the pq/pqh, ratio and occupancy of photosystem 11-qb site by

THE JOURNAL 0 1991 by The American Society for Biochemistry

OF BIOLOGICAL CHEMISTRY and Molecular Biology, Inc

Vol. 266, No. 31, Issue of November ,5, PP .21293-21299,1991 Printed in U.S.A.

The PQ/PQH, Ratio and Occupancy of Photosystem 11-QB Site by Plastoquinone Control the Degradation of Dl Protein during Photoinhibition in Vivo*

(Received for publication, May 30, 1991)

Huashi Gong$ and Itzhak Ohad From the Department of Biological Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel and the $Department of Biology, the Phytotron, University of Oslo, P. 0. Box 1066, Blindern, 0316 Oslo 3, Norway

Photoinactivation of photosystem I1 (PSII) and light- dependent degradation of the reaction center I1 (RCII) protein D l have been investigated in Chlamydomonas reinhardtii mutants D6, AC208, and B4 deficient in cytochrome ba/f, plastocyanin, and photosystem I (PSI) activity, respectively. These mutants possess active PSII and reduce plastoquinone (PQ) but cannot oxidize plastoquinol (PQH,) via light-dependent reduction of NADP. In light-exposed cells a high ratio PQH,/PQ and a low turnover of PQ/PQHz at the RCII-QB site are maintained. In all mutants photoinactivation of RCII was slower as compared to the wild-type (wt) cells, and D l degradation was drastically decreased. The degradation of D l was also lower in the wt cells under anaerobic conditions and presence of ascorbate, while raising the concentration of dissolved oxygen increased the degradation of the D l protein in the AC208 mutant. Photoinactivation and light-dependent degradation of the D l protein were drastically increased in the Scenedesmus obliquus LF- 1 mutant cells altered in its PSII manganese binding and thus unable to reduce PQ using water as an electron donor. Diuron inhibited the light-dependent degradation of D l protein in both the LF-1 mutant and wt cells. Based on these results we propose that availability of PQ at the QB site is required for (i) the photoinactivation process of the RCII acceptor side followed by inactivation of the donor side leading to the generation of harmful cation radicals (Z+, P680+, chl,+) which damage the D l protein, and (ii) the accessibility of the cleavage site of the damaged D l protein to proteolytic degradation.

The oxygen-evolving photochemical reaction center of photosynthetic membrane (RCII)’ is inactivated when exposed to light intensities above saturation of electron flow (1-5). This

*This work is part of H. G.’s Ph.D. thesis and was supported partially by a fellowship (awarded to H. G.) by the University of Oslo, Norway, by a grant (awarded to I. 0.) by the Israeli Academy of Sciences, the BARD Binational Israel-American Agricultural R and D Foundation in cooperation with W. Vermaas, Arizona State Uni- versity, Tempe, AR, and a grant awarded by the German-Israel Foundation (to I. 0.) in cooperation with Dr. D. Godde, Ruhr Uni- versity, Bochum, Federal Republic of Germany. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertise- ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: RCII, reaction center 11; Cyt, cytochrome; Diuron, 3-(3’4’-dichlorophenyl)-l,l-dimethylurea; LHCII, light-harvesting chlorophyll protein complex; PSI and PSII, photosystem I and 11; PQ and PQH,, plastoquinone and plastoquinob wt, wild type; PFD, photon flux density.

process is referred to as photoinhibition and has been studied in much detail using both in vitro and i n viuo systems (1-8). During photoinhibition the D l protein of RCII is rapidly degraded and replaced by a newly synthesized molecule (4,5). This process has been studied extensively since the D l and D2 proteins form the RCII heterodimer which binds the chlorophyll, pheophytin, quinones, and Fe atom participating in the primary photochemical charge separation and the electron acceptor side of RCII (9-11). The D l protein contains also the primary electron donor, 2, identified as D l amino acid Tyr16’ (12, 13) and participates in the formation of the manganese binding site required for the water oxidation and oxygen evolution (14). The secondary electron acceptor quinone is bound in a hydrophobic niche formed by the transmembrane helices IV and V and the interconnecting surface exposed segment of the D l protein, the QB site (15,16). Upon double reduction and protonation, PQHz leaves the QB site and is replaced by a PQ molecule from the plastoquinone pool in the photosynthetic membrane.

Photoinhibition can be resolved in at least two (8, 17) and possibly three phases (18), including reversible photoinactivation of RCII (8, 17, 18, 19) followed by irreversible damage and degradation of the D l protein. Recovery of electron flow activity at the latter stage requires replacement of the D l protein (8, 17, 19).

Light-dependent degradation of D l protein occurs also at low light intensities at which no detectable photoinhibition occurs. The relation between the D l protein degradation induced at low light intensity and the process of photoinactivation is not yet clear, since these two processes can be separated by the light intensity and quality at which they occur (20, 21). It is now well established that i n vitro photoinactivation is enhanced under strict anaerobic conditions (18, 22) while degradation of the D l protein does not occur unless at least traces of oxygen are present (22). It was also proposed that oxygen or hydroxyl radical (3, 23) formed during the high light illumination may cause the inactivation of PSII (24) and induce the degradation of the D l protein (23).

We have proposed before that alterations induced in the acceptor side of RCII during rapid turnover of PQ/PQH2 at the QB site may be essential in the triggering of the photoinactivation and D l degradation processes (5, 8, 25). This view is still controversial. Evidence obtained by other labo- ratories suggests that loss of QA- oxidation (7, 18), loss of P68O-pheophytin charge separation (26), and particularly the inactivation of the RCII donor side (6, 27, 28, 29) are the primary targets of photoinhibition.

The degradation of D l protein is prevented both i n vivo and i n vitro by herbicides such as Diuron or Atrazine which

21293

21294 PQIPQH, in Photoinhibiton and Dl Protein Degradation

compete with PQ for the QB binding site (20,21,30). Tryptic proteolysis of D l in vitro is accelerated when the PQ pool is removed from the membranes and inhibited by the above herbicides. It was therefore suggested that the occupancy of the QB site either by herbicides or PQ may play a protective role in this process while an unoccupied site may become accessible to proteolytic D l degradation (30). However, the light-dependent degradation of D l was significantly prevented in a cytochrome b6/f-deficient mutant of Chlamydo- monas reinhurdtii in which the PQ pool is mostly reduced in light (31). To establish the role of the turnover of PQ/PQH2 at the QB site in the process of RCII photoinactivation and Dl degradation, we have analyzed these processes in vivo in Chlamydomonas and Scemdsmus mutants deficient in PQH, oxidation or electron donation to RCII. In such mutants the ratio PQ/PQH, and thus occ,upancy of the QB site by PQ or PQHz in high light-exposed cells is drastically changed as compared to the wild type cells.

The results clearly demonstrate that the ratio PQ/PQH, is a key factor in the process of D l degradation.

MATERIALS AND METHODS

Cell Growth-C. reinhurdtii cells were grown in white fluorescent light in a semicontinuous culture apparatus using a mineral medium supplemented with sodium acetate as a carbon source (32). The y-1 mutant is identical with the wild type when grown in the light and therefore served as a control (32). The mutants D6 and Ac208 are defective in cytochrome b6/f (33, 34) and plastocyanin, respectively (35,36). The B4 mutant defective in PSI activity was kindly supplied by Dr. L. Metz. Scenedesmus obliquus wild type and LF-1 mutant were cultivated as described by Bishop and Senger (37). The LF-1 mutant is defective in the water-splitting activity and does not evolve oxygen (14, 38) but can reduce PQ using artificial electron donors to RCII.

Photoinhibitory Treatment and Measurements of Photosynthetic Actiuity-Cells were harvested at the end of the exponential phase of growth and resuspended in fresh growth medium at a final chlorophyll concentration of 30 Fg/ml. Unless otherwise specified, the cell suspension was exposed to a PFD of 1500 pmol m-* s" provided by a projector equipped with a tungsten halogen lamp (650 watts) filtered through a UV and a heat filter. Incubation was carried out in glass cylinders of 3-cm internal diameter a t 25 "C (17). Light intensities and incubation time were as indicated in the different experiments.

Fluorescence induction was measured on intact cells as described before, using a computer-assisted homemade fluorimeter (8,31). The chlorophyll concentration during these measurements was 5-10 pg/ ml. The fast and slow phases of fluorescence rise (8) were resolved by recording the initial phase at 10,000-20,000 points/s and the slow phase at <200 points/s. Oxygen evolution in intact cells was assayed using a Clark-type oxygen electrode.

Thylakoid Membrane Preparation and Measurement of PSZZ Poly- peptide Degradation-Cells were collected by centrifugation and resuspended in Tricine-HC1 buffer (50 mM pH 7.8) containing 5 mM MgC12 and 10 mM NaCl (TMN buffer (39)). C. reinhurdtii cells were broken in an ice-cooled French press operated at 6000 p.s.i. S. obliquus cells were broken by shaking with glass beads (0.2-0.3-mm diameter), 2 X 1 min, at 0 "C. The homogenates were centrifuged at 3000 X g for 5 min to remove unbroken cells and large debris, and a thylakoid- enriched membrane fraction was collected from the supernatant by centrifugation at 10,000 X g for 10 min at "C and suspended in TMN buffer (39). For analysis of D l and CP47 polypeptides degradation, chloramphenicol (Sigma, D-threo form, 200 pg/ml) was added to prevent de nouo chloroplast protein synthesis. This procedure did not alter the results as indicated by experiments in which protein degradation was tested by radioactive pulse-chase (Ref. 17, also this work, data not shown). The content of the above polypeptides was estimated by the Western blotting technique (40). The thylakoid membrane proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using equal amount of membranes on a chlorophyll basis (31). The resolved polypeptides were electrotransferred to ni- trocellulose paper and detected with anti-Dl or anti-CP47 antisera followed by decoration with lZ5I-protein A and exposure to x-ray film (40).

Other Procedures-Chlorophyll was determined in 80% acetone

extracts of whole cells or membranes using the metho&of Arnon (41). Fluorescence emission spectra at 77K were recorded using a Perkin- Elmer spectrofluorimeter (MP4) as described before (42). All reagents used in this work were of analytical grade.

RESULTS

C. reinhardtii Mutants Lacking Cytochrome b6/L Plastocy- anin, or PSI Are Partially Resistant to Photoinactivation of PSII-Variable fluorescence can be used as an indicator of RCII activity and its loss following photoinhibition (3, 43, 44). The variable fluorescence on a chlorophyll basis of the D6, AC208, and B4 mutants indicates that these cells possess active reaction center I1 (Table I). The amount of active RCII/chl in the D6 mutant was similar to that of the Y-1 cells, lower by about 35% in the AC208 and higher by about 50% in the B4 mutant. Reduction of the plastoquinone pool by electron donation from PSII was not impaired in these mutants as judged from comparing the kinetics of fluorescence rise in intact cells in the absence or presence of Diuron. Cells exposed to a PFD of 1500 pmol m-'s-l progressively lost the variable fluorescence activity ((F,,, - Fo)/Fo)) (Fig. 1). This was mostly due to a rise in the F, level and a decrease of the F,,, level as a function of exposure time as reported before (8, 17). However, the changes in these parameters were significantly slower and occurred to a lesser extent in the mutants as compared with the control y-1 cells. Among the mutants, D6 was slightly more sensitive to the light treatment and the B4 the least affected (Fig. 1). Thus while F, decreased by 85% after 30 min of light exposure in the control y-1 cells and to 70% in the D6 cells, the B4 and AC208 mutants retained 45-50% of their initial activity. Even after 4 h of light exposure, the F, of the mutants remained between 20- 45% of the initial value while that of the control y-1 cells was reduced to bellow 10% (Fig. 1).

The relative resistance of the mutants to photoinactivation could be partially due to an inefficient energy transfer from the antennae to the reaction centers of the mutants used in this work. However, Fig. 2 shows that this is not the case. Measurements of initial F,,, (in presence of Diuron) fluorescence rise versus actinic light intensity which reflects the amount of light energy absorbed by or transferred to RCII show that the AC208 mutant is comparable to y-1 control cells while the D6 and B4 mutants are more efficient in energization of PSII (Fig. 2). A better coupling of the LHCII to RCII could be expected in the cytochrome bs/f-leSS mutant (D6) since, in absence of this complex, the mobile component of LHCII is dephosphorylated and bound to RCII (45-47). The fluorescence emission spectra at 77K indicated that the chlorophyll complexes emitting at 686,695, and 715 nm were present in all mutants, however the 715-nm peak was broad and partially obstructed the 695-nm emission band in the D6 and AC208 mutants (data not shown).

TABLE I Relative content of active RC ZZ in the C. reinhardtii

nonphotosynthetic mutants The fluorescence parameters were measured in the absence ( F J or

presence (FmJ of 10 PM Diuron. The chlorophyll concentration was 10 pg/ml. The variable fluorescence, F,, was not normalized to F, since all measurements were carried out at the same chlorophyll concentration and data are given in this table as Fmax - Fo.

Cells Parameters Y-1 D6 Ac208 B4

(control) (-cytochrome bJj) (-plastocyanin) (-PSI) mV

FmaX 1622 2266 1634 4426 F" 1319 1289 839 2090

PQ/PQH2 in Photoinhibiton and Dl Protein Degradation 21295

'0 €0 120 180 240 Time ( m i n )

FIG. 1. Kinetics of photoinactivation of C. reinhardtii y-1 (control) and mutant cells. Cell suspension was exposed to high light and at times as indicated samples of equal chlorophyll concentration were taken and fluorescence induction-measured. Fmax was measured in the presence of 5 WM Diuron. The 100% values for F, were 303, 977, 795, and 2336 mv for the y-1, D6, Ac208, and B4 mutants, respectively; the Fmax values are given in Table 1. Variable fluorescence data are normalized to F,.

Since these mutants cannot transfer electrons to NADP and reduce inorganic carbon, the rate of oxidation of the plastoquinol formed by electron donation from PSI1 could be expected to be the same both in the light and dark. Measure- ments of the plastoquinol reoxidation in the dark indicate an initial fast rate with a tlIz of about 5 s followed by a slow rate with a tlIz of about 15-20 s (Fig. 3). In cells exposed to photoinhibitory light for 30 min the initial fast reoxidation phase is apparently lost and only the phase with a t112 of about 20 s can be observed (Fig. 3). These rates are slower by two orders of magnitude as compared to those occurring in cells which oxidize plastoquinol by the light-driven linear electron flow via PSI.

Light-dependent Degradation of Dl Protein Is Drastically Decreased in C. reinhurdtii Nonphotosynthetic Mutants-It has been demonstrated before that the degradation of D l is related to the process of RCII photoinactivation (8, 17, 31). Since the rate and extent of photoinactivation of C. reinhardtii nonphotosynthetic mutants were lower than that of the control cells (Fig. l ) , we have measured the rate of D l degradation in these mutants. The results show that D l degradation is extremely low in the mutant cells exposed to a PFD of 1500 pmol m-'s" for up to 8 h as compared to the control y-1 cells (Fig. 4, A and B) . Thus the tlIP of D l protein in the AC208 mutant is slightly longer than 4 h, over 8 h in the D6 mutant (Fig. a), and practically not measurable in the B4 mutant (Fig. 4B) as compared with a tlI2 of about 2 h in the control y-1 cells.

The degradation of the CP47 polypeptide was measured as a control for thylakoid proteins which are not affected by light (39, 48). The results show that the CP47 polypeptide is significantly more stable as compared with D l in the control y-1 cells and practically not affected by the high light treat-

310

275 24 I

190

142

- D6 PFD

( pno l m 190 - 7.3rnS

160

142

105

74

-2 ,

60

50

-

-

40 - 30

20

10-

- -

A

I;O 2dO 2AO 3AO 3?, PFD ( p m o l m-2 s" 1

FIG. 2. Rise of F,,, fluorescence as a function of actinic light intensity in C. reinhardtii control and mutant cells. Fluorescence rise was measured in presence of 5 pM Diuron during the initial linear 4-13-ms period after the complete opening of the exciting light shutter (Uniblitz, 2 ms). Actinic light was filtered through a Corning 4-96 blue filter and measured with a LI-COR Inc. radiometer. A, computer records of the fluorescence rise for the control y-1 and D6 mutant; B, rate of fluorescence rise calculated from records similar to those shown in A.

ment in the mutants (Fig. 4, A and B) . The results presented so far indicate that the resistance to

photoinactivation and high light-induced D l protein degradation in the mutants is related to the maintenance of a reduced plastoquinol pool in light. To further substantiate this interpretation, the oxygen tension during light exposure was increased by oxygen bubbling. The results of such an experiment are shown in Fig. 5. Indeed, bubbling of oxygen through the cell suspension during the light exposure accelerates somewhat D l protein degradation in the AC208 cells.

As an additional indication for the role of plastoquinone reduction in protecting the D l protein from the light induced degradation, cells of Oscillatoria limnetica, a cyanophyte which can perform anoxygenic photosynthesis when grown in the presence of Na2S and anaerobic conditions (49,50), were also assayed. The results show that the D l turnover in these cells is significantly lower under anoxygenic condition (Fig. 5) when the plastoquinone pool is mostly reduced by electron donation from sulfide (50). About 70% of the Dl content was degraded in the oxygenic cells after 3 h of light exposure while practically no change occurred in the anoxygenic cells as estimated from scanning radiograms exposed so as to permit quantitative measurements (data not shown). The effect of low oxygen tension and reducing conditions on the photoinactivation and D l protein degradation was tested on the control y-1 cells as well. The results (Fig. 6), show that addition of ascorbate and lowering the oxygen tension by

2 1296 PQ/PQH2 in Photoinhibiton and D l Protein Degradation

Ac. 208

= 0- 0 IO 20 30 40 50 60

Time (sec) FIG. 3. Rate of PQH2 reoxidation in dark incubated C. rein-

hardtii Ac208 mutant cells before and after exposure to high light treatment. Cells were either kept a t growth light intensity (30 pmol m-'s"), (control), or exposed to high light for 30 min (HL). Samples of cell suspension at equal chlorophyll concentration were then placed in the fluorimeter, dark adapted for 45 s, exposed to 10 s of continuous actinic light, and fluorescence induction was measured immediately (<0.5 s) or after further periods of dark readaptation as shown. The relative residual plastoquinol pool a t each time point was estimated from measurements of the area above the induction curve. The area at the end of the dark adaptation period was considered as representing the 100% reduced PQ pool and was 27.4 and 7.2 mv/s in control and HL cells, respectively. The reduction in the plastoquinone pool in the HL sample is only apparent since the FmaX is slightly lowered and F, increased (compare with Fig. 1) as a result of the changes in RCII activity and thus the area above the induction curve is reduced as well. The PFD of the actinic light intensity during the measurements was 300 pmol m-*s-'.

Time 0 4 8 0 4 8 0 4 0 0 4 8 (hr)

FIG. 4. Light-dependent degradation of D l protein is drastically reduced in C. reinhardtii B4 , D6, and Ac208 as compared to control y-1 cells. Cell suspensions were exposed to high light for 8 h, and samples were taken at times as indicated. Thylakoid membranes were prepared and their electrophoretic pattern was resolved as described. The Dl and CP47 content of the membranes was detected by Western blotting; A, two different experiments, including the control y-1 and the D6 or AC208 mutant cells, respectively; B, an experiment including the control y-1 and the B4 mutant cells; CBB, Coomassie Brilliant Blue-stained gels; WB, Western blots; MW, molecular weight markers. Note that the polypeptide pattern is not changed significantly during the light treatment.

bubbling N2 diminished the photoinactivation as assayed by fluorescence kinetics measurements. Under these conditions the t 1 /2 of the D l protein was increased from about 0.5 h to more than 1.5 h in high light-exposed cells (Fig. 6). The CP47 polypeptide was not degraded significantly either in the air- exposed or in the ascorbate-treated cells (data not shown).

PSII Photoinactivation and D l Degradation Are Enhanced

"""

- A i r l L + 0 2 -

0. Limnetic0

"" "lLAnox.J

Time 0 1.5 3 0 1.5 3 (hr )

FIG. 5. Effect of oxygen tension on the light-dependent degradation of D l protein in C. reinhardtii Ac208 and 0. limnetica cells. Cell suspensions were exposed to high light as described. For adaptation to anoxygenic conditions (Anox), the Oscillutoria cells were preincubated in growth medium for 2 h with addition of 3.5 mM Na2S and the suspension was bubbled with NP in presence of 5 mM NaHC03 (50). For oxygenic photosynthesis (Ox) , the cells were preincubated for the same time in a similar medium in air without addition of Na2S before exposure to high light. Thylakoids were prepared from the 0.limnetica cells as described before (50). The AC208 mutant cells suspension was bubbled with 0 2 or incubated in air during exposure to high light intensity. D l protein content was estimated by Western blotting. No significant changes were observed in the protein pattern as examined by protein staining (data not shown).

- >. I l:pT I20

0 30 60 90 Time (min)

I ;;i~; 0 0.5 1.5 0 0.5 1.5 I 0 +Ascorbate 0 Control

40

20

'0 30 60 90 Time (min)

FIG. 6. Effect of ascorbate on photoinactivation (A , C, and D ) and degradation of D l protein ( B ) . C. reinhardtii y-1 cells were photoinhibited by incubation at a PFD of 5,000 pmol rn-'s-l for 90 min. A , C, and D, the cell suspension was either kept in air throughout the experiment (control) or bubbled with Nz for 30 min in low light, and then 10 mM sodium ascorbate was added and the suspension was sealed and exposed to the high light treatment (+ ascorbate); B, the cell suspension was either incubated in air without or with addition of sodium ascorbate or bubbled with N P , supplemented with 10 mM sodium ascorbate, and sealed before exposure to the light treatment; detection of D l polypeptide was carried out by Western blotting as described under "Materials and Methods."

in the Scenedesmus LF-1 Mutant Unable to Evolve Oxygen and Reduce PQ-The results presented so far indicate that maintenance of a high PQH2/PQ ratio and slow turnover of PQ/PQH2 at the QB site partially protects PSII from photoinactivation and prevents the light-induced degradation of the D l protein. If this interpretation of the data is correct one would expect that the degradation of D l protein will be enhanced if the PQ pool could be maintained in its oxidized form in vivo. Such a situation occurs in the Scenedesmus LF- 1 mutant lacking water-splitting activity (14, 38) and thus unable to reduce plastoquinone. However, all other compo- nents of the photosynthetic electron transport chain are func- tional in this mutant, and thus in light-exposed cells the PQ pool will be oxidized and the PQ/PQH, turnover very limited.

The results of an experiment in which the light-dependent

PQlPQH, in Photoinhibiton and Dl Protein Degradation 2 1297

photoinactivation and degradation of the Dl protein were assayed in the S. obliguus LF-1 mutant cells as compared to the wt cells are shown in Fig. 7. Wild type Scenedesmus cells exposed to a PFD of 1500 pmol rn-'s-' show an initial fast rate of variable fluorescence loss decreasing to 50% of its initial value within 15 min, followed by a stabilization and even partial recovery during the next 4 h of light exposure (Fig. 7A). The tlP2 of the Dl protein degradation in the wt Scenedesmus cells is about 3 h (Fig. 7R). Under similar experimental conditions the photoinactivation of the LF-1 mutant is slower and does not show the initial fast phase. However the variable fluorescence is completely lost in these cells after 2 h of light exposure. In accordance, the degradation of the Dl protein in the LF-1 mutant cells is significantly accelerated, and its t lIZ shorter than about 1.5 h (Fig. 7). The degradation of the Dl protein is completely protected by addition of Diuron in both the wild type and LF-1 mutant of Scenedesmus cells (Fig. 7R).

DISCUSSION

Based on the results obtained in this work we propose that in photosynthetic cells the ratio of PQ/PQH, in the PQ pool and the occupancy of the QB site control the light-dependent degradation of D l protein. This conclusion is supported by the drastically decreased degradation of the Dl protein in high light-exposed C. reinhardtii mutants which do not oxidize PQH, as well as by the enhanced degradation of the Dl protein in the S. obliqu~ls LF-1 mutant in which the PQ pool reduction is inhibited.

The lower degradation of D l in 0. limnetica under anoxygenic conditions in presence of Na,S in which the plastoquinone pool is reduced via the SQR protein by exogenous sulfide (50) is in agreement with this hypothesis. The protective effect of ascorbate against D l degradation in the control C. reinhardtii y-1 cells and the increased degradation of D l in the AC208 mutant exposed to a high oxygen tension concur with the concept that reducing conditions favoring a high ratio PQH,/PQ protect D l from degradation during photoinhibition in vioo. Furthermore, the fact that under oxidizing condition D l is also degraded in the AC208 mutant indicates that the mutant does not lack the degrading protease.

' 0 60 I20 I80 240

" - 14.4 -.. - "-

. -mm.LIm =e= Tlrne 0 2 4 0 2 '1 0 1 4 0 2 4 ihr)

FIG. 7. Light-dependent degradation of the D l protein is accelerated in the Scenedesrnus LF-1 mutant defective in oxygen evolution. Cell suspensions of S. ohliquus wild type (wt) and LF-I mutant were exposed to high light for up to 4 h. Diuron (5 pM) was added to part of the cell suspension used for measuring degradation of the D l protein. Samples were taken at times as indicated for measurements of fluorescence induction ( A ) and Dl protein content detected I)y Western blotting ( H ) .

We have reported before that alteration of the QR hinding site occupied by PQ induced at high rates of energization of RCII (8) precedes and may he a prerequisite step for the photoinactivation of RCII, damage to its donor side, and irreversible modification of the Dl protein (8, 1'7). The data presented here support this concept as evidenced by the lower rate of photoinactivation of RCII in the C. reinhardtii mutants which do not oxidize plastoquinol. Furthermore, the results showing that Dl degradation is prevented when PQ is not available to hind to the QB site due to a low ratio PQ/PQH? may be one of the possible reasons why oxygen is required for the degradation of D l protein in isolated thylakoid during photoinhibition in vitro in absence of electron acceptors (22, 23). In this case the plastoquinone pool will be completely reduced unless some reoxidation may occur in presence of oxygen. The fast rate of D l degradation in this system a t air oxygen tension which can cause only a slow plastoquinol reoxidation could he ascribed to an accelerated damage to the donor side in the isolated thylakoids which may he less stable as compared with the in vioo system. In a similar way, the far-red light-induced degradation of D l in oivo when the rate of electron flow via RCII is significantly lowered (20) can he explained at least partially in the frame of this h-ypothesis since the far-red light effectively reoxidizes the plastoquinone pool and maintains a high PQ/PQH, ratio.

In the AC208 mutant lacking plastocyanin, the rate of reoxidation of PQH, is similar in both light and dark. In this as well as the thylakoids of the other mutants unable to reduce NADP, PQH, can he slowly oxidized by oxygen or eventually more rapidly by the process of chlororespiration (51, 52). The biphasic kinetics of PQH, oxidation in the AC208 cells (Fig. 3 ) is compatible with this explanation. The loss of the initial, more rapid reoxidation phase in the AC20R cells exposed to high light intensity could indicate that the chlororespiration activity may be lost during the photoinhibition process. Similar results were obtained in the other C. reinhardtii mutant R6 (31) and D6 (data not shown) both deficient in cytochrome b6/f. Both mutants exhihit significantly lower rates of D l protein degradation when exposed to high light intensity (31 and this work, Fig. 4). The loss of the initial rapid phase of PQH, oxidation during photoinhihition could further increase the ratio PQH2/PQ and account for both the slow-down of the photoinactivation process and Dl degradation observed in these cells. One should, however, mention that both the slower and faster rates of oxygen- dependent PQH, oxidation in the AC208 and the other C. reinhardtii mutants used in this work are at least two orders of magnitude slower than in cells able to oxidize PQH? hy light-driven linear electron flow.

The enhanced Dl degradation in S. ohliquus LF-1 mutant as compared to that of the wt cells is in agreement with the results of experiments carried out in vitro with donor side- inactivated thylakoid membranes (28). In this case following charge separation and reduction of the QR quinone, the electron donor Z and P680 remain oxidized (Z'and P680') and the chl,' radical can he formed as well (27,28, 29,54).

The formation of these radicals in the donor side-inactivated RCII has a high quantum yield (5.5). The irreversihle covalent modification of D l (8) targeting i t for degradation could he due to the activity of these cation radicals possihly mediated or enhanced via formation of singlet oxygen radicals. This explains the role of the QR site occupancy by PQ in the degradation of D l in donor side-inactivated RCII. In agreement with this view, occupancy of the site by Diuron which cannot accept electrons from P680 should prevent D l degradation in donor side-inactivated RCII exposed to the light.

21298 PQ/PQH2 in Photoinhibiton and Dl Protein Degradation

Indeed Diuron prevents the degradation of D l in the donor side-inactivated LF-1 mutant cells in vivo as shown in this work.

We have proposed before that acceptor side inactivation in membranes exposed to high light and high turnover of PQ/ PQH, at the QB site induce inactivation of the donor side of RCII (Ref. 8; see also Ref. 5). Thus QB occupancy by PQ is also essential for the process of donor side inactivation of RCII and the ensuing events as described above. The QB site can be occupied by PQ or PQH, (53). In the C. reinhardtii mutant cells deficient in the light-driven oxidation of PQH, the QB site will be occupied mostly by PQH, which cannot accept electrons and may prevent the sequence of events leading to donor side inactivation. This is considered to be the main reason for the stability of the D l protein in these light-exposed mutant cells.

The experimental results presented here demonstrate that occupancy of the QB site by PQ acting as a two-electron acceptor at the QB site is essential for the light-induced degradation of the D l protein. However the QB occupancy by PQ may be required for the actual D l protein degradation for additional reasons. The first cleavage site in the light-dependent process of D l degradation is located within the D l segment formed by the parallel helix connecting transmembrane helices IV and V of D l which also contain the QB and herbicide binding sites (16, 56). However certain phenol type herbicides binding at the QB site and blocking electron flow and thus permitting the formation of the harmful cation radicals do not protect D l from light-induced degradation (30). This strongly suggests that the exact positioning of different ligands within the QB niche may affect the interac- tion of this D l segment with its surroundings, particularly with the homologous segment of the D2 protein forming the QA binding site (16). The ligand may thus influence the availability of the cleavage site to the protease. It is possible that the presence of PQH, at the site may act in this mode as well, i.e. interfere with the proteolysis while occupancy by PQ may allow the degradation of the modified D l protein. This is indicated by the protective effect of Diuron on the light- dependent degradation of the D l protein in the Scenedesmus LF-1 mutant cells. In these cells the donor side of RCII is already inactivated, and thus, chances are increased for the formation of the cation radicals at high light intensity. Since in this mutant the light-dependent oxidation of the plastoquinol pool is considerably higher than its reduction by electron flow from PSII the QB site can be occupied by PQ. Displacement of PQ from the QB binding site by Diuron prevents the light-dependent D l degradation as shown in this work.

The equilibrium constant for the reaction QA-QB + QAQB- is estimated to be close to 20 (57) while that of the reaction QB'-+PQ + QB+PQ" was shown to be close to 1 (58). Based on measurements of the oxidation/reduction kinetics of the quinone acceptor complex of PSII and a calculated value of 5 mM and 0.7 nM for the concentration of PQ and PSII, respectively, the equilibrium constant for the reaction QA-+PQ $ QA-QB was estimated to be about 35 M" and only about 15% of PSII have the QB site occupied by PQ in dark adapted chloroplast (59). In chloroplasts in which the PQ pool is fully reduced, the QB site in practically all centers capable of evolving oxygen at the third flash is occupied by PQ2- (57). According to recent considerations, equilibration of the membrane PQ pool and accessibility of PSII to PQ are diffusion restricted within domains containing clusters of PSII estimated to four to eight centers per domain (60). While it is difficult to estimate the proportion of centers in which

the QB site is occupied by either PQ or PQH, in thylakoids exposed to continuous light, it is certain that a significant fraction of the centers is populated by PQHz when the pool is completely reduced as it is the case in the NADP nonreducing C. reinhardtii mutants.

Obviously at any given time the QB site in some of the centers could be vacated (57,59). Light-induced charge separation and reduction of QA will not result in PQ reduction but in back-electron flow as in centers occupied by a herbicide. Centers which do not reduce PQ are considered to be resistant to photoinhibition, however, there is no information on the D l degradation in such reaction centers. Depletion of PQ by hexane extraction of isolated thylakoids increases the D l sensitivity to trypsin digestion (30).

Occupancy of the QB site by PQ fulfills two roles; (i) electron acceptance, thus permitting transient persistence of the harmful RCII cation radicals; (ii) a steric role which presently can only be defined operationally by its effect on the light induced D l degradation. Evidence that in vitro proteolysis of the D l protein in isolated thylakoids is prevented by urea or triazine type PSII herbicides but not by certain phenolic herbicides (30) and the increase in the D l light induced degradation in mutants impaired in PQ reduction (25)' further supports this concept.

CONCLUSIONS

In this work we have addressed the question whether the QB site plays a regulatory role in the process of photoinactivation of RCII and the light-dependent degradation of its D l protein during photoinhibition in vivo. Presence of PQ at the QB binding site accelerates the photoinactivation of RCII and is required for the degradation of the D l protein. These phenomena are explained by: (i) the turnover of PQ/PQH, at the QB site which affects the properties of the reducing side of RCII and promotes the inactivation of the donor side (5, 8) and (ii) by accepting two electrons thus mediating formation of harmful cation radicals damaging the Dl protein when the activity of the donor side of RCII becomes gradually impaired. The accessibility of the D l cleavage site(s) to proteolytic activity is affected by the binding of various ligands at the QB site. Occupancy of the site by PQ does not hinder and may even promote the cleavage of the D l protein following its targeting for degradation. The mechanism whereby photoinactivation of the acceptor side affects the activity of the donor side of RCII remains to be further investigated.

Acknowkdgments-We thank Dr. B. Arieli, Institute of Life Sci- ence, the Hebrew University, for preparing the cultures of 0. limnetica and helping us to perform the respective experiment and Dr. L. Mets, University of Chicago, for his generous gift of the D6 and B4 Chlum- ydomonas mutant cells, Dr. F. A. Wollman, Institute of Biological Physical Chemistry, Paris, France, for his generous gift of Chlumydo- m o m AC208 mutant, and Dr. M. Seibert, Solar Energy Research Institute Golden Colorado, for his gift of the Scenedesmu wt and LF-1 mutant cells.

REFERENCES 1. Powles, S. B. (1984) Annu. Rev. Plant Physiol. 36, 15-44 2. Kyle, D. J., Ohad, I. & Arntzen, C. J. (1984) Proc. Natl. A d .

Sci. U. S. A. 81,4070-4074 3. Kyle, D. J. (1987) in Topics in Photosynthesis (Kyle, D. J.,

Osmond, C. B. & Arntzen, C. J., ed) Vol. 9, pp. 197-226, Elsevier Science Publishers, Amsterdam, The Netherlands

4. Andersson, B. & Styring, S. (1991) in Current Topics in Bioen- ergetics (Lee, C. P., ed) Vol. 16, pp.1-81, Academic Press, New York

5. Prasil, O., Adir, N. & Ohad, I. (1991) in Topics in Photosynthesis

N.Ohad and J. Hirschberg, submitted for publication.

PQ/PQH2 in Photoinhibiton (Barber, J., ed) Vol. 11, Elsevier Science Publishers, Amster- dam, The Netherlands

6. Theg, S. M., Filar, L. J. & Dilley, R. A. (1986) Biochim. Biophys.

7. Styring, S., Virgin, I., Ehrenberg, A. & Andersson, B. (1990)

8. Ohad, I., Adir, N., Koike, H., Kyle, D. J. & Inoue, Y. (1990) J.

9. Nanba, 0. & Satoh, K. (1987) Proc. Nutl. Acud. Sci. U. S. A. 84,

10. M&nne-Loccoz, P., Robert, B., & Lutz, M. (1989) Biochemistry

11. Mathis, P. & Rutherford, A. W. (1987) in Photosynthesis: New Comprehensive Biochemistry (Amesz, J., ed) Vol. 15, pp. 63-96, Elsevier Science Publishers, Amsterdam, The Netherlands

12. Debus, R. J., Barry, B. A., Sithole, I., Babcock, G. T. & McIntosh,

13. Metz, J. G., Nixon, P. J., Rogner, M., Brudvig, G. W. & Diner,

14. Metz, J. G., Bricker, T. M. & Seibert, M. (1985) FEBS Lett. 186,

15. Hirschberg, J., Bleecker, A., Kyle, D., McIntosh, L. & Arntzen,

16. Trebst, A. (1987) 2. Nuturforsch. 42c, 742-750 17. Ohad, I., Koike, H., Shochat, S. & Inoue, Y. (1988) Biochim.

Bwphys. Acta 933,288-298 18. Setlik, I., Allakhverdiev, S. I., Nedbal, L., Setlikova, E. & Klimov,

V. (1990) Photosynth. Res. 23, 39-48 19. Kirilovsky, D., Ducruet, J.-M. & Etienne, A.-L. (1990) Biochim.

Biophys. Acta 1020,87-93 20. Gaba, V., Marder, J. B., Greenberg, B. M., Mattoo, A. K. &

Edelman, M., 1987) Plant Physwl. 84,348-352 21. Greenberg, B. M., Gaba, V., Canaani, O., Malkin, S., Mattoo, A.

K. & Edelman, M. (1989) Proc. Nutl. Acud. Sci. U. S. A. 86,

Acta 849,104-111

Biochim. Biophys. Acta 1016,269-278

Bwl. Chem. 266,1972-1979

109-112

28,3641-3645

L. (1988) Biochemistry 27,9071-9074

B. A. (1989) Biochemistry 28,6960-6969

191-196

C. J. (1984) 2. Naturforsch. 39c, 412-420

6617-6620 22. Arntz, B. & Trebst, A. (1986) FEBS Lett. 194,43-49 23. Richter, M., Ruhle, W. & Wild, A. (1990) Photosynth. Res. 24,

24. Krause, G. H. & Comic, G. (1987) in Topics in Photosynthesis (Kyle, D. J., Osmond, C. B. & Arntzen, C. J., ed) Vol. 9, pp. 169-196, Elsevier Science Publishers, Amsterdam, The Neth- erlands

25. Ohad, N., Amir-Shapira, D., Inoue, Y., Koike, H., Ohad, I. & Hirschberg, Y. (1990) 2. Nuturforsch. 45c, 402-408

26. Demeter, S., Neale, P. J. & Melis, A. (1987) FEBS Lett. 214,

27. Callahan, F. E., Becker, D. W. & Cheniae, G. M. (1986) Plant

28. Jegerschold, C., Virgin, I. & Styring, S. (1990) Biochmistry 29,

29. Blubaugh, D. J. & Cheniae, G. M. (1990) Biochemistry 29,5109-

30. Trebst, A., Debka, B., Kraft, B. & Johanningmeier, U. (1988)

237-243

370-374

Physiol. 82,261-269

6179-6186

5118

and Dl Protein Degradation 21299

Photosynth. Res. 18,163-177

(1990) 2. Nuturforsch. 46c, 395-401 31. Shochat, S., Adir, N., Gal, A., Inoue, Y., Mets, L. & Ohad, I.

32. Ohad, I., Siekevitz, P. & Palade, G. E. (1967) J. Cell Biol. 36,

33. Schmidt, G. W., Matlin, K. S. & Chua, N.-H. (1977) Proc. Nutl. Acud. Sci. U. S. A. 74,610-614

34. Wurtz, E. A., Sears, B. B., Rabert, D. K., Shepherd, H. S., Gillham, N. W. & Boynton, J. E. (1979) Mol. Gen. Genet. 170,

521-562

235-242 35. Levine, R. P. (1969) Annu. Reu. Plant Physwl. 20, 523-540 36. Levine, R. P. & Goodenough, U. W. (1970) Ann. Rev. Gen. 4,

37. Bishop, N. I. & Senger, H. (1971) Methods Enzymol. 23,53-66 38. Taylor, M. A., Packer, J. C. L. & Bowyer, J. R. (1988) FEBS

39. Schuster, G., Timberg, R. & Ohad, I. (1988) Eur. J . Biochem.

40. Schuster, G., Even, D., Kloppstech, K. & Ohad, I. (1988) EMBO

41. Arnon, D. I. (1949) Plant Physwl. 24, 1-15 42. Schuster, G., Owens, G. C., Cohen, Y., & Ohad, I. (1984) Biochim.

43. Satoh, K. (1971) Plant Cell Physiol. 12, 13-27 44. Krause, G. H. & Weis, E. (1984) Photosynth. Res. 6, 139-157 45. Gal, A., Shahak, Y., Schuster, G. & Ohad, I. (1989) FEBS Lett.

46. Gal, A., Hauska, G., Herrmann, R. & Ohad, I. (1990) J. Biol.

47. Bennett, J., Shaw, E. K. & Michel, H. M. (1988) Eur. J . Biochem.

48. Adir, N., Shochat, S. & Ohad, I. (1990) J. Biol. Chem. 266,

49. Oren, A. & Padan, E. (1978) J. Bucterwl. 133,558-563 50. Arieli, B., Padan, A., & Shahak, Y. (1991) J. Biol. Chem. 266,

51. Bennoun, P. (1982) Proc. Nutl. Acud. Sci. U. S. A. 79,4352-4356 52. Maione, T. E. & Gibbs, M. (1986) Plant Physiol. 80, 364-368 53. Diner, B. A., Schenck, C. C. & De Vitry, C. (1984) Biochim.

54. Thompson, L. K. & Brudvig, G. W. (1988) Biochemistry 27,

55. Eckert, H. J., Geiken, B., Bernarding, J., Napiwotzki, A., Eichler, H. J. & Renger, G. (1991) Photosynth. Res. 27,97-108

56. Greenberg, B. M., Gaba, V., Mattoo, A. K. & Edelman, M. (1987)

57. Diner, B. A. (1977) Biochim. Bwphys. Acta 460,247-258 58. Diner, B. A. (1975) in Proceedings of the ZZZd International

Congress on Photosynthesis (Avron, M., ed) pp. 589-601, Elsev- ier, Amsterdam

59. Robinson, H. H. & Crofts, R. A. (1983) FEBS Lett. 163, 221- 226

60. Lavergne, J. & Joliot, P. (1991) Trends Biochem. Sci. 16, 129- 134

397-408

Lett. 237, 229-233

177,403-410

J. 7, 1-6

Biophys. Acto 767, 596-605

221,210-212

Chem. 266,9742-19749

171991-100

12563-12568

104-1 11

Biophys. Acta 766,9-20

6653-6658

EMBO J. 6,2865-2869

the pq/pqh, ratio and occupancy of photosystem 11-qb site by

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