the chromophores as endogenous sensitizers involved in the photogeneration of singlet oxygen in...

Photochemistry and Photobiology Vol. 52 , No. 5 , pp. 1003-1009, 1990 Printed in Great Britain. All rights reserved

0031 -8655190 $03 .oO+O.oO Copyright 0 1990 Pergamon Press plc

THE CHROMOPHORES AS ENDOGENOUS SENSITIZERS INVOLVED IN THE PHOTOGENERATION OF SINGLET

OXYGEN IN SPINACH THYLAKOIDS JIN JUNG* and HWAN-SU KIM

Department of Agricultural Chemistry, Seoul National University, Suwon 440-744, Korea

(Received 20 Februury 1990: accepted 30 April 1990)

Abstract-The photogeneration of singlet oxygen ( loz) from thylakoids and the chromophores involved as endogenous sensitizers were investigated using chloroplasts and thylakoids isolated from spinach. The blue light-induced inhibition kinetics of photosynthetic electron transport and that of CF,,-CF, ATPase were also studied. The spectral dependence of the generation of (0, from thylakoid membranes, measured by the imidazole plus RNO method, clearly demonstrated that the Fe-S centers play an important role in IO, generation, acting as sensitizers in thylakoids. The photoinhibition of the electron transport in isolated chloroplasts was strikingly depressed by a lipid-soluble '0, quencher and enhanced by deuterium oxide substitution, indicating that the inhibition processes are mainly mediated by '0, which is produced tiu photodynamic activation. The involvement of chloroplast cytochromes in the production of lo, was deduced from the action spectrum for the photodynamic inhibition of the electron carrier chain. The results obtained from the kinetic studies appear consistent with the involvement of some components such as the Fe-S centers and cytochrome chromophores of the carrier chain in the generation of '02.

INTRODUCTION

Working under severe conditions regarding the production of active oxygen species, chloroplasts are organelles especially exposed to oxygen toxicity. Various mechanisms for oxygen activation by the photosynthetic apparatus have been proposed, one of which is the production of singlet oxygen ('Ag, lo2), especially in situations where photosynthetic electron transport is impaired (Elstner, 1982, 1987; Asada and Takahashi, 1987).

The production of '02 in irradiated thylakoids has been deduced from the peroxidation of thylakoid membranes in the presence and absence of lo2 quenchers and deuterium oxide (Takahama and Nishimura, 1975,1976; Takahama, 1979). However, '02 has not been directly detected in illuminated chloroplasts. Chlorophyll in the free state is a well- known photodynamic sensitizer. In the photosystem of chloroplasts, however, the energy transfer and the primary photoprocess proceed so fast (in sub- nanoseconds and picoseconds) that the "chance" to

_ ~ ~ _ _ _ _ _ ~~~

*To whom correspondence should be addressed. fAbbret8iutions: CF,,-CF,, chloroplast ATPase: Chl, chloro-

phyll: DBMIB, 7,5-dibromo-3-methyl-6-isopropyl-p- benzoquinone; DCIP, 2,6-dichlorophenolindophenol; DCMU, 3-(3,4-dichlorophenyI)-l,l-dimethylurea: D20, deuterium oxide: DPBF, 2,5-diphenyl-3,4-isobenzo- furan; EDTA, ethylenediamine tetraacetic acid; HEPES, N-[2-hydroxyethyl]piperazine-N'-[2-ethanoI- sulfonic acid]; NHIP, nonheme iron protein: PSI, photosystem I; PSII, photosystem 11; RNO, N,N,-dimethyl-p-nitrosoaniline; SOD, superoxide dismutase: TRICINE, N-[2-hydroxy-l, I-bis( hydroxymethy1)- ethyllgl ycine.

populate which is produced ria the reaction, 3Chl + 302 4 Chl + lo2, is effectively eliminated. Meanwhile, even in a system which lacks these fast photoprocesses (i.e. the excited photosystem in a situation where electron transport is limited), the Chl photosensitization is immediately quenched by carotenoids (Koka and Song, 1978). Thus, although the possibility of thylakoid chlorophyll being a photosensitizing agent cannot be excluded unless evidence against it is found, the probability of chlorophyll in situ taking part in the photoproduc- tion of lo2 might be small. If such is the case, and if '02 is indeed produced via photodynamic activation, it seems reasonable to expect that there is at least a pigment, which is more efficient in lo2

generation than chlorophyll, in thylakoid membranes acting as an endogenous sensitizer.

Recently, this laboratory has reported that the mitochondria1 inner membrane generates lo2 on exposure to blue light. It also appears evident that the Fe-S centers of membrane-bound nonheme iron proteins (NH1P)t play a crucial role in the photogeneration of '02 (Jung et af., 1990). Because thylakoid membranes contain NHIP, it would not be unreasonable to assume that the Fe-S centers of chloroplast NHIP photosensitize to produce lo2, which may exert various deleterious effects on the photosynthetic apparatus. This paper presents experimental evidence supporting our assumption. In addition, some results obtained in the present study indicate that chloroplast cytochromes also play a role, acting as endogenous sensitizers, in the photodynamic inhibition of photosynthetic electron transport.

1003

1004 JIN JUNG and HWAN-SU KIM

MATERIALS AND METHODS

Preparation of chloroplasts and thylakoids. Intact chloroplasts and thylakoid membranes were prepared from spinach (Spinacia oleracea L.) leaves as described by Lee- good and Malkin (1986) with a slight modification. The leaves were ground briefly in a blender using an ice cold grinding medium [0.3 M sucrose, 50 mM HEPES (pH 7.6), 20 mM NaCl, and 5 mM MgClJ. The chloroplasts were pelleted by centrifugation at 3500 g for 1 min and washed twice at 3500 gfor 1 min after suspending carefully in a suspension medium [0.3 M sucrose, 10 mM mannitol, 5 mM MgCI,, 5 mM NaCl, 50 mM HEPES (pH 7.6), and 2 mM EDTA]. Thylakoid membranes were isolated from the osmotically ruptured chloroplasts; the chloroplasts were ruptured by the treatment of isotonic solution (10 mM NaCI) in the suspension medium, and centrifuged for 1 min at 3500 g to prepare thylakoids. Chlorophyll contained in chloroplasts was extracted into 80% acetone and determined by the method of Arnon (1949). All pro- cedures were carried out at 4°C under a safety light.

Measurement of photosynthetic electron transport. The rate of photosynthetic electron transport from PSI1 to PSI was measured by the DCIP photoreduction method described by Izawa (1980) and Hieke and Rodes (1986). The DCIP reduction in chloroplast suspension was measured spectrophotometrically with continuous illumination of red actinic light (wavelength > 600 nm) at 30 W/mz. The light source was a 500 W tungsten-halogen lamp (Seiko Special lamps, Tokyo) attached to a slide projector. The actinic light was introduced to the chloroplasts at a right angle to the measuring beam using a 5 mm (i.d.) flexible fiber-optic light guide.

Measurement of CF,-CF, A TPase activity. The activity of CF,,-CF, ATPase was assayed fluorometrically, as in Mills (1986). The thiol-modulation to activate thylakoid ATPase was achieved by the illumination of thylakoids with red actinic light at 30 W/m2 for 3 min in a lysis medium (10 mM dithiothreitol, 0.33 M sorbitol, 6 mM MgC12, 24 mM KCI, 0.12 mM methylviologen. 750 units/ me catalase, and 30 mM Tricine-KOH, adjusted to pH 8.0). In the case where thylakoid membranes were kept in the dark for more than 30 min, they were reactivated by illuminating with red actinic light at 150 Wlm' for 15 s prior to use. 9-Aminoacridine (9-AA) was used as a fluorescence probe to estimate the transmembrane pH gradient of thylakoids suspended in an assay medium (0.33 M sorbitol, 5 mM MgCL, 20 mM KCI, 20 pM 9- AA, 30 mM Tricine-KOH, adjusted to pH 8.0).

Photolysis. Chloroplasts and thylakoids in the suspension medium were irradiated at 15°C in a long necked quartz cuvette (10 mm lightpath) with a water jacket. Irradiation was performed with monochromatic and blue light. The monochromatic light was isolated from a 750 W Xe arc lamp (Shanghai bulb factoring No.3, China) using a f/3.4 grating monochromator (Applied Photophysics, London, England) with slits set at 10 mm. The blue light was also obtained from the Xe lamp using an absorption filter with a maximum transmittance at 425 nm and an effective bandwidth of 98 nm. During irradiation, samples were bubbled gently with air.

Detection of singlet oxygen. The imidazole plus N,N- dimethyl-4-nitrosoaniline (RNO) (imidazole-RNO) method developed by Kraljif and Mohsni (1978) was used to measure the photogeneration of '02 from thylakoid membranes. The 'O2-rnediated oxidation reaction was monitored spectrophotornetrically by measuring the decrease in absorbance at 440 nm of RNO. The reaction mixtures for this experiment contained thylakoids (7.5 pg Chl/mQ), imidazole (8 mM), and RNO (5 p M ) in the suspension medium. (Hereafter we will call this the thylakoid-imidazole-RNO system.)

Chemicals and instruments. Enzymes and biochemicals were purchased from Sigma Chemical Co. (St. Louis,

MO). All other chemicals were of reagent grade and obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan). These were used without further purification. Throughout the work, absorbance and fluorescence were measured using a Cary 118 C spectrophotometer (Varian Assoc. Pala Alto, CA) and a spectrofluoropho- tometer model RF 500 (Schimazu, Seisakusho, Japan) respectively. A Macam quantum/radiometer/photometer Q. 101 (Macam Photometrics, Livingstone, England) was used to measure the light fluence rates.

RESULTS AND DISCUSSION

Photogeneration of singlet oxygen f r o m thylakoid membranes

In the imidazole-RNO system, '02 first reacts with imidazole and produces an unstable intermedi- ate, trans-annular peroxide, which in turn reacts with RNO, bleaching it (Kraljit and Mohsni, 1978). It has been noted that R N O also reacts with the hydroxyl radical. However, imidazole acts as an effective hydroxyl scavenger even at a low concentration (Sharpatyi and Kraljit, 1978). Besides, mannitol contained in the reaction system is also known as a hydroxyl radical scavanger (Kralji? and Trum- bore, 1965). Therefore, the probability that the hydroxyl radical takes part in the R N O bleaching in this experiment would be extremely low. The concentration of imidazole in the thylakoids- imidazole-RNO system was maintained at 8 mM to attain the maximum efficiency of the RNO bleaching (Kralji? and Mohsni, 1978; Krishna et al . , 1987; Jung et al . , 1990).

If thylakoid membranes produce '02 upon exposure to light, the RNO bleaching will occur in the thylakoid-imidazole-RNO system. From the results shown in Fig. 1, it appears evident that '02 is photogenerated from thylakoids. The affirmative data were obtained from a study on the effects of azide and D 2 0 on RNO bleaching. When sodium azide was added to the thylakoid-imidazole-RNO system, the bleaching was substantially diminished. In contrast, DzO substitution resulted in a consider- able enhancement of the bleaching. Azide quenches lo2 and D 2 0 substitution increases the lifetime of '02 (Spikes, 1989), so these observations provide strong evidence that lo2 is photogenerated from thylakoid membranes.

'02 can be formed by dismutation of superoxide (03, although this has been questioned by Nilsson and Kearns (1974) and King el al. (1975). Thus, if the R N O bleaching is presumably mediated by the superoxide-originated lo2, it should be diminished in the presence of a superoxide scavenger, SOD. In fact, SOD protected R N O against the bleaching to a certain extent. However, its efficiency for protection was distinctly lower than that of azide (Fig. 1); the R N O bleaching was not depressed any more with further increase in the activity unit of SOD contained in the photolysis system (data not shown). From this observation, it seems apparent that the

Singlet oxygen in thylakoids 1005

350 400 450 500 Wavelength (nrn 1

Figure 1. Effects of azide, SOD and D,O on the bleaching of R N O in the thylakoid-imidazole-RNO system by blue light irradiation. The difference spectra of the thylakoid-imidazole-RNO systems containing 20 mM azide (a), 50 u n i t s h f SOD (b) and 20% D,O (d) are compared with the difference spectrum of the system without inclusion of these additives (c). AA represents the difference in absorption of irradiated and nonirradiated (control) samples. Samples were irradiated under aerobic conditions at 15°C with blue light (A,,, = 425 nm, effective bandwidth = 98 nm, and fluence rate = 750 Wim')

for 10 min.

bleaching of R N O is mainly caused by the '02 directly photogenerated from thylakoid membranes, but not by the '02 formed through the dismutation reaction of superoxide.

R N O bleaching at 440 nm in the thylakoids-imidazole-RNO system by monochromatic light was measured in the ranges from 360 to 700 nm, and its dependence on the irradiating wavelength was plotted as shown in Fig. 2(A). Because the bleaching of R N O is directly related to the generation of lo2 in the system, as discussed by Kraljii. and Mohsni (1978). this should represent the spectral dependence of lo2 generation from thylakoids. The resulting action spectrum appears to be very similar, if not identical, to that for '02 generation from mitochondria1 membranes (Jung et a l . , 1990), resembling the absorption spectra of the oxidized Fe-S centers of membrane-bound NHIP. This tempted us to speculate that the thylakoid Fe- S centers may be the chromophores involved in the production of lo2. In order to prove this, the effect of mersalyl acid (MA) treatment of thylakoids on the bleaching of R N O was studied. As expected, the treatment resulted in a substantial diminution of R N O bleaching in the thylakoid-imidazole-RNO system [Fig. 2(A)]. Because MA destructs the Fe- S centers in thylakoids (Kojima et al . , 1987), the remarkable reduction of the RNO bleaching is a good indication that the Fe-S centers are most likely involved in the generation of lo2 from thylakoids.

Subtracting the R N O bleaching in the MA treated thylakoid-imidazole-RNO system from that in the

PAP 52:s-F

"I

N 400 500 600 71

400 5 00 600 700 Wavelength (nrn 1

Figure 2. Spectral dependence of the photogeneration of '02 from thylakoid membranes. (A) '0, generation from intact thylakoids (0-0) compared with that from the MA treated-thylakoids (M); ( B ) '02 generation from the thylakoid Fe-S centers estimated from the data pre- sented in (A). Irradiation conditions are same as in Fig. 1 except that samples were irradiated with monochromatic

light for 20 min.

untreated thylakoid-imidazole-RNO system, we estimated the contribution of thylakoid Fe-S centers in the R N O bleaching at various wavelengths and constructed a spectrum which demonstrates the participation of the Fe-S center in lo2 photogeneration from thylakoids [Fig. 2(B)]. Although the spectro- scopic properties of thylakoid Fe-S centers have not been well characterized yet, except for ferredoxin, the photosynthetic Fe-S centers all seem to absorb at around 430 nm, as with other Fe-S centers (Andreassan and Vangird, 1988). Hence, the action spectrum for '02 generation was compared wih the absorption spectrum of spinach ferredoxin measured by Tagawa and Arnon (1963). The peaks at 420 nm coincide; another peak of a ferredoxin in the visible region at 463 nm seems to find the counterpart in this spectrum, a shoulder around 360 nm; a broad shoulder of the action spectrum in the U V region between 360 and 380 nm might correspond to a U V absorption peak of the ferredoxin at around 330 nm. In all respects, it seems reasonable t o suggest that the photosynthetic Fe-S centers, acting as endogenous photodynamic sensitizers, play an important role in the generation of

from thylakoid membranes.

1006 JIN JUNC and HWAN-SU KIM

As shown in Fig. 2(A), other chromophores besides Fe-S centers are probably involved in photosensitization, although they appear to contribute a minor share to '02 generation. The action spectrum for lo2 generation from the MA-treated thylakoids exhibits two bands, one with a peak at about 390 nm and the other in the region between 410 and 460 nm. Several chromophores, such as porphyrins (chlorophyll, pheophytin and cytochrome), carotenoids and flavins, present in thylakoids, absorb blue light. Nevertheless, none of the absorption spectra of these chromophores match the action spectrum. However, the shape of the action spectrum, in general, resembles the absorption spectra of flavins. In addition, the possibility of the involvement of chlorophyll and/or pheophytin in loz generation may not be excluded, as the action spectrum seemingly shows a small peak at around 660 nm; in this respect, the action band with a peak at 430 nm in the 410-460 nm region may be attributed, at least in part, to these phorphyrins. But any conclusion about the nature of other chromphores should be reserved until a further study is carried out.

'02-mediated inhibition of photosynthetic electron transport and chloroplast A TPase

The rate of photosynthetic electron transport in chloroplasts was measured using DCIP as a PSI acceptor. Although DCIP was often treated as a PSII electron acceptor in fragmented chloroplasts, it has been shown to behave more like a PSI electron acceptor when used with unfragmented chloroplasts (Izawa, 1980; Hieke and Rod&, 1986). The samples used in our experiments were unfragmented chloroplasts, as established using DBMIB, a PSI electron transport inhibitor (data not shown). Fig- ure 3 shows the effect of blue light irradiation of chloroplasts on the rate of photosynthetic electron transport and the effects of DPBF, SOD and D 2 0 substitution on the rate in blue light-irradiated chloroplasts. The chloroplasts on exposure to blue light at 750 W/m2 for 10 min suffered severe damage in their electron transport activity, losing as much as 80% of the activity. The photoinhibition of electron transport was enhanced by D 2 0 substitution and markedly suppressed by the lipid soluble lo2 quencher DPBF. These results clearly indicate that the photoinhibition processes are mainly mediated by lo2. Superoxide anion radical (03 also seems to be involved in the inhibition processes, as indi- cated by the protection effect of SOD against the photoinhibition. But the efficiency of SOD for the protection was much lower than that of DPBF. The low efficiency of SOD may be interpreted in terms of the limited accessibility of the enzyme to the generation sites of 0; in chloroplasts. Another possible interpretation for the low efficiency is that the chloroplasts are so well equipped with an efficient self-protection mechanism against 0; (Rabinowitch and Fridovich, 1983; Hayakawa et al.,

I -

O.*O t +-

0 20 40 60 T i m e ( s e c 1

Figure 3. Photosynthetic electron transport activities of chloroplasts irradiated in the presence of 20% D,O (a), 50 unitdmk' SOD (c). and 50 nmol/mZ DPBF (d), as compared with the activity of chloroplasts irradiated in the absence of these additives (b) and that of chloroplasts kept in the dark (e). The activity of photosynthetic electron transport (PSII 3 PSI) was measured by monitoring DCIP photo-reduction at 600 nm. The measurements were performed at 25°C. Electron transport was initiated by red actinic light at 30 W/m2. The reaction mixtures contained 45 p V f DCIP and 15 p M Chl/me chloroplasts in the suspension medium. Irradiation conditions were same as in

Fig. 1.

1984) that the extraneous SOD cannot contribute much to the protection. Nonetheless, the pro- nounced effect exerted by DPBF for the protection of photosynthetic electron transport implies that the photogeneration of '02 is a major cause of the blue light-induced inhibition of electron transport activity in chloroplasts. Note that chloroplasts even when exposed to blue light at 750 W/m2 for 10 min retained 77% of the electron transport activity in the presence of DPBF at 50 nmol/mf; this is a striking contrast to a situation where chloroplasts were subjected to the photoinhibitory treatment in the absence of the '02 quencher.

Spectral dependence of the photoinhibition of chloroplast electron transport is illustrated in Fig. 4, showing several peaks in the ranges from 360 to 700 nm. In this action spectrum compared with the spectra shown in Fig. 2, the action peaks at 420 nm and around 460 nm seem to arise from the Fe-S centers, as expected. A rather faint action peak around 670 nm may be due to porphyrin (probably chlorophyll)-photosensitization. The appearance of a peak at about 550 nm (or probably two peaks in the regions of 540-570 nm) may arise from chloroplast cytochromes, as these heme proteins have absorption peaks in the a-band region of 550-570 nm (Bendall and Rolfe, 1987). If such is the case, the contribution by the y-bands of the cytochromes, which have been found to arise in the 400-430 nm region (Molnar et al., 1987), to the action spectrum must be buried in the band with a 420 nm peak which was primarily assigned to the Fe-S centers. Actually, the ratio of the height of the 420 nm peak to that of the 460 nm peak in the action spectrum


a 0 40 T ime( m i n )

400 500 600 I00 Wavelength (nm)

Figure 4. Action spectrum for the photoinhibition of the photosynthetic electron transport in chloroplasts. The photoinhibition at each irradiating wavelength was measured from the rate of DCIP photoreduction in the reaction mixture irradiated compared to that in the nonirradiated. Inset: Typical traces of the DCIP photoreduction of the reaction mixtures irradiated by monochromatic light of 420 nm (a) and 500 nm (b). Photon hence rates of 420 and 500 nm were 0.316 and 0.548 mmol m-? s- ' respectively. Irradiation conditions were same as in Fig. 2.

for the photoinhibition (Fig. 4) is about twice as large as that in the spectrum for I 0 2 production by the Fe-S centers [Fig. 2(B)]; this may imply that the action band with a 420 nm peak represents the combined action of the Fe-S centers and the cytochromes for the photoinhibition. Based on this result, we tentatively suggest that not only the Fe-S centers but also the chromophores of chloroplast cytochromes, acting as endogenous sensitizers, take part in photosensitization. This suggestion may be partly supported by the report of Will 111 et al. (1985), in which the involvement of cytochrome c as a potential endogenous sensitizer in the photodynamic kill- ing of the smut fungus Ustilago violacea has been investigated. The greater the cytochrome c content relative to carotene, the more sensitive the cells are to high intensity incandescent radiation; the survival kinetics for a strain lacking carotene in response to radiation shows a typical exponential decay. This may be taken as an indication that a heme chromophore can be an endogenous photodynamic sensitizer in cells.

If the photoinhibition of electron transport is caused primarily by lo2, as discussed earlier, a ques- tion arises. Why was the involvement of cytochromes not observed in the action spectrum for '02 generation from thylakoids as shown in Fig. 2? Due to lack of useful information this may be answered only by inference. The physical and chemical environments of the cytochrome chromophores in thylakoid membranes are distinct from those of the Fe-S centers such that 'Or produced by the excited heme chromphores is effectively removed by some intrinsic scavengers before it diffuses to reach the

surfaces of thylakoid membranes. In such a situation, the generation of '02 from the membranes cannot be detected by the imidazole-RNO method. If, however, some components of the photosynthetic electron transport chain play a role in '02 scavenging, the chain can be damaged, as a non- enzymatic 'Or scavenging reaction is a chemical process by nature. Once '02 is photoproduced by a chromophore, the primary targets of '02 attack should be the substrates located at the very vicinity of the sites of I02 generation. In this respect, it may be assumed that certain components of the electron transport chain which are in close contact with the chromophore are rather vulnerable to the lo2- mediated photooxidation. As the case may be, the chromophore itself can be the target of '02 attack: it has been observed that the main cause of photoinhibition of PSI is the destruction of the Fe-S centers by some species of active oxygen produced by illuminated class I1 chloroplasts (Inoue et al., 1986). This is probably an implication that the chromophores participating in photosensitization are also involved in the lo2 scavenging processes leading to their own destruction, as the Fe-S centers were found to be involved in the generation of '02 (Fig. 2 ) . It seems worthwhile to mention here that the lipid soluble '02 quencher DPBF which was used in the experiment for the photoinhibition of electron transport activity (Fig. 3) should have easy access to the '02 production sites presumably located at the aprotic interior of membranes to quench immediately the active oxygen species.

Chloroplasts are organelles especially exposed to oxygen toxicity, so a variety of protection systems exist to protect them from the deleterious effects resulting from oxygen activation. These include antioxygenic enzymes such as SOD and peroxidase, and small organic substances such as glutathione, ascorbate, carotenoids and a-tocopherol. All of the a-tocopherol and all of the p-carotene of plant leaves are localized in chloroplast membranes (Bucke, 1968; Grumbach, 1983) where they scavenge reactive oxygen species. Under such circumstances '02 may hardly reach to the substrates located at a distance from the sites of production. However, the scavengers are consumed during the scavenging processes. Therefore, the rate of '02 production can exceed the rate of '02 scavenging at a certain point, especially under high irradiance conditions, if the scavengers are not regenerated fast enough to counter a continuous attack of lo2. In this situation, even a biological apparatus located at a distance could not be effectively protected any more. The kinetic study of the photoinhibition of CFo-CFI ATPase produced a result which seems consistent with this conjecture. As shown in Fig. 5, the H+-translocating enzyme remained almost intact through up to 30 min of the photoinhibitory treatment with blue light at 750 W/rn2. However, the prolonged exposure to light of thylakoids finally resulted in inactivation of the enzyme, producing a

1008 J IN JUNG and HWAN-SU KIM

0 50 100 150 200

Figure 5 . Photoinhibition of CF,,-CF, ATPase. (A) Fluo- rescence quenching by the ATPase activity of thylakoids irradiated (light) for 70 min, compared with that of nonirradiated (dark). (B) Time course of the inhibition of CF,- CF, ATPase in irradiated (Q-0) and nonirradiated (M) thylakoids. The measuring wavelengths of 9-AA fluorescence were 360 nm for excitation and 490 nm for emission. Ten pY of 1 mM DCMU was added to 2.9 mY of thylakoid suspension (6.9 pg Chllmt' in the assay medium containing 9-AA), and the suspension was kept in the dark for 1 min to attain the signal stabilization of fluorescence. Reaction was initiated by 100 pt? of 10 mM ATP and terminated by 30 pt? of 1 M NHCI. The measurements were carried out at 25°C. Irradiation conditions were same as in Fig. 1 . Other experimental details are described in

Materials and Methods.

Time(min)

sigmoid curve of the time sequential inactivation. The rather long resisting lag time of CFo-CFI may not only reflect a high efficiency of the intrinsic protection systems present in thylakoid membranes, but also imply that the active oxygen species involved in the inactivation of CFo-CFI was gener- ated by chromophore(s) located at a distance from the enzyme.

The photoinhibition kinetics of photosynthetic electron transport in chloroplasts (Fig. 6) is in contrast to that of CFo-CFI ATPase. The time course with no lag time and a much faster rate of inhibition appeared to fit first order kinetics. The electron transport system lost 50% of its activity in just 5 min of the photoinhibitory treatment with blue light at 750 W/m2. The lack of resisting lag time, the rela- tively fast rate of inhibition, and the typical exponential decay of activity clearly demonstrate that the electron transport chain is not protected at all from photosensitization, as well as that the chain is photo- dynamically sensitive to blue light. It is striking that the electron transport system is not in the least protected under the circumstances where the protec-

100

:: 20 W a

c .? 50 a u d

B 5 10 15

I I I I

5 10 15 20 25 30 T i m e (minl

Figure 6. Photoinhibition kinetics of photosynthetic electron transport activity in chloroplasts irradiated with blue light. The electron transport activity was measwed as in

Fig. 3. Irradiation conditions were same as in Fig. 1.

tion systems present in chloroplasts are apparently working to scavenge the reactive intermediates of photosensitized reaction, as seen in CF0-CFI. A plausible interpretation for this is that active oxygen species (mostly lo2) when produced by the excited chromophores of some components of photosynthetic electron carrier system immediately react with the components of the very system resulting in inactivation of the electron transport.

CONCLUSION

The present study provides direct evidence that lo2 is produced in thylakoids through photodynamic activation of molecular oxygen by endogenous sensitizers present in the membranes; it appears evident that the potential sensitizers involved are thylakoid Fe-S centers and also likely cytochrome chromophores.

The involvement of the Fe-S centers was proved by the measurement of the action spectra for '02 generation from intact thylakoids as well as from the Fe-S center-destroyed thylakoids. The production of '02 by the sensitization of cytochrome chromophores was deduced from the action spectrum for the photodynamic inhibition, which appeared to occur mainly via the type I1 processes, of photosynthetic electron transport.

The time course of the photoinactivation of CFo- CFI ATPase in thylakoids showed a rather long resisting lag time implying that the effective self- protection systems against the active oxygen- mediated inhibition of photosynthetic apparatus was apparently working in the membranes. Even under such circumstances, the photoinhibition kinetics of the electron transport exhibited a typical exponential decay curve without a lag time. This is taken as an indication that lo2 produced by some components such as the Fe-S centers and cytochrome chromophores of the photosynthetic electron carrier system attack immediately to inactivate the system itself before being scavenged.


Acknowledgements-This work was supported by Korea Science and Engineering Foundation Grant No. 870509. We thank Jin-Man Kim for his help in the preparation of the manuscript and also Yong-Uk Kim for his technical assistance.

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