Isolated Photosynthetic Reaction Center of Photosystem I1 as a Sensitizer for the Formation of Singlet Oxygen DETECTION AND QUANTUM YIELD DETERMINATION USING A CHEMICAL TRAPPING TECHNIQUE*

Singlet oxygen formation by photosystem I1 reaction centers isolated from Pieum sativum has been detected by two chemical trapping techniques: histidine-depend- ent oxygen uptake and bleaching of p-nitrosodimethyl-aniline by the intermediary endoperoxide of histidine. The quantum yield of singlet oxygen formation determined by these methods was estimated to be 0.16 by comparison with the known quantum yields of standard singlet oxygen sensitizers. Singlet oxygen was formed on illumination of reaction centers under conditions that lead to formation of the triplet state of the primary electron donor, P680. Experiments with deuterated buffer and active oxygen scavengers indicated that singlet oxygen was the only active oxygen species produced by this reaction. Neither azide nor histidine, which are scavengers of singlet oxygen, protected against photobleaching of the chlorophyll of reaction centers that oc- curs concomitantly with singlet oxygen formation, suggesting that bleaching involves singlet oxygen generated within the protein matrix of the complex. Singlet oxygen sensitized exogenously by rose bengal (when excited specifically at 650 nm) was also found to bleach reaction center chlorophyll in a manner similar to the intrinsic mechanism. We conclude that singlet oxygen formed within the hydrophobic interior of the reaction center attacks the chlorophylls of

the light-harvesting systems of photosynthetic organisms are normally protected from damage by '0, (and also other photooxidative effects) by carotenoids (3)(4)(5)(6)(7). This is demonstrated by the greater susceptibility to oxygen-dependent photodamage of carotenoid-less mutants of higher plants (81, algae (91, and bacteria (10). Despite the effectiveness of carotenoids in the protection of photosynthetic organisms, high light intensities do bring about loss of photosynthetic activity in oxygenic organisms as reflected by the physiological phenomenon of photoinhibition (see Ref 11). This phenomenon has been localized mainly to the photosynthetic reaction center of photosystem I1 (PSII). High light initially causes a decrease in the rate of electron transport through PSII and a preferential degradation of t h e D l protein. Restoration of activity requires protein synthesis. Oxygen has been implicated in photoinhibition (see Refs. 12 and 13), and the production of damaging oxygen species may possibly be a mechanism that activates D l protein degradation.
The structure of the purple bacterial reaction center and the close homology of the chromophore-binding sites in polypeptides L and M of this complex to regions of polypeptides D l and D2 of the PSII reaction center have led to an understanding of the electron transfer processes occurring in oxygenic organisms (14). The electron transfer reactions occurring within the PSII complex are as follows (Equation 1): hu H,O -j 'Qrz + P680 -j Pheo + QA --* Q, --* PQw, (Eq. 1) where Tyrz is tyrosine 161 of t h e D l protein, P680 is the chlorophyll a molecules of the primary electron donor, Pheo is the pheophytin molecule of the primary electron acceptor, QA and QB are the bound plastoquinone molecules of the secondary quinone electron acceptors, and PQ, , is the pool of plastoquinone molecules that freely diffuses in the lipid bilayer.
There has been much discussion of the processes involved in photoinhibition, which has focused on differentiating between whether the loss of activity occurs on the acceptor or donor side of the initial charge transfer reaction between P680 and pheophytin (which yields P680+Pheo-). It is possible that both of these mechanisms operate in vivo, with the relative balance being dependent on other factors in addition to light intensity (13,15).
One suggestion is that under high light conditions, the plastoquinone pool may become fully reduced if the light reactions tosystem 11; QA and Q,, first and second quinone electron acceptors in photosystem 11; RC, isolated reaction centeds) of PSI1 consisting of the D l and D2 proteins, a and p subunits of cytochrome b, , , and the I protein; RNO, p-nitrosodimethylaniline; AIPcS,, aluminum phthalocyanine disulfonate; TPPS,, mesotetra-(4-~ulfonatophenyl)porphine; pE, microeinsteins; BA, singlet oxygen quantum yield; RB, rose bengal; SiMo, silicomolybdate. of PSII exceed the rate of plastoquinol oxidation. As there will be no oxidized plastoquinone available, the Q,-binding site, on the D l protein, will be empty. This in turn will lead to the possibility of Q,, which is normally a single electron acceptor, becoming doubly reduced and protonated (16). In its fully protonated state, this quinone may vacate the Q,-binding site on the D2 protein. Under these conditions, primary charge separation can occur to give P680+Pheo-, but the radical pair will simply recombine again, with a high probability of the triplet state of P680 being formed. Indeed, this has been seen by Vass et al. (17) when isolated PSII-enriched preparations were subjected to very high light intensities under anaerobic conditions. Under aerobic conditions, however, these authors found that light treatment led to an irreversible loss of electron transport and that no P680 triplet was detected. They suggested that the triplet was quenched by molecular oxygen to form singlet oxygen, which in turn caused the irreversible damage.
Isolated PSII reaction centers (RC) have not only lost water splitting activity, but do not retain the quinone acceptors, QA and Q, (18,19). They are, however, capable of primary charge separation, which consequently results in recombination and formation of the triplet state of P680, with a quantum yield of 0.3 (20)(21)(22) Despite the fact that the isolated RC normally binds two p-carotene molecules (23,241 this preparation shows only a low quantum yield of carotenoid triplet, -0.03 (21,22). This inability of p-carotene to quench the 3P680 state has also been noted in more intact PSII complexes and seems to be an intrinsic feature of the PSII reaction center (25). The detection of oxygen-dependent irreversible bleaching of pigments when isolated PSII reaction centers are illuminated has been suggested to be due to '0, formation according to Equation 3 (26). Indeed, the dramatic shortening of the 3P680 lifetime under aerobic conditions, from 1 ms to 33 ps, supports this hypothesis (21,22).
Recently, we detected the photoinduced formation of '0, by isolated PSII reaction centers using both steady-state and time-resolved measurements of its emission at 1270 nm (27). This was probably the first direct observation of '0, luminescence sensitized by an intrinsically bound chromophore in a defined biological system as opposed to sensitizer-doped biological material (e.g. Ref. 28). We have now conducted further studies on PSII reaction center-sensitized '0, formation using chemical trapping techniques. We have employed an oxygen uptake method (29) in which histidine (or imidazole) reacts with '0, to form an intermediate product, a trans-annular peroxide, [Hiso,], which then rearranges or decomposes into a final oxygenation product, HisO, (see Equation 4). We have also used the technique of Kraljic and El Mohsni (30), which is based on the bleaching of p-nitrosodimethylaniline (RNO) to the nitro form caused by the trans-annular peroxide product of the '0, reaction with either histidine (His) or imidazole (see Equation 5). Chemical trapping of '0, is fraught with difficulties (see Refs. 31 and 32); therefore, in the experiments reported here, we have used a number of controls. These include the investigation of the effect of '0, quenchers and oxygen radical scav-engers. We have also compared our data obtained with PSII reaction centers with effects seen with well known '0,-sensitizing dyes. We conclude that '0, is the product formed when isolated PSII reaction centers are illuminated and is the cause of irreversible bleaching of the chlorophyll bound to this complex. We further conclude that this process can occur in vivo under conditions that favor the formation of 3P680 and that the detrimental reaction occurs because of the ineffectiveness of the carotenoid quenching mechanism.
MATERIALS AND METHODS PSII reaction centers were isolated from pea plants (Pisurn satiuurn var. Feltham First) as described previously (33) and stored at -80 "C. Samples were thawed at 4 "C, stored on ice, and protected from light before use. For measurements of oxygen uptake and RNO bleaching, they were diluted into 50 rn Tris-HC1, pH 7.2, and 2 m~ n-dodecyl P-D-maltoside to the chlorophyll concentrations given in the figure legends. The chlorophyll concentration was determined by the method described by De Las Rivas et al. (34).
Light-induced oxygen uptake was measured with a Hansatech oxygen electrode, and light-induced absorption difference spectra were measured with an SL"AMINC0 DW2000 dual beaddual wavelength spectrophotometer at 10 "C. In both cases, illumination was provided with a tungsten iodine light source. The intensity and wavelength were controlled with Schott neutral density glass cut-off or interference filters supplied by Precision Optical Instruments. When reversible lightinduced absorption changes at 460 nm were measured, the photomultiplier was protected by a 4-mm BG18 and a 2-mm BG38 Schott glass filter, and excitation was with an RG645 glass cut-off filter (1000 pE m-' s-1).
For singlet oxygen quantum yield (aA), calculations from oxygen uptake experiment data were obtained for samples that were selectively excited by light of a particular wavelength using either a 424or 666-nm interference filter. AlPcS, and TPPS, were used as @A standards at 666 and 424 nm, respectively. The from P680 (@yo) was calculated as follows (Equation 6): where is the singlet oxygen quantum yield of the standard, R , is the initial rate of oxygen uptake, and I is the integrated absorption spectrum over the transmission range of the filter that is used to normalize the number of photons absorbed by the sample and standard.

RESULTS
Oxygen Uptake-Initially, we tested whether, under conditions expected to allow the formation of '0, by isolated PSII reaction centers, i.e. illumination under aerobic conditions in the absence of added electron acceptors, the presence of imidazole derivatives catalyzed oxygen uptake. Fig. 1 shows that, indeed, light-dependent uptake of oxygen by RC is seen in the presence of histidine or imidazole, whereas in the absence of any additions, there is essentially none. The maximum rate of oxygen uptake was >4000 pmol of oxygedmg of chlorophyllh, which is in the same order of magnitude seen for photoinduced electron transport rates from Mn2+ to silicomolybdate measured with RC (36,371. These results are consistent with the view that illumination of RC causes '0, formation and that the uptake of oxygen is due to the interaction of this species with histidine or imidazole to form a dioxygen complex (see Equation 4).
We then compared the histidine dependence for RC-photoinduced oxygen uptake with that for known '0, generators, Al-PcS, and TPPS,. In Fig. 2a, it can be seen that AlPcS,, TPPS,, and RC have the same concentration requirement for histidinecatalyzed photoinduced oxygen uptake (and imidazole (data not shown)). This supports our conclusion that '0, production is catalyzed by RC. suggested that this indicates that only lo, participates in histidine photooxidation, azide is also known to quench hydroxyl radicals more efficiently than '0, (rate constants of 1.1 x 10'' and -10' M-' s-l, respectively) and also superoxide radicals, although less efficiently (rate constant of <lo6 M-' s-') (39). Therefore, we cannot, from our results with azide, rule out the participation of other radicals in the photooxidation of histidine (however, see "Discussion").
We found a considerable acceleration of histidine-dependent oxygen uptake by both RC and AlPcS, in the presence of D,O compared with H,O (-1. 6 times in both cases (data not shown)). This D,O effect is also usually thought to be indicative of '0, formation as the lifetime of '0, in D,O is -20 times that in H,O (40). Again, this is not definitive proof of '0, formation as superoxide has also been shown to have a longer lifetime in D,O than in H,O (41). However, because of the similarity between the RC and AlPcS, data, it seems likely that '0, is the major oxygen species involved in histidine photooxidation and hence the major product when PSII reaction centers are illuminated.
Quantum Yield of '0, Formation: Oxygen Uptake Method-If we assume that '0, is the only toxic oxygen species produced on illumination of RC under aerobic conditions, it should be possible to estimate the @A by comparing the relative rates of oxygen uptake seen in the presence of histidine with those seen with dyes for which the quantum yields are known. Fig. 2u shows the relative yield of '0, formed by AlPcS, and RC. In these experiments, samples were illuminated with 666-nm light, and the absorption of this excitation by the two sensitizers was matched as described under "Materials and Methods." Fig. 2a also shows similar data obtained by illumination of RC and another dye, TPPS,, with 424-nm light (see Fig. 3 for a comparison of the absorption spectra of RC and the '0,-sensitizing dyes used in these experiments). The data show oxygen uptake in arbitrary units that has been corrected for the different absorption at the two wavelengths employed. Table I shows the @A values for the different sensitizers calculated from the data of Fig peroxide of histidine, [Hiso,], or imidazole causes the bleaching of RNO, which is normally followed at its absorption maximum (440 nm). This method has been used for detection of '0, sensitized by a number of dyes, including eosin and rhodamine B (30) and porphyrins (29). It has also been used to monitor '0, formation by crude coal tar extracts (47), by the nitrophenyl ether herbicide oxyfluorfen in conjunction with isolated chloroplast membranes (481, and more recently, by thylakoid membranes alone (49). Fig. 3 compares the absorption spectrum of RC with that of RNO and of the various dyes that have been used in the experiments reported here. As can be seen in Fig. 3, AlPcS, absorbs in the red region, as does the Qy band of the chlorophylls and pheophytin of the RC. TPPS, absorbs mainly in the blue region, as does the Soret band of the RC chlorophyll and pheophytin. Rose bengal (RB) dissolved in buffer has its major absorbance at 550 nm; however, when measured in the presence of RC, we found that there was a 14-nm red shift of this maximum (data not shown), which indicates binding of RB to the RC. Both these maxima are in the region of the lowest absorbance by the RC. Absorbance by RNO peaks at 440 nm,   HCl, pH 7.2, and 2 m~ n-dodecyl P-D-mdtoside at room temperature. Spectra were normalized at their maximum absorbance to the maximum absorbance by RC in either the blue or red region of the spectmm. which overlaps with a major absorbance band of the RC. It should be noted that RNO still absorbs strongly at 460 nm, where the main absorbance by the RC is by p-carotene (18).
In our initial experiments, we compared RNO bleaching sensitized by RB, AIPcS,, and RC. The three sensitizers were illuminated with white light in the presence of histidine and RNO. The light minus dark absorption difference spectra of the irreversible bleaching brought about by 0.5, 1, and 2 min of illumination are shown in Fig. 4. As seen by others, both RB (30) and AlPcS, (51) sensitized a rapid, continuous bleaching centered at 440 nm, with only minor changes at their own absorbance maxima (550 and 674 nm, respectively) (compare Figs. 3 and 4). RC were also bleached, but the difference spectra were rather more complex than those seen with RB and AlPcS,. The RC showed bleaching at 440 nm, which could be due to loss of RNO. However, there was also bleaching of the chlorophyll absorption (at 417 and 680 nm), which is normally seen in the absence of RNO and histidine (50) and is thought to be caused by degradation of chlorophyll by '0, (52).
To determine whether some of the bleaching at 440 nm seen with RC is due to loss of RNO, we carried out a number of controls. Proof that the histidine-dependent and azidesensitive bleaching at 460 nm is due to loss of RNO is seen in traces e and f, which are traces d minus b and d minus c, respectively. These difference spectra ( A A , , = 440 nm) are essentially the same as the inverted spectrum of RNO alone (trace g ) . This shows that on illumination, isolated PSII reaction centers sensitize histidine-dependent RNO bleaching. We conclude that this is most likely due to '0, formation as OH' (531, other reactive species (30), or highly oxidizing conditions (32) would be expected to bleach RNO in the absence of histidine. Fig. 5 also shows that the chlorophyll photobleaching of RC seen in the Qy region (centered at 680 nm) is to the same extent whether or not '0, scavengers (histidine or azide) are present. This indicates, as noted previously for azide (271, that '0, scavengers are unable to protect against photodamage to the chlorophylls bound to the RC. To confirm that the RNO bleaching was due to IO2, we have compared the rate of irreversible bleaching in deuterated and water buffers (Fig. 6). D,O increases the rate three times, which is consistent with the primary product being '0,. The bleaching seen in H,O buffer was totally dependent on the presence of histidine, but some histidine-independent bleaching was observed with D,O buffer. Bleaching was inhibited by azide (data not shown); however, this effect was masked by an azide-induced bleaching of p-carotene, which was independent of the presence of RNO and histidine. Quantum Yield of '0, Formation: RNO Bleaching Method-We have also used the RNO bleaching method to estimate the relative '0, quantum yield of illuminated RC. Fig.  7a compares the rate of irreversible bleaching at 460 nm seen with RC and AlPcS,. Bleaching was induced by 666-nm light, and samples were matched for equal absorption of this excitation. Initially, the rate of bleaching of RNO in the presence of AlPcS, was slightly faster than that seen with RC. However, with increased time of illumination, there was a slight decline in the rate with AlPcS,, whereas the RC-induced rate increased. This is emphasized in Fig. 7b, which shows the ratio of the rates of RNO bleaching induced by RC and AlPcS, as a function of illumination time. During the first few minutes of illumination, the ratio, which represents the relative a&, was -0 . W . 7 . This initial ratio is similar to that determined by the oxygen uptake method (Table I), for which measurements were made over the first few minutes of illumination. As the values of @A obtained for RC by the oxygen uptake and RNO bleaching methods during the initial illumination period are similar, this reinforces the validity of the '0, quantum yields determined by these methods. The gradual increase in the relative yield of '0, with illumination time seen using the RNO bleaching method correlates with the loss of absorbance at 680 nm, which indicates photoinactivation of charge separation (Fig. 7c). Note that the data in Fig. 7 ( a and b ) were not corrected for the loss of absorbance of the 666-nm excitation light due to photodamage, and therefore, the later measurements with time are an underestimation of the relative quantum yield of '0, formation by RC as compared with AlPcS,. P680' Does Not Cause Bleaching of RNO-It has been suggested (32) that the RNO bleaching method for '0, detection is unsuitable under highly oxidizing conditions. As the redox potential of P680' at 1.17 V (54) is probably the most highly oxidizing potential occurring in biological systems, it is possible that it is this species (P680') rather than '0, that bleaches RNO. To test this, we illuminated samples in the presence of a n electron acceptor, silicomolybdate (SiMo), which accepts electrons from reduced pheophytin and allows the accumulation of P680' (19). SiMo prevents the back-reaction between P680'Pheo-, hence stopping the formation of 3P680 and the potential for '0, production (see Equations 2 and 3 and Refs. 55 and 56). Fig. 8 shows that if SiMo is present during illumination of RC together with RNO and histidine, there is substantial bleaching in the region where RNO absorbs, which is mainly due to irreversible bleaching of P-carotene (maximum bleaching seen at 486 nm; see ref. 50). Detailed examination of the difference spectra shows that there is also some RNO bleaching. However, as the bleaching attributed to RNO is completely independent of histidine (traces b and c), it must be due to an oxidation effect involving the long-lived P680' state and not to '0,. Indeed, addition of Mn" as an electron donor, which is known to prevent P680' from accumulating (501, inhibits the SiMo-dependent bleaching of RNO (trace a ) , although there is still some loss of absorption by carotenoids. Thus, we can conclude that unless the P680+ state is stabilized by the presence of an electron acceptor, there is no bleaching of RNO due to high oxidizing potential.
Are Other Active Oxygen Species Causing Photodamage to PSII Reaction Center?-Although the above experiments show that '0, is formed via the triplet state of P680, it is possible that other toxic species such as OH', H,O,, or 0, are also produced by the reaction center when it is illuminated in the presence of oxygen. However, it is clear that OH' is not produced, otherwise RNO would have been bleached in the absence of histidine (Fig.   5). In the case of H,O, and O,, we carried out experiments to test if these species were formed during histidine-induced oxygen uptake by RC. Table I1 shows that neither catalase nor superoxide dismutase had an effect on the rate of histidinedependent oxygen uptake, indicating no significant formation of H,O, or O,, respectively. However, we did detect light-induced oxygen uptake by RC in the presence of ascorbate (Table  11) at the same rate as that seen with histidine. Ascorbate is likely to form H,O, and O,, but it is also known to scavenge '0, (57). We found that the rate of ascorbate-dependent oxygen uptake was decreased by about one-third with catalase, but there was no significant effect with superoxide dismutase (Table 11). This indicates that H,O, is a major product of this reaction. The most likely explanation for the effect of ascorbate

Effect of superoxide disrnutase and catalase on histidineand ascorbate-catalyzed light-induced oxygen uptake by isolated PSII reaction centers
Methods." Additions were as follows: 600 unitdml superoxide dis-Oxygen uptake was measured as described under "Materials and mutase, 400 unitdml catalase, 10 m~ histidine, and 2 m~ ascorbate. is that it scavenges '0, in a process that results in the formation of O,, possibly by a secondary reaction, which in turn dismutates to H,O,. If this dismutation is sufficiently rapid, it would explain the lack of inhibition of oxygen uptake by superoxide dismutase. Experiments with superoxide dismutase and catalase did not show any protection against light-induced irreversible bleaching of RC chlorophyll, nor was there any significant effect on the rate of pigment bleaching when ascorbate was present during illumination of RC (data not shown). It therefore seems unlikely that any active oxygen species other than '0, is responsible for the photoinduced bleaching of chlorophyll when RC are illuminated in the absence of an electron acceptor.

'0, Generated by Rose Bengal Bleaches PSII Reaction
Centers-The evidence presented here and by Macpherson et al. (27) shows that illuminated RC can generate '0,; however, there is only indirect evidence that it causes the irreversible bleaching of the RC chlorophyll. Thus, to investigate the effect of '0, on the pigments of RC, a known '0, generator, RB, was used. '0, specifically sensitized by RB was generated by excitation at 550 nm, where absorption by RB is considerable, whereas that by RC is very low (see Fig. 3). Fig. 9A shows the absorption difference spectra in the Qy band of RC after excitation with 550-nm light for 2, 4, 6, and 8 min. A marked increase in the bleaching of RC was observed when RB was present (Fig. 9A, traces a and b). However, if the chlorophylls of RC were excited directly with 666-nm light, the presence of RB had essentially no effect on the rate of bleaching (Fig. 9A, traces  c and d ) . We also found that the RB-sensitized bleaching of RC (using 550-nm light) was markedly enhanced in deuterated buffer (Fig. 9B). Thus, we conclude that '0, sensitized by RB can diffuse into the protein core of the reaction center and induce pigment bleaching.
It is significant that the pigments bleached by RB-sensitized '0, appear to be the same as those bleached by '0, produced by the RC itself. However, we did find that there was a broadening of the irreversible bleaching in deuterated buffer, i.e. monomeric chlorophylls were also bleached. Therefore, we suggest that the chlorophylls of P680 are the pigments that are most sensitive to '0, damage and that a greater proportion of the accessory chlorophylls can be bleached only when its lifetime is extended by the presence of D,O. The fact that exogenously formed '0, brought about the same type of irreversible bleaching of RC as is seen under conditions that favor formation of 3P680 is strong evidence that '0, is the species responsible for oxygen-dependent bleaching of this complex. Despite finding that RB-sensitized bleaching of the chlorophyll of reaction centers was enhanced by D,O, we did not find any inhibition of this RB-independent bleaching by azide (data not shown). However, we noted that RB underwent a red shift in its absorption maximum in the presence of RC, suggesting binding of this dye to the protein complex. Such a binding has also been shown by others for isolated thylakoids (58). In our preparations, this '0, generator may be closely associated with the protein of the RC or may be located in the detergent associated with the complex. Therefore, we suggest that lo, is both being generated and causing damage in a hydrophobic environment inaccessible to the negatively charged azide anion. We also presume that binding of RB to the RC means that this sensitizer is more effective at bringing about photodamage to the complex. Indeed, Kim et al. (58) saw an effect like this when they found that photoinhibition of thylakoids induced by white light treatment was enhanced more by the presence of RB than by less lipophilic '0, sensitizers. DISCUSSION We have shown here that '0, produced by illumination of RC can be detected either by chemical trapping with histidine, to form [HisO,], which results in consumption of oxygen, or in a secondary reaction in which [HisO,] bleaches RNO. '0, formation by RC was also recently demonstrated directly by the detection of its luminescence at 1270 nm (27). The initial route for the generation of this '0, is via the P680 triplet state formed by the recombination of the radical pair P680+Pheo-. Although two p-carotene molecules are bound to the reaction center complex, there is no evidence for significant quenching of 3P680 by the carotene. The obvious consequence to the RC of the formation of '0, is therefore its vulnerability to photochemical damage. Indeed, under aerobic conditions, chlorophyll is irreversibly bleached (261, and there is loss of the ability to form 3P680 (22), followed by the breakdown of the two major proteins of the complex, polypeptides D l and D2 (59). In these studies, this photodamage has been attributed to the action of lo,.
It is well known that photodynamic action can proceed via two basic reactions (see Ref. 32). The excited sensitizer may react directly with a substrate (Type I) or with oxygen (Type 11). Type I reactions lead to the formation of radicals or their ions, which then react with ground state oxygen, whereas Type I1 reactions lead to '0, formation by energy transfer, which may subsequently react with the substrate to yield oxygenated products. Although homogeneous solution environments may favor Type I1 reactions, it has been suggested that in biological systems, where the sensitizer may be bound to the substrate, Type I reactions would be more likely to occur (see Ref. 32). However, there are reports of '0, formation by biological material that has been doped with sensitizer (28,60) and also of '0, being formed on excitation of chloroplast lamellae in the presence of the nitrodiphenyl ether herbicide oxyfluorfen (48). As discussed above, it seems highly likely that '0, causes the photoinactivation of the isolated PSII reaction center, i.e. a Type I1 reaction is occurring. It has also been suggested that '0, is formed in vivo during "acceptor side" photoinhibition and that '0, may trigger the degradationhepair cycle of PSII, which underlies the rapid turnover of t h e D l protein (15,17). However, as Foote (32) has warned, "detection of a species does not demonstrate its intermediacy in a process." In the field of photodynamic therapy, it has been very difficult to prove definitively that '0, is the major reactive species that causes damage to biological material and brings about cell death. Although we have been able to demonstrate '0, formation by the RC, it is possible that other active oxygen species may be formed as well. Thus, it may be these species that cause the bleaching of chlorophyll and initiate protein breakdown. However, we have found no evidence for this alternative explanation in the case of pigment bleaching and attribute the damage directly to '0,.
RC-sensitized oxygen uptake seen in the presence of histidine (or imidazole) and also the inhibition of this reaction by azide ( Figs. 1 and 2) followed the same concentration dependences as found previously in experiments that were concluded to demonstrate '0, formation (29,301. The essentially complete inhibition by azide shows that hydroxyl radicals are not being formed because although azide can quench hydroxyl radicals (39), oxygen would still be consumed as the quenching products are OHand the azide radical (Equation 7). Quenching of '0, by azide, on the other hand, is probably a charge transfer process so that molecular oxygen would be released again. Our results are therefore consistent with '0, formation. This conclusion is supported by the RNO bleaching data, which showed a n absolute histidine dependence, indicating that only '0, is involved (see Ref. 29). It also seems unlikely that significant levels of 0, or H,O, are formed by illumination of RC because of the lack of effect of the presence of catalase or superoxide dismutase on histidinecatalyzed oxygen uptake (Table 11). Oxygen uptake is also seen in the presence of ascorbate, which is apparently due to its ability to scavenge lo2. In support of this conclusion is the fact that at the limiting light intensity used, histidine and ascorbate catalyzed the same rate of oxygen uptake. An alternative explanation is that ascorbate is acting as an electron donor to P680+ (possibly through cytochrome b,,,, which ascorbate reduces in the dark (61)), allowing oxygen to act as an electron acceptor (50 The question arises as to whether the rate of oxygen uptake seen in the presence of histidine would account for the expected rate of '0, formation by the RC. At high excitation intensity, we saw a n oxygen consumption rate of >4000 pmoVmg of chlorophyllh, which is consistent with its formation via the P680 triplet. The quantum yield of '0, formation by RC measured by the chemical trapping techniques (as soon after commencement of excitation as possible) was found to be -0.16, which is of similar magnitude to the previous estimate of 0.1 reported by Macpherson et al. (27) using steady-state luminescence measurements. This yield is also consistent with the quantum yield of the P680 triplet (0.3) assuming some internal quenching by the protein matrix of the RC. Although even the most carefully prepared RC have some free chlorophyll, the estimated amount is only -6% of the total chlorophyll present (62), which is too low to account for the levels of '0, detected in this work. However, continued illumination under aerobic conditions leads to an increase in the yield of lo,, as was reported by Macpherson et al. (27) and is confirmed here in the experiment of Fig. 7. This increase in yield can be attributed to triplet states generated by intersystem crossing between chlorophyll molecules no longer able to transfer energy to P680, the proportion of which will gradually increase as the primary donor is inactivated by light treatment.
The results obtained with the RNO bleaching method are also consistent with '0, being the only active oxygen species generated when the P680 triplet state is formed by RC on illumination under aerobic conditions. This method is very useful as it allows a simultaneous measurement of the effect of illumination on RNO and the chlorophylls of the RC. In agreement with Macpherson et al. (271, we did not see any protection of the chlorophylls from photobleaching by the presence of lo, quenchers. It therefore seems likely that internal quenching mechanisms can compete very effectively with externally added quenchers. Indeed, p-carotene appears to be such a quencher as the '0, yield is considerably higher in RC preparations from which one of the two p-carotenes has been re-moved (by the method of De Las Rivas et al. (34)) as compared with the normal preparation.' In the light-harvesting systems of photosynthetic organisms, carotenoids protect by directly quenching chlorophyll triplets (see Ref. 7), but it is known that in thylakoids and PSI1 particles, carotenoid can be oxidized under conditions where electron donation from water is interrupted (63, 64). To prevent rapid oxidation by P680+, the distance between the carotenoid and P680 must presumably be more than the van der Waals distance, thus preventing direct quenching of 3P680. The main role of /3-carotene in the reaction center therefore may be to quench '0, rather than the P680 triplet state.
In addition to /3-carotene, a number of amino acids (in particular, histidine, tryptophan, and methionine) are known to react directly with '0, (65,66), and as has been demonstrated here, the reaction of '0, with added histidine is very rapid. Reaction of '0, with the RC protein would lead to oxidation products and might be particularly significant in the case of histidines as these form the ligands for the binding of the P680 chlorophylls. It is therefore possible that '0, oxidation of the reaction center proteins could cause conformational changes, which in turn activate proteolytic reactions.
Finally, direct evidence that '0, is the species that causes the bleaching of the RC chlorophylls is shown by the experiments wherein '0, formation was sensitized specifically by a n added dye, rose bengal. Dye-sensitized photobleaching of the RC was very similar to that seen when the chlorophylls were excited directly, i.e. '0, produced by the exogenous sensitizer appears to diffuse into the protein matrix of the RC and preferentially causes bleaching of P680, the primary electron donor chlorophylls. This is surprising as exogenously generated '0, might have been expected to bleach all of the pigments equally. It is possible either that there is easier access of '0, to P680 from the aqueous medium as compared with the other chlorophylls in the RC or that /3-carotene within the RC may be more effective at protecting the accessory chlorophylls than those of the primary donor, which seems to be the case in the reaction center of purple bacteria (67). In addition, the stimulation of bleaching of the accessory chlorophylls in D,O when the lifetime of '0, is lengthened suggests perhaps that P680 is particularly susceptible to the effect of this reactive species. It should be noted, too, that the stimulation of chlorophyll bleaching by D,O indicates that this is a n effect of '0, formed by RB itself and does not involve singlet energy transfer from excited RB to chlorophyll.
It has been suggested (49, 58) that photoinhibition in vivo might be caused by '0, formation sensitized by the non-heme iron involved in electron transfer from QA to QB or even iron sulfur centers or cytochromes. In these studies, '0, formation was detected in a suspension of isolated thylakoids using the RNO bleaching method. However, it should be noted that in our experiments, we use isolated RC, which lose their non-heme iron during preparation and bind no iron sulfur centers. We found that '0, is formed by the RC on excitation with red light as well as blue light, which is consistent with chlorophyll being the sensitizer and not, for example, the bound cytochrome b,,,. Our data are therefore more compatible with the acceptor side model for photoinhibition ( X ) , which invokes double reduction of QA and P680 triplet formation due to radical pair recombination (16,17). This leads to generation of '0, and thus presumably initiates the processes that lead to breakdown of the reaction center complex. cussions and Susan van Acker and Caroline Woollin for expert technical