Destruction of a Single Chlorophyll Is Correlated with the Photoinhibition of Photosystem II with a Transiently Inactive Donor Side (Photodamage/Oxygen Evolution)

Proc. Natl. Acad. Sci. USA Vol. 92, pp. 12195-12199, December 1995 Biochemistry

Destruction of a single chlorophyll is correlated with the photoinhibition of photosystem II with a transiently inactive donor side (photodamage/oxygen evolution)

DIRK BUMANN AND DIETER OESTERHELT* Department of Membrane Biochemistry, Max Planck Institute for Biochemistry, Am Klopferspitz 18a, 82152 Martinsried, Germany Communicated by George Feher, University of California, San Diego, La Jolla, CA, August 7, 1995 (received for review January 30, 1995)

ABSTRACT Pigments destroyed during photoinhibition Experimental Procedures of water-splitting photosystem II core complexes from the green alga Chiamydomonas reinhardtii were studied. Under Water-splitting PSII core complexes containing about 40 Chl conditions of a transiently inactivated donor side, illumina- a were isolated from the green alga Chiamydomonas reinhardtii tion leads to an irreversible inhibition of the electron transfer as described (28). These complexes have a somewhat altered at the donor side that is paralleled by the destruction of QB side. Since the QA side seems to be intact and is accessible chlorophylls a absorbing maximally around 674 and 682 nm. for ferricyanide, the acceptor side allows efficient electron The observed stochiometry of 1 + 0.1 destroyed chlorophyll flow out of PSII in the presence of this electron acceptor. per inhibited photosystem II suggests that chlorophyll de- For photoinhibition experiments frozen samples were struction could be the primary photodamage causing the thawed on ice and diluted in 20 mM morpholineethanesulfonic inhibition of photosystem II under these conditions. acid/KOH, pH 6.5/0.03% dodecyl maltoside/2 mM ferricyanide (final PSII concentration, 0.2 ,tM; Ca2+ concentration = 0 mM, Cl- concentration < 0.1 mM) and illuminated in a In photosynthesis light energy is converted to chemical energy. fluorescence cuvette (pathlength, 10 mm; volume, 1.2 ml) with However, side reactions can lead to considerable destruction light from a xenon lamp transmitted through a Schott RG630 of the photosynthetic apparatus and a concomitant loss of cutoff filter, a KG1 heat filter, and 4 cm of water at a photosynthetic activity in a process called photoinhibition (1). temperature of 4°C for time intervals from 1 to 30 s (80 In oxygenic photosynthesis the main target for photoinhibition mJ cm-2 in the range of 630-720 nm). During illumination the is photosystem II (PSII) (2). In intact PSII the excited primary sample was slowly stirred to obtain a uniform photoinhibition. donor P680 reduces plastoquinone. The oxidized primary Bleaching spectra (dark minus illuminated) were recorded on donor P680+ is then reduced by water via an redoxactive an Aminco DW2000 spectrophotometer (spectral bandwidth, tyrosine "Z" (3). In case of a transient malfunction of the 2 nm) after a dark period of 5 min to eliminate reversible water-splitting reaction, P680+ has an extended lifetime during signals. which it can degrade (4) or damage other PSII components, To estimate the number of destroyed Chl per PSII we including carotenoids, chlorophylls (Chl), possibly Z, the man- assumed that all 40 Chl a of PSII have similar oscillator ganese binding sites, and other amino acids of the PSII proteins strengths. The same assumption has been made for the analysis (5-10). One or several of these damages result in an irrevers- of PSII-RC absorption spectra (22, 29-31) and seems reason- ible inhibition of the electron transfer from Z to P680+ and able since the protein environment modulates the Qy(0-0)- hence in an irreversible loss of the water-splitting activity (6, absorption properties of Chl a only weakly as compared to Chl 7, 10-14). Despite many investigations it is still not clear which a in acetone (shift of absorption maximum by <20 nm, increase of the various damages actually causes this inhibition. We of the oscillator strength by about 10%; D.B., unpublished investigated this problem by correlating pigment destruction results). The 2 pheo and 3 Chl b bound to PSII core complexes with the loss of oxygen evolving activity. (28) were assumed to have absorptions in the region 650-720 Quantitative observations of pigment destruction in vivo or nm equivalent to about 3 Chl a giving a total absorption of all in grana membranes are difficult due to the high number of chlorins equivalent to 43 Chl a. Based on these assumptions we pigments per PSII (200-700) (15, 16). PSII reaction center estimated that a 1% absorption decrease in the region 650-720 preparations (PSII-RC) contain only 4-6 Chl, 2 pheophytins nm corresponds to an average destruction of 0.43 Chl a per (pheo), and 1-2 carotenoids (car) per PSII (17-21). Hence, PSII. destruction of less than one pigment per PSII can be Linear dichroism spectra were recorded with PSII embed- easily ded in acrylamide gels [final concentrations: 12.5% (wt/vol) observed in these particles (4, 22-24). However, since the acrylamide, 0.4% (wt/vol) N,N-methylenbisacrylamide, 0.03% electron transfer from Z to P680+ is already strongly impaired (vol/vol) N,N,N',N'-tetramethylparaphenylenediamine, in freshly isolated PSII-RC (25-27), these particles cannot be 0.05% (wt/vol) ammonium persulfate, 10 mM morpho- used for the investigation of the first irreversibly inactivating lineethanesulfonic acid/KOH, pH 6.5; the volume was 0.7 ml] reaction of photoinhibition. We therefore used PSII core (32, 33). The gels were polymerized for 1 h at 4°C in the dark complexes, which have an intermediate pigment content and and then washed for several hours in 20 mM morpho- are capable of water splitting (28). With these particles it is lineethanesulfonic acid/KOH, pH 6.5. The gels were then possible to observe the destruction of less than one Chl per soaked in 20 mM morpholineethanesulfonic acid/KOH, pH PSII and to measure the loss of water-splitting activity (in- 6.5/2 mM ferricyanide. We had to soak the gels in acceptor cluding the electron transfer from Z to P680+) in parallel. solution after polymerization since ferricyanide reacted with

The publication costs of this article were defrayed in part by page charge Abbreviations: Chl, chlorophyll(s); PS, photosystem; RC, reaction payment. This article must therefore be hereby marked "advertisement" in center; car, carotenoid(s); pheo, pheophytin(s). accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 12195 Downloaded by guest on September 24, 2021 12196 Biochemistry: Bumann and Oesterhelt Proc. Natl. Acad. Sci. USA 92 (1995) the polymerization reagents. The embedded PSII were ori- known cofactors of the donor side (41). In their absence the ented in a home-built linear dichroism cuvette by compressing electron transfer from Z to P680+ is inactive (42, 43). This the gels in one direction by a factor of 1.5 and expanding them inactivation is reversible since addition of CaC12 restores full in another direction. The dimension of the gels did not change activity. However, illumination under these conditions leads to in the direction of the optical path (9 mm). Isotropic spectra an irreversible loss of the water-splitting activity that cannot be showed no differences to PSII in solution, indicating that the restored by the addition of CaC12. A comparison of the pigments were not distorted by the embedding procedure. light-saturation behavior of intact and partially inhibited PSII Spectra with linearly polarized light (parallel to the direction revealed that only the light-saturated activity decreases while of compression or expansion) and comparison with published the light intensity necessary for 50% saturation is not affected spectra of other PSII core complexes (34,35) revealed that our (Fig. 1). This indicates that the inhibition is caused by a block PSII was similarly oriented with their plane corresponding to of the electron transfer while the energy transfer from the the former membrane plane parallel to the direction of the antenna to P680 is not affected. expansion. The maximum dichroic ratio (absorbance differ- To further characterize the defective electron transfer we ence between the two light polarizations divided by the measured flash-induced absorption changes of intact and isotropic absorbance) was 0.2-0.3 at 687 nm depending on the partially inhibited PSII in the absence of Ca2+ and Cl- (Fig. 2). sample. Linear dichroism bleaching spectra were measured Under these conditions the fast reduction of P680+ by Z is after illumination of the gels at 10°C. blocked (42, 43). Both samples exhibited an absorption in- Water-splitting activity was measured as oxygen evolution crease at 820 nm, which decayed multiphasically with a dom- with a Clarke-type electrode in 20 mM morpholineethanesul- inant lifetime of 500-600 ,us. This signal can be assigned to fonic acid/KOH, pH 6.5/2 mM ferricyanide/50 mM CaCl2 at long-living Chl cation radicals that are probably slowly reduced 20°C with light from a diaprojector filtered by a Schott RG630 by electron back flow from QA- (44, 45). Both samples filter. The maximum activity was 1000-1200 ,umol of 02 (mg generated a similar amount of long-living cations, indicating of Chl)-1h-1. Light saturation was measured by varying the that the primary charge separation from P680 to the primary light intensity with neutral glass filters (6.5%, 12.5%, 27%, quinone is- still intact after partial inhibition of the water- 48%, 68% transmission in the range 660-690 nm). During a splitting activity. The loss ofwater-splitting activity is therefore period of several hours the light intensity of the diaprojector due to an irreversible damage of the donor side in agreement varied by up to 5%. Therefore, we measured the light intensity with earlier results (6, 7, 10-14). at the end of each experiment (Optometer, United Detector A second flash-induced absorption increase occurred in the Technology, Santa Monica, CA), which was on average 150 near-infrared (absorption maximum higher than 960 nm), mJ cm-2 in the range 630-720 nm. The light-saturation be- which decayed biexponentially with lifetimes of 4 and 18 ms havior was modeled assuming a Michaelis-Menten-type ki- (Fig. 2). This signal could be assigned to carotenoid cations netic (36-38). Photoinhibited PSII was kept on ice until assay In inhibited PSII of the much of activity to minimize Dl protein cleavage (39). During the (23, 46). partially (53% activity) assay the suspension was first kept for 4 min at 20°C in the dark less carotenoid cations were generated, indicating an almost to obtain a constant drift. During that time the Dl protein completely blocked electron transfer from carotenoid to should be cleaved in <10% of the damaged particles (39). P680+. However, such PSII can still split water to a consider- Flash-induced transient absorption changes [excimer-dye able extent, suggesting that this electron transfer is not nec- laser system EMG100, FL 3002, Lambda Physics (Acton, MA); essary for the water-splitting activity as was already observed with dye rhodamine 101 LC 660; pulse centered at 645 nm; in higher plants (46). pulse duration, 18 ns; power, 1 mJ] were measured in the Parallel to the loss of water-splitting activity, an absorption absence of Ca2+ and Cl- (2.4 ,uM PSII in 20 mM morpho- decrease occurred in the chlorin Qy(0-0)-band (Fig. 3). lineethanesulfonic acid/KOH, pH 6.5/2 mM ferricyanide) at Chlorin destruction in PSII with a transiently inactive donor 20°C with a multidiode array spectrophotometer of local side caused by P680+ oxidizing the Chl followed by chlorin ring design (pathlength, 10 mm; range, 800-960 nm; spectral opening and further decay (23). Singlet oxygen proposed to be resolution, 10 nm; time resolution, 128,us) (40). The measuring the major damaging agent under conditions of an inactive beam, from a xenon source, was passed through a Schott acceptor side is not involved in the photoinhibition ofPSII with RG780 filter to avoid any actinic effect. Signals are the average a transiently inactive donor side (4). Since chlorins decay to of 15 flashes at 0.2 Hz. Decays were fitted with an increasing products without significant absorption in the visible region number of exponentials until no systematically oscillating (23, 47), the difference spectra probably represent the native residual could be observed. spectra of chlorins destroyed during photoinhibition (4). PSII The correlation between Chl destruction and loss of activity was simulated assuming that the destruction of a single Chl leads to a complete loss of activity and Chl of already inhibited 100 *. PSII is destroyed with the same quantum yield as Chl of intact PSII according to the recursive formula: 80 1 PSIlinactive(n + 1) = PSIlinactive(n) + [N - PSIIinactive(n)]/N, I 60 , where PSIlinactive(n) is the number of inhibited PSII particles -0 after the destruction of n Chl of the sample and N is the total 40 -. number of PSII particles. The assumption of equal destruction 20j of Chl in intact and inhibited PSII has not been experimentally 20 - ° verified but we simulated only values up to 1.1 destroyed Chl minor deviations would have no per PSII. Therefore, signifi- O X 7-X ---r- / I/ cant impact on the results. Results shown were obtained for N 0 20 40 60 80 100 0O = 108, where convergence at the 0.01% level was reached. Relative light intensity Results FIG. 1. Light intensity dependencies of water-splitting activity for intact (-) and partially photoinhibited (0) samples in the presence of PSII core complexes isolated from the green alga C. reinhardtii CaC12. The data were fit with a Michaelis-Menten-type kinetic with split water only in the presence of Ca2+ and Cl- (28), which are photons as the substrate. The dashed lines represent 50% saturation. Downloaded by guest on September 24, 2021 Biochemistry: Bumann and Oesterhelt Proc. Natl. Acad. Sci. USA 92 (1995) 12197

960 0 820 .0&.1 .0 ,/1 0 5 10 Time, ms a) .00o v) 660 680 700 660 680 700 2 ItOD Wavelength, nm FIG. 4. (A) Second derivative of a bleaching spectrum. Positive peaks correspond to negative absorption differences (bleaching). (B) Bleaching spectra of PSII core complexes (0.2 ,uM corresponding to 8 mM Chl a) oriented in squeezed gels. The spectra were measured with 800 850 900 950 light linearly polarized in the direction of expansion (Exp.) and compression (Comp.) of the gel. The isotropic Qy(0-0)-absorption Wavelength, nm decrease was 0.9%. FIG. 2. Flash-induced transient absorbance difference spectra at correlated with the loss of about half of the water-splitting 128-,s delay of intact (solid line) and partially photoinhibited (dashed line) PSII core complexes (2.4 ,uM corresponding to 96 ,uM Chl a) in activity, suggesting that the destruction of a single Chl results the absence of CaCl2 and the presence of 2 mM ferricyanide. (Inset) in complete inhibition. As inhibition proceeds one would Decay at 820 and 960 nm for intact complexes. expect further Chl destruction in already inhibited PSII, leading to higher ratios of destroyed Chl per inhibited PSII. To core complexes from Chlamydomonas contain the chlorins Chl obtain exact stochiometries we therefore extraplated this ratio a, Chl b, and pheo (28). Chl b is probably bound to peripheral (destroyed Chl/inhibited PSII) to an undamaged sample (Fig. antenna complexes (48) and, therefore, unlikely to be de- 5B) and determined that the loss of-1.0 ± 0.1 Chl a is correlated stroyed by reactive species generated in the center of PSII. with a complete inhibition of a PSII particle. Theoretical Pheo is not attacked by P680+ in PSII-RC (23) and probably simulations of this relationship (see Experimental Procedures) also not in PSII core complexes because of the high redox are in good agreement with the experimental data (Fig. 5). potential of pheo (in vitro 1.3-1.4 V) (49). Hence, the bleaching in the region 650-720 nm purely represents Chl a destruction. Discussion The second derivative of the difference spectra revealed at least two destroyable Chl a species with absorption maxima Photoinhibition of PSII with a transiently inactivated donor around 674 and 682 nm (Fig. 4A). Bleaching spectra of PSII side is caused by an irreversible block of electron transfer from oriented in squeezed acrylamide gels showed that both de- water to P680, as was shown in several studies (6, 7, 10-14) and stroyable Chl a have Qy transition moments almost parallel to confirmed in our experiments (Fig. 2). Potential targets for the the membrane plane (Fig. 4B). The dichroic ratios at 674 and inhibiting photodamage are the redoxactive components Z, 682 nm were similar (about 0.6), indicating that both Chl have Mn ions, and possibly amino acids, which all participate in the similar orientations. electron transfer from water to P680. However, all of these Alternatively, the bimodal bleaching spectrum could be components are not expected to be destroyed under the explained by the destruction of a single Chl that is excitonically conditions used since P680+ is not able to oxidize any of them coupled to another Chl. In this case both exciton bands of this due to the inactivated electron transfer from Z to P680 in the Chl pair would bleach while a new absorption would rise due absence of Ca2+ and Cl- (42, 43). Therefore, other compo- to the remaining undamaged second Chl of the pair yielding a nents of PSII not directly involved in the electron transfer are complex bleaching spectrum. probably responsible for the loss of activity. Besides Z, cyto- The spectral resolution does not permit us to decide between chrome b559, car, and Chl are species known to be oxidized by the both possibilities. long-living P680+ (46,50). Cytochrome b559 is already oxidized To investigate the influence of Chl destruction on the in our intact particles (28) and further oxidation of already water-splitting activity we estimated the Chl a destruction per oxidized cytochrome b559 has not been observed (50). The PSII from the absorption decrease in the region 650-720 nm results obtained here show that car destruction is not the cause and compared it with the loss of light-saturated water-splitting of inhibition (Fig. 2), in agreement with earlier studies (46). On activity (Fig. 5A). The average loss of 0.5 Chl a per PSII was the other hand, the stochiometry of one destroyed Chl per inhibited PSII indicates that Chl destruction and photoinhibi- 01

5- E40 1-4 U.1laj- 20 I ~~~~~1.0 10 0 0.0 0.4 0.8 0.0 0.4 0.80 660 680 700 Destroyed Chl, no. per complex Wavelength, nm FIG. 5. (A) Comparison of Chl destruction and loss of water- FIG. 3. Photobleaching of PSII core complexes with an inactive splitting activity. (B) Number of destroyed Chl per inhibited PSII for donor side (difference spectra: untreated minus illuminated). Shown samples photoinhibited to different extents. The lines represent the- are bleaching spectra with 0.35%, 1.0%, and 1.3% decrease of oretical values based on the assumption that the destruction of one Chl Qy(0-0)-absorption (0.2 ,lM PSII corresponding to 8 mM Chl a). is correlated with a complete loss of water-splitting activity. Downloaded by guest on September 24, 2021 12198 Biochemistry: Bumann and Oesterhelt Proc. Natl. Acad. Sci. USA 92 (1995) tion proceed at the same rate (Fig. 5). This suggests that Chl oxidizable Chl has been shown to have a distance of 4 nm to destruction could be the first irreversibly inhibiting step of the non-heme iron ion of PSII that is compatible with a binding photoinhibition under these conditions. We cannot exclude of this Chl to RC polypeptides (70). Indeed, an oxidizable Chl that other PSII components are also destroyed with the same different from P680 is present in PSII-RC (23, 55). Interest- rate as that of the Chl and that this damage, instead of Chl ingly, our bleaching spectra are rather similar to those obtained destruction, is the actual cause of inhibition. However, since no for PSII-RC (23). Spectroscopic evidence seems to indicate the other species oxidized by P680+ have been observed, species presence of chlorins different from P680 with absorption being attacked and destroyed by P680+ with the same rate as maxima near 674 and 681 nm and transition moments almost that of the Chl are highly unlikely to exist. parallel to the membrane plane in the PSII-RC (59,67, 68, 71). Chl destruction was already proposed to occur early in This evidence seems to suggest that the destroyable Chl could photoinhibition. Thompson and Brudvig (50) assumed that be bound to the RC polypeptides, although it is also possible specific antenna Chl not essential for the electron transfer are that they are bound to other polypeptides of the core complex. destroyed first. However, light saturation measurements Site-directed mutagenesis of potential Chl ligands combined yielded no evidence for a destruction of antenna Chl in PSII with photoinhibition experiments might eventually resolve this from Chlamydomonas (this work) or in grana membranes from subject. higher plants (7, 12, 51). On the other hand, Telfer et al. (23) proposed that the destruction of P680 is the cause of photo- We thank two anonymous reviewers for their help in improving the inhibition in PSII-RC. However, a number of investigations manuscript. D.B. was supported by Stiftung-Stipendien-Fonds des with larger particles and in vivo demonstrated that, under Verbandes der Chemischen Industrie and Stiftung Volkswagenwerk in conditions of an inactive donor side, P680 remains intact even terms of a Kekule fellowship. after loss of the water-splitting activity (6, 7, 10-14). Possibly P680 is destroyed in later steps of photoinhibition that might 1. Ogren, E. & Rosenqvist, E. (1992) Photosynth. Res. 33, 63-71. be important for the final destruction of inhibited PSII but this 2. Prasil, O., Adir, N. & Ohad, I. (1992) in The Photosystems: is not relevant for the loss of water-splitting activity since it Structure, Function and MolecularBiology, ed. Barber, J. (Elsevier affects only already inhibited PSII. Science, Amsterdam), pp. 295-348. Taken together both previous models do not seem to 3. Debus, R. J., Barry, B. A., Sithole, I., Babcock, G. T. & McIn- correctly explain the role of Chl destruction in photoinhibition tosh, L. (1988) Biochemistry 27, 9071-9074. 4. Telfer, A., He, W.-Z. & Barber, J. (1990) Biochim. Biophys. Acta under conditions of a transiently inactive donor side. Instead, 1017, 143-151. Chl different from P680 and essential for the electron transfer 5. Klimov, V. V., Shafiev, M. A. & Allakhverdiev, S. I. (1990) from water to P680 seem to be destroyed in the inhibiting step. Photosynth. Res. 23, 59-65. We cannot decide whether two Chl are destroyed in parallel 6. Blubaugh, D. J., Atamian, M., Babcock, G. T., Golbeck, J. H. & or one Chl of an excitonically coupled pair of Chl is destroyed Cheniae, G. M. (1991) Biochemistry 30, 7586-7597. since both would produce a bimodal bleaching spectrum. Due 7. Chen, G.-X., Kazimir, J. & Cheniae, G. M. (1992) Biochemistry to the stochiometry of one destroyed Chl per inhibited PSII, 31, 11072-11083. two different Chl would be essential for the water-splitting 8. Ohad, I., Adir, N., Koike, H., Kyle, D. & Inoue, Y. (1991)J. Biol. activity in the first case, whereas only one Chl would be Chem. 265, 1972-1979. essential in the second case. 9. Ono, T.-A. & Inoue, Y. (1991) Biochemistry 30, 6183-6188. Apparently the destroyable one or two Chl are essential for 10. Jeferschold, C., Agren, H. & Styring, S. (1992) in Research in the electron transfer from water to P680+. Possibly they Photosynthesis, ed. Murata, N. (Kluwer, Dordrecht, The Neth- erlands), Vol. 2, pp. 421-424. control the native conformation of PSII or modulate the redox 11. Theg, S. M., Filar, L. J. & Dilley, R. A. (1986) Biochim. Biophys. properties of the components participating in electron trans- Acta 849, 104-111. fer. Previous studies revealed that most likely the electron 12. Eckert, H.-J., Geiken, B., Bernarding, J., Napiwotzki, A., Eichler, transfer between Z and P680+ is affected (12). Indeed, this H.-J. & Renger, G. (1991) Photosynth. Res. 27, 97-108. electron transfer is highly sensitive to a disturbed environment 13. Jegerschold, C. & Styring, S. (1991) FEBS Lett. 280, 87-90. as in RCs (25-27). Alternatively, the destroyable Chl could 14. Rova, M., Franzen, L.-G., Frederiksson, P.-O. & Styring, S. directly participate in the electron transfer since they are able (1994) Photosynth. Res. 39, 75-83. to donate electrons to P680+ as it is indicated by their 15. Dunahay, T. G., Staehelin, L. A., Seibert, M., Ogilvie, P. D. & destruction and various spectroscopic evidence (4, 22, 27, 50, Berg, S. P. (1984) Biochim. Biophys. Acta 764, 179-193. 52-55). No experimental evidence exists that these Chl cations 16. Neale, P. J. & Melis, A. (1986) J. Phycol. 22, 531-538. are able to accept electrons from Z but at least the various 17. Gounaris, K., Chapman, D. J., Booth, P., Crystall, B., Giorgi, of "P680" do not rule out this L. B., Klug, D. R., Porter, G. & Barber, J. (1990) FEBS Lett. 265, conflicting spectra possibility 88-92. since they all show considerable deviations from a simple 18. Kobayashi, M., Maeda, H., Watanabe, T., Nakane, H. & Satoh, Gaussian absorption peak (22, 27, 30, 56-67). The species K. (1990) FEBS Lett. 260, 138-140. absorbing maximally at 682 nm might be especially difficult to 19. Montoya, G., de las Rivas, J., Booth, P. J., Giorgi, L. B., Klug, separate from P680 if it is really directly involved in electron D. R., Porter, G., Barber, J. & Picorel, R. (1994) Biochim. transfer. Moreover, a participation of different Chl cations in Biophys. Acta 1185, 85-91. electron transfer could explain the fundamentally conflicting 20. Yruela, I., van Kan, P. J. M., Muller, M. G. & Holzwarth, A. R. properties of P680 (68). Indeed, a putative Chl donor has (1994) FEBS Lett. 359, 25-30. already been proposed as a possibility among others based on 21. Aured, M., Moliner, E., Alfonso, M., Yruela, I., Toon, S., Seibert, spectroscopic evidence and structural considerations (69), but M. & Picorel, R. (1994) Biochim. Biophys. Acta 1187, 187-190. more detailed spectroscopic evidence is required to clarify this 22. Telfer, A. & Barber, J. (1989) FEBS Lett. 246, 223-228. point. 23. Telfer, A., de las Rivas, J. & Barber, J. (1991) Biochim. Biophys. PSII core complexes contain 40 Chl a including many with Acta 1060, 106-114. 24. de las Rivas, J., Telfer, A. & Barber, J. (1993) Biochim. Biophys. absorption properties similar to the destroyable Chl. Any Acta 1142, 155-164. structural assignment of the destroyable Chl is therefore 25. Mathis, P., Satoh, K. & Hansson, 0. (1989) FEBS Lett. 251, difficult. Possible binding sites could be located on the core 241-244. antenna Chl proteins CP 47 and CP 43 as well as on the RC 26. Nugent, J. H. A., Telfer, A., Demetriou, C. & Barber, J. (1989) polypeptides Dl protein and D2 protein. The oxidation of the FEBS Lett. 255, 53-58. destroyable Chl by P680 and their impact on electron transfer 27. Takahashi, Y., Satoh, K. & Itoh, S. (1989) FEBS Lett. 255, reactions to P680 might indicate a position near P680. An 133-138. Downloaded by guest on September 24, 2021 Biochemistry: Bumann and Oesterhelt Proc. Natl. Acad. Sci. USA 92 (1995) 12199 28. Bumann, D. & Oesterhelt, D. (1994) Biochemistry 33, 10906- 50. Thompson, L. K. & Brudvig, G. W. (1988) Biochemistry 27, 10910. 6653-6658. 29. Braun, P., Greenberg, B. M. & Scherz, A. (1990) Biochemistry 29, 51. Blubaugh, D. J. & Cheniae, G. M. (1990) Biochemistry 29, 5109- 10376-10387. 5118. 30. Van Kan, P. J. M., Otte, S. C. M., Kleinherenbrink, F. A. M., 52. Visser, J. W. M., Rijgersberg, C. P. & Gast, P. (1977) Biochim. Nieveen, M. C., Aartsma, T. J. & van Gorkom, H. (1990) Bio- Biophys. Acta 460, 36-46. chim. Biophys. Acta 1020, 146-152. 53. Nugent, J. H. A., Evans, M. C. W. & Diner, B. A. (1982) Biochim. 31. Durrant, J. R., Giorgi, L. B., Barber, J., Klug, D. R. & Porter, G. Biophys. Acta 682, 106-114. (1990) Biochim. Biophys. Acta 1017, 167-175. 54. De Paula, J. C., Innes, J. B. & Brudvig, G. W. (1985) Biochemistry 32. Abdourakhmanov, I. A., Ganago, A. O., Erokhin, Y. E., Solovev, 24, 8114-8120. A. A. & Chugunov, V. A. (1979) Biochim. Biophys. Acta 546, 55. S. E. J., J. H. A. & O'Malley, P. J. (1994) Bio- 183-186. Rigby, Nugent, 33. Breton, J. & Vermeglio, A. (1982) in Photosynthesis: Energy chemistry 33, 10043-10050. Conversion by Plants and Bacteria, ed. Govindjee (Academic, 56. Doring, G., Renger, G., Vater, J. & Witt, H. T. (1969) Z. New York), pp. 153-194. Naturforsch. B 24, 1139-1143. 34. Tapie, P., Choquet, Y., Wollman, F.-A., Diner, B. & Breton, J. 57. van Gorkom, H., Pulles, M. P. J. & Wessels, J. S. C. (1975) (1986) Biochim. Biophys. Acta 850, 156-161. Biochim. Biophys. Acta 408, 331-339. 35. Van Dorssen, R. J., Plijter, J. J., Dekker, J. P., den Ouden, A., 58. Eckert, H.-J., Renger, G. & Witt, H. T. (1984) FEBS Lett. 167, Amesz, J. & van Gorkom, H. J. (1987) Biochim. Biophys. Acta 316-320. 890, 134-143. 59. Nujis, A. M., van Gorkom, H. J., Plijter, J. J. & Duysens, L. N. M. 36. Miyao, M. & Murata, N. (1983) Biochim. Biophys. Acta 725, (1986) Biochim. Biophys. Acta 848, 167-175. 87-93. 60. Wasielewski, M. R., Johnson, D. G., Seibert, M. & Govindjee, 37. Franzen, L.-G., Styring, S., Etienne, A.-L., Hansson, 0. & (1989) Proc. Natl. Acad. Sci. USA 86, 524-528. Vernotte, C. (1986) Photochem. Photobiophys. 13, 15-28. 61. Telfer, A., Durrant, J. & Barber, J. (1990) Biochim. Biophys. Acta 38. Maenpaa, P., Andersson, B. & Sundby, C. (1987) FEBS Lett. 215, 1018, 168-172. 31-36. 62. Durrant, J. R., Hastings, G., Hong, Q., Barber, J., Porter, G. & 39. Aro, E.-M., Hundal, T., Carlberg, I. & Andersson, B. (1990) Klug, D. R. (1992) Chem. Phys. Lett. 188, 54-60. Biochim. Biophys. Acta 1019, 269-275. 63. Hastings, G., Durrant, J. R., Barber, J., Porter, G. & Klug, D. R. 40. Uhl, R., Meyer, B. & Desel, H. (1985) J. Biochem. Biophys. (1992) Biochemistry 31, 7638-7647. Methods 10, 35-48. 64. Seibert, M., Toon, S., Govindjee, O'Neil, M. P. & Wasielewski, 41. Rutherford, A. W., Zimmermann, J.-L. & Boussac, A. (1992) in M. R. (1992) in Research in Photosynthesis, ed. Murata, N. The Photosystems: Structure, Function and Molecular Biology, ed. (Kluwer, Dordrecht, The Netherlands), Vol. 2, pp. 41-44. Barber, J. (Elsevier, Amsterdam), pp. 179-229. 65. Van der Vos, R., van Leeuwen, P. J., Braun, P. & Hoff, A. J. 42. Boussac, A., Setif, P. & Rutherford, A. W. (1992) Biochemistry (1992) Biochim. Biophys. Acta 1140, 184-198. 31, 1224-1234. J. P. & 43. Boussac, A. & Rutherford, A. W. (1994) J. Biol. Chem. 269, 66. Roelofs, T. A., Kwa, S. L. S., van Grondelle, R., Dekker, 12462-12467. Holzwarth, A. R. (1993) Biochim. Biophys. Acta 1143, 147-157. 44. Van Best, J. A. & Mathis, P. (1978) Biochim. Biophys. Acta 503, 67. Schelvis, J. P. M., van Noort, P. I., Aartsma, T. J. & van Gorkom, 178-188. H. J. (1994) Biochim. Biophys. Acta 1184, 242-250. 45. Mathis, P. & Setif, P. (1981) Isr. J. Chem. 21, 316-320. 68. van Gorkom, H. J. & Schelvis, J. P. M. (1993) Photosynth. Res. 38, 46. Schenck, C. C., Diner, B., Mathis, P. & Satoh, K. (1982) Biochim. 297-301. Biophys. Acta 680, 216-227. 69. Van Mieghem, F. J. E. & Rutherford, A. W. (1994) Biochem. 47. Brown, S. B., Houghton, J. D. & Henry, G. F. (1991) in Chloro- Soc. Trans. 21, 986-991. phylls, ed. Scheer, H. (CRC, Boca Raton, FL), pp. 465-492. 70. Koulougliotis, D., Innes, J. B. & Brudvig, G. W. (1994) Biochem- 48. Bassi, R. & Wollman, F.-A. (1991) Planta 183, 423-433. istry 33, 11814-11822. 49. Watanabe, T. & Kobayashi, M. (1991) in Chlorophylls, ed. Scheer, 71. Otte, S. C. M., van der Vos, R. & van Gorkom, H. J. (1992) J. H. (CRC, Boca Raton, FL), pp. 287-316. Photochem. Photobiol. B: Biol. 15, 5-14. Downloaded by guest on September 24, 2021