in sample types of different structural complexity. This problem was addressed by: a) comparative measurements in thylakoids, PSII membrane fragments and PSII corecomplexes which exhibit significant differences around the QB site already in the untreated control as reflected by the Q~ reoxidation kinetics (this study) and the atrazinebinding properties (Renger et al. 1986) b) Photo-inhibitory treatment at 0 °C and pH = 6.5 in order to minimize possible interference by endoproteolytic polypeptide degradation, especially of D1 (Kuhn 1989, Aro et al. 1990, Virgin et al. 1990). The results obtained indicate: (i) the very fast QA reoxidation supported by the oxidized Fe 3+ form of the endogenous non-heme iron center located between QA and QB is highly susceptible to deleterious light effects, if the illumination is performed in the presence of an exogenous electron acceptor; (ii) the variable fluorescence as a qualitative indicator of the photochemical activity exhibits a biphasic dependence on the time of photoinhibition with a more pronounced fast decline under anaerobic conditions; (iii) in sam-

123 ples with the non-heme iron center in its normal reduced Fe 2+ state, the rate constant of the fast component of QA reoxidation becomes diminished upon photoinhibition treatment, and (iv) the redox equilibrium between QA and QB(Qa) does not drastically change in photo-inhibited samples, which is reflected by the oscillation pattern of the oxygen evolution induced by a flash train. The basic conclusion gathered from these findings is the possibility of a photoinhibitory functional modification of acceptor-side reactions prior to any significant protein degradation. This is in line with recent observations of Ohad et al. (1990) and Kirilovsky et al. (1990) who have presented indirect evidence for a rapid structural change around the QB site. Our data suggest that photoinhibition can lead to modifications which specifically affect the properties of the high-spin Fe 2+. The results of Figs. 3 and 4 are explainable by two alternative models: (1) elimination of the Ka[Fe(CN)6 ] induced Fea+-formation (as an indispensible prerequisite for the very fast QA reoxidation) either by a shift of the oxidation potential or a largely increased shielding of the endogenous non-heme iron center, (2) drastic retardation of the electron transfer from QA to Fe 3+ due to increase of the effective distance of the redox centers and/or change of the reorganization energy. Our present data do not permit an unequivocal distinction. Regardless of this mechanistic problem, the endogenous high spin Fe E+ and its micro-environment appear to act as a primary target for photoinhibition under certain conditions. This effect appears to be independent of the fine structure of the QB site, because the elimination of the very fast Ka[Fe(CN)6 ]induced QA reoxidation after the first flash (which requires formation of Fe 3+ and its proper coupling with QA) occurred in thylakoids, PS IImembrane fragments and PS II core complexes with a similar dependence on the photoinhibition time. As the high spin Fe 2+ coordinates with four histidines (two in polypeptides D1 and D2, respectively), it appears attractive to speculate that a modification in this region could act as a trigger signal for an enhanced susceptibility of polypeptide(s) DI(D2?) to endoproteolytic degradation. In addition to that, an attack on the non-heme iron and its microenvironment by light

stress might also be related to the recently discovered role of H C O 3 in the process of photoinhibition (Sundby 1990). Likewise, it appears interesting to speculate whether a modified nonheme iron center could interact with peroxide, formed at the PS II acceptor side under aerobic conditions (Schr6der and Akerlund 1990). This reaction might give rise to highly reactive oxygen radicals which are assumed to cause degradation of polypeptide(s) DI(D2) (Kuhn and B6ger 1990, Sapory et al. 1990, Casado et al. 1990, Hundall et al. 1990). Previous studies revealed that the susceptibility to proteolytic degradation markedly increases in proteins that are oxidatively modified by 0 2 in the presence of Fe 3+ or Cu E+ (for recent review, see Stadtman and Oliver 1991). An additional acceptor-side modification by photoinhibition is the retardation of the fast QA reoxidation by endogenous plastoquinone (QB / QB) or further electron acceptors other than the oxidized Fe 3+ form of the endogenous non-heme iron center. This phenomenon clearly differs from the Fe 3+ related effect in its dependence on the sample type and its independence of the absence or presence of Ka[Fe(CN)6 ] in the suspension exposed to illumination. A comparison of the data in Fig. 4 and Table 1 suggests that the modification of the non-heme iron properties by photoinhibition (as reflected by the suppression of the very fast QA reoxidation after the first flash in Ka[Fe(CN)6]-treated samples) does not affect the kinetics of the electron transfer from QA to Qa(QB). This finding also suggests that the non-heme iron center is not required for the functional connection between QA and the QB site. Analogously, previous studies with anoxygenic purple bacteria unambiguously showed that even removal of the iron from the reaction centers does not markedly change the electron transfer rate of QA reoxidation by QB (Debus et al. 1986). It has to be emphasized that, in control samples, a retardation of the fast QA reoxidation by endogenous acceptors other than the oxidized Fe 3+ form of the endogenous non-heme iron center already arises as a consequence of the detergent treatment during the isolation of PS IImembrane fragments and PS II core complexes. This reflects the sensitivity of the QA reoxidation to structural changes in the protein matrix of the

124 acceptor side as proposed previously (Renger 1976, Renger et al. 1981). The differences in the finetuning of the reactive properties of the endogenous non-heme iron center and of QB (QA) are not surprising because effects that are assumed to be predominantly related to the structure of the QB-site already can exhibit remarkable variations. This is clearly illustrated by quite dramatic consequences of some single site mutations of the D1 polypeptide for the binding properties of QB and various herbicides (Gleiter et al. 1990). Another striking phenomenon observed here is the biphasic decline of the normalized variable fluorescence induced by the second (and subsequent) flash(es). At first glance, this effect might be due to the different susceptibilities of PSII~ and PSII~ centers to photoinhibition (M/iennp~ia et al. 1987). However, this interpretation appears to be unlikely because the biphasicity also arises in PS II-core complexes which are expected to originate predominantly from PS II~ centers. A more likely explanation could be based on the existence of different kinetics of photoinhibition (as suggested recently by Setlik et al. 1990) together with a heterogeneity of the PS II centers that does not markedly depend on the sample type (thylakoids, PS II-membrane fragments or PS II core complexes). The more pronounced fast phase in anaerobically photoinhibited samples suggests that a functional heterogeneity correlated with the different modes of photoinhibition. Likewise, the dependence of the fluorescence quantum yield on the redox state of Q A (and other centers) and non-photochemical quenching processes is also responsible for the different degradation kinetics of the oxygen evolution and P680 turnover (Eckert et al. 1991) and the variable fluorescence, respectively. The results of this study revealed that the pattern of the photo-inhibitory effects at the PS II acceptor side is a general phenomenon of PS II which does not markedly depend on the natural lipidic environment (compare thylakoids and PS II membrane fragments) or the antenna size (compare PS II membrane fragments and PS II core complexes). Furthermore, the results of this study confirm that the process of photoinhibition is not simply triggered by the modifica-

tion of a single unique target site but comprises different sites depending on the functional integrity of PS II.

Acknowledgements The authors would like to thank Dr T. Wydrzynski for critical reading of the manuscript, M. M/iller for skilful technical assistance and S. Hohm-Veit for drawing the figures. The financial support by ERP-Sonderverm6gen (ERP 2603) is gratefully acknowledged.

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