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 ...
THEJOURNAL OF BIOWICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 269,No. 18, Issue of May 6,pp. 13244-13253, 1994 Printed in U.S.A.
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* (Received forpublication, November 17, 1993, and in revised form, February 18, 1994)
Alison TelferSP, Steven M. Bishop1 1, David Phillipsn, and JamesBarber* From the $Agricultural and Food Research Council Photosynthesis Research Group, Wolfson Laboratories, Department of Biochemistry and the Wepartmentof Chemistry, Imperial College of Science, Technology, and Medicine, London SW72AI: United Kingdom
Singlet oxygen formation by photosystem I1 reaction centers isolated fromPieum sativum has been detected by twochemical trapping techniques: histidine-dependent oxygen uptake and bleaching of p-nitrosodimethylaniline 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 withthe 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. Experimentswith deuterated buffer and active oxygen scavengers indicated that singlet oxygen wasthe only active oxygen species produced by this reaction. Neither azidenor histidine, which are scavengers of singlet oxygen, protected against photobleaching of the chlorophyll of reaction centers that occurs concomitantly with singletoxygen 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 singletoxygenformed within the hydrophobic interior of the reaction center attacks the chlorophylls of P680, and presumably also amino acids in the vicinity, and that only the singlet oxygen that escapes to the medium is affected by added scavengers or deuterated medium. These experiments extend our earlier reportof the detection of singlet oxygen by its luminescence at 1270 nm when isolated photosystem I1 reaction centers are illuminated (Macpherson, A. N., Telfer, A., Barber, J., and Truscott, T.G. (1993) Biochim. Biophys. Acta 1143,301-309).Moreover, our results support the hypothesis that production of singlet oxygen underlies the vulnerability of photosystem I1 to photodamage and hence necessitates the rapid turnover of the D l protein of the reaction center.
One of the most abundant photosensitizersof singlet oxygen (IOJ1 formation in biology is chlorophyll (1,2). Consequently,
* This work was supported by grants from the Agricultural and Food Research Council (to J. B. and A. T.) and the Science and Engineering Research Council (to D. P. and S. M. B.). “he costs of publication of this article were defrayed in part by the payment of page charges. This article must thereforebe hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 8 To whom correspondence should be addressed.Tel.: 44-71-589-5111; Fax: 44-71-225-0960.
jl Present address: Biochemistry Dept., 322 Choppin Hall, Louisiana State University, Baton Rouge, LA 70803. The abbreviations used are:lo,, singlet oxygen (02(’Ag)); PSII, pho-
the light-harvesting systems of photosynthetic organisms are normally protected from damage by ‘0, (and also other photooxidative effects) by carotenoids (3-7). This is demonstrated by the greater susceptibility oxygen-dependent to 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 lprotein. Restoration of activity requires proteinsynthesis. Oxygen has beenimplicated 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 structureof the purple bacterialreaction 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 haveled to an understandingof the electron transfer processes occurringin 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 161of t h e D lprotein, P680 is the chlorophyll a molecules of the primary electron donor, Pheo is the pheophytin molecule of the primary electronacceptor, 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 theloss of activity occurs on the acceptor or donor side of the initial charge transferreaction 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 lightconditions, 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 reactioncenteds) 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 phthalocyapE, ninedisulfonate; TPPS,, mesotetra-(4-~ulfonatophenyl)porphine; microeinsteins; BA,singlet oxygen quantum yield; RB, rose bengal; SiMo, silicomolybdate.
13244
Quantum Yield of Singlet Oxygen Production by Photosystem ZZ of PSII exceed the rateof 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 ofQ,, which is normally a single electron acceptor, becoming doubly reduced and protonated (16). In itsfully 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 hasbeen 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 (20-22) (Equation 2). P680Pheo
t
-
hu
-
'[P68O+Pheo-]
3[P680+Pheo-]+ 3P680
(Eq. 2)
1
Despite the fact that the isolated RC normally binds two p-carotene molecules (23,241this preparation shows onlya low quantum yield of carotenoid triplet, -0.03 (21,22). This inability of p-carotene to quench the 3P680 state hasalso 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 1ms to 33 ps,supports this hypothesis (21, 22). 3P680+ 30,+ P680 + '0,
'0,+ His "-* [HisO,] + HisO, [HisO,] + RNO + RNO,
+ products
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 isthe 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 3P680and that the detrimental reaction occurs because of the ineffectiveness of the carotenoid quenching mechanism. MATERIALSANDMETHODS 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 a t 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). Aluminum phthalocyanine disulfonate (AlPcS,) was synthesized using the method of Ambroz et al. (35). Mesotetra-(4-sulfonatophenyl)porphine (TPPS,) tetrasodium salt was purchased from Porphyrin Products and was used as received. Other chemicals were obtained from Sigma. 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 a t 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 424- or 666-nm interference filter. AlPcS, and TPPS, were used as @A standards at 666 and 424 nm, respectively. The fromP680 (@yo) was calculated as follows (Equation 6):
(&)
@F=@$g)
(Eq. 3)
Recently, we detected the photoinduced formation of '0, by isolated PSII reaction centers using both steady-stateand time-resolved measurements of its emission a t 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 to form an intermediate product, a trans-annular perwith '0, oxide, [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). (Eq. 4) (Eq. 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-
13245
(Eq. 6)
is the singlet oxygen quantum yield of the standard, R , is where 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 to silicomolybdatemeasured electron transport ratesfrom Mn2+ with RC (36,371.These results are consistent with the view that illumination of RC causes '0, formation and that theuptake 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, AlPcS, and TPPS,. In Fig. 2a, it can be seen that AlPcS,, TPPS,, and RC have the same concentration requirement for histidinecatalyzed photoinduced oxygen uptake (andimidazole (data not
13246
Quantum Yield of Singlet Oxygen Production by Photosystem II
compared with H,O(-1.6 timesin both cases(datanot be indicative shown)). This D,O effect is also usually thought to 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 b 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 C major oxygen species involved in histidine photooxidation and d hence the major product when PSII reaction centers are illuminated. Formation: Oxygen Uptake Method-If Quantum Yield of '0, we assume that'0, is theonly 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 theabsorption of this excitation by the two sensitizers was matchedas described under "Materials and Methods." Fig. 2a also showssimilar 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. 2a, in which it is assumed that theyield for TPPS, is 0.62 (the value found by Verlhac et al. (29)). The quantum yields obtained for RC are very similar whether they were excited in the Soret band(424 nm) or the Qyregion (666 nm) of chlorophyll a absorption (0.155 and 0.164, respectively). The yield for AlPcS, is somewhat lower than other published values for similar phthalocyanine compounds, i.e. 0.30 for AlPcS, in monodeuterated methanol (42) and 0.34 for AlPcS, in oxygen-saturated aqueous (D,O) phosphate solution, pH 7.0 (43). However, it is more similar to that found by Bishop (44) for AlPcS, in phosphatebuffer (D,O) using time-resolved emission = 0.18) and is consistent with triplet quantum methods (aA yield data for AlPcS, in the same solvent system (aT= 0.22) FIG.1. Histidine and imidazole catalyze light-dependentoxygen uptakeby isolated PSII reaction centers.RC were suspended determined by Beeby et al. (45, 46). Bleaching of p-Nitrosodimethylaniline" very sensitive at 2 pg/ml chlorophyll in 50 m~ "is-HC1, pH 7.2, and 2 m~ n-dodecyl P-D-maltoside with the following additions: truce a,none; trace b, 1 m~ method for detection of 'O,, developed by Kraljic and ElMohsni histidine; truce c, 10 m~ imidazole; truce d, 10 m~ histidine. Samples (30), is based on the secondary bleachingof RNO induced by the were maintained at 20 "C and irradiated with -2000 1.E of white light intermediate oxidation product of the reaction of '0, with hism-2 s-l . Relative rates of oxygen uptake are shown as numbers by the traces. The rate of oxygen uptake with 10 m~ histidine (truce d ) was tidine or imidazole (see Equations4 and 5). The trans-annular peroxide of histidine, [Hiso,], or imidazole causes thebleaching 4718 pmoVmg of chlorophyllh. of RNO, which is normally followed at its absorption maximum (440nm). This method has been used for detection of '0, senshown)). This supports our conclusion that '0, production is sitized by a number of dyes, including eosin and rhodamineB (30) and porphyrins (29). It has also been used to monitor '0, catalyzed by RC. Fig. 2b shows that theazide ion (Ni), a known '0, quencher formation by crude coal tar extracts (47), by the nitrophenyl (see Ref.381, inhibits histidine-dependent oxygen uptake by ether herbicide oxyfluorfen in conjunction with isolated chloroRC. Nearly complete inhibition of histidine-catalyzed 0, up- plast membranes (481, and more recently, by thylakoid memtake is found at lo-' M azide. This was the same concentration branes alone (49). Fig. 3 compares the absorption spectrum of RC with that of range for inhibition by azide seen by Verlhac et al. (29) using porphyrin dyes as '0, sensitizers. Although these authors have RNO and of the various dyes that have been used in the exsuggested that this indicates thatonly lo, participates inhis- periments reported here. As can be seen in Fig. 3, AlPcS, abtidine photooxidation, azide is also known to quench hydroxyl sorbs in the redregion, as does the Qy band of the chlorophylls and pheophytin of the RC. TPPS, absorbs mainly in the blue radicals more efficiently than '0, (rate constants of 1.1 x 10'' region, as does the Soret bandof the RC chlorophyll and pheand -10' M-' s-l, respectively) and also superoxide radicals, although less efficiently (rate constant of
