Proc. Natl. Acad. Sci. USA Vol. 90, pp. 10942-10946, December 1993 Biochemistry A functional model for the role of b559 in the protection against donor and acceptor side photoinhibition (functional model) JAMES BARBER AND JAVIER DE LAS RIVAS* Agricultural and Food Research Council Photosynthesis Research Group, Wolfson Laboratories, Department of Biochemistry, Imperial Coliege of Science, Technology and Medicine, London SW7 2AY, United Kingdom Communicated by Daniel I. Arnon, July 6, 1993

ABSTRACT A quinone-independent photoreduction ofthe oxidizing potentials of long-lived species (7, 8). Donor side low potential form of cytochrome b55, has been studied using photoinhibition is readily seen when the -splitting re- isolated reaction centers of photosystem II. Under anaerobic actions have been inhibited (9-11). Acceptor side photoin- conditions, the cytochrome can be fully reduced by exposure to hibition occurs when there is an overreduction ofthe quinone strong illumination without the addition of any media- pool and QA becomes doubly reduced (12, 13). In this event, tors. Under high light conditions, the extent and rate of the there is an increase in the probability ofcharge recombination reduction is unaffected by addition of the exogenous electron between the primary radical pair P680+ Pheo- leading to the donor Mn2+ and, during this process, no irreversible damage triplet state of P680. This triplet state can interact with occurs to the reaction center. However, prolonged mlumination and form highly reactive singlet oxygen. in strong light brings about irreversible bleaching of chloro- Support for these mechanisms comes mainly from studies phyll, indicative of photoinhibitory damage. When the cy- using in vitro systems. A particularly useful experimental tochrome is fully reduced and excess Mn2+ is present, the effect system has been the isolated reaction center of PSII. This of moderate light is to facilitate the photoaccumulation of complex consists of the Dl and D2 (products of the reduced pheophytin. The dark reoxidation of the reduced psbA and psbD genes, respectively), the a and p apoproteins cytochrome is very slow under anaerobic conditions but sig- of cyt b559, and the product of the psbl gene (3, 14). This nificantly speeded up on addition of oxidized 2,5-dibromo-3- isolated reaction center does not contain the secondary methyl-6-isopropyl-p-benzoquinone. From these results it is quinone electron acceptors QA and QB and therefore is suggested that the low potential form of cytochrome b5sg can restricted in its photochemical activities to radical pair for- accept electrons directly from reduced pheophytin and in so mation and recombination (15-17). Addition of exogenous doing help to protect the reaction center against acceptor side electron donors and acceptors, however, allows secondary photoinhibition as suggested by Nedbal et al. [Nedbal, J., electron flow reactions to occur (14). Of relevance to the Samson, G. & Whitmarsh, J. (1992)Proc. Natl. Acad. Sci. USA ideas presented in this present paper was the finding that both 89, 7929-7933]. This conclusion has been incorporated into a acceptor and donor side photoinhibition can occur in this model that further suggests that in its high potential form the the cytochrome primarily acts to protect against donor side pho- relatively simple system. When no additions are made, toinhibition due to increased lifetime of highly oxidized species acceptor side mechanism occurs since singlet oxygen is as previously proposed by Thompson and Brudvig [Thompson, formed as a consequence of radical pair recombination (18). L. & Brudvig, G. W. (1988) Biochemistry 27, 6653-6658]. The The generation of this highly toxic species causes initially a particular feature of our scheme is that it incorporates revers- selective and irreversible bleaching of the that ible interconversion between the two redox forms so as to constitute P680 (ref. 19) and a breakdown of the Dl protect against either type of photoinhibition. to a 23-kDa fragment containing the N terminus of the complete protein (20). This fragment is possibly the same as et al. and seems to Cytochrome b559 (cyt b559) is closely associated with the that observed in vivo by Greenberg (21) reaction center of photosystem II (PSII) as judged from be due to a proteolytic cleavage in the loop joining the kinetic (1, 2), structural (3-5), and molecular biological (6) putative transmembrane helical segments four and five in the studies. Ofthe many different suggestions for its function, the region of the QB binding site. If, however, an electron most popular is that it helps to protect PSII against photo- acceptor is present, the P680+ lifetime is increased, and induced damage (7). In this paper we show that cyt b559 can irreversible bleaching of carotenoid and occurs, be directly reduced by pheophytin (Pheo) and propose a which is independent of oxygen (19). Under these conditions scheme by which cyt b559 could prevent photoinhibitory the donor side mechanism prevails, and breakdown ofthe Dl damage of PSII due either to "acceptor" or "donor" side protein leads to a 24-kDa fragment of C-terminal origin (22, events (8). The proposed scheme requires cyt b559 to be 23). The cleavage site in this case is likely to be on the lumenal reversibly convertible between its high-potential (Em +400 side of the complex, probably in the loop joining transmem- mV) and low-potential (E = +60-80 mV) forms. brane segments one and two. Similar conclusions have been An understanding of the distinction between donor and drawn from experiments utilizing isolated oxygen-evolving acceptor side photoinhibition is relatively new (8). Donor side PSII cores (24). photoinhibition occurs when the rate of electrons leaving the There are many reports showing that the high-potential PSII reaction center is greater than the rate of donation. form of cyt b559 (cyt b559Hp) can be converted to a low- Consequently the lifetime of the primary electron donor potential form of cyt b559 (cyt b559Lp) (25). This conversion is P680+, and other secondary oxidized species, increases. The damage incurred by this effect is thought to be due to the high Abbreviations: cyt b559, cytochrome b559; cyt b559Hp and cyt b559Lp, high- and low-potential forms of cyt b559; DBMIB, 2,5-dibromo-3- methyl-6-isopropyl-p-benzoquinone; Pheo, pheophytin; PSII, pho- The publication costs ofthis article were defrayed in part by page charge tosystem II. payment. This article must therefore be hereby marked "advertisement" *Present address: Department of Biochemistry, Faculty of Science, in accordance with 18 U.S.C. §1734 solely to indicate this fact. University of the Basque Country, P.O. Box 644, Bilbao, Spain. 10942 Downloaded by guest on October 2, 2021 Biochemistry: Barber and De Las Rivas Proc. Natl. Acad. Sci. USA 90 (1993) 10943 readily observed when PSII particles are isolated by using Absorption spectra were measured at 10°C in a dual beam detergents (e.g., ref. 26). But the conversion also occurs spectrophotometer (Aminco, SLM Instrument; model no. when isolated membranes are subjected to various treat- DW2000) set with a bandwidth of2 nm. Where indicated, the ments not involving detergents, such as exposure to photo- reaction center solutions were preilluminated with heat- inhibitory light (27) or removal ofthe extrinsic proteins ofthe filtered white light with intensities and times as described in water-splitting complex (e.g., ref. 28). Importantly it has been the figure legends. Photoinduced absorbance changes at shown that cyt b559Lp can be converted back to cyt b559Hp by, different wavelengths (559, 450, and 422 nm) were measured for example, the addition ofcertain lipids (29) or rebinding of with the same spectrophotometer by using side excitation of extrinsic PSII proteins (30). There is also good evidence that a 1-ml, 1-cm path length sample cuvette with heat-filtered red cyt b559Hp and cyt b559Lp exist in vivo, and indeed the light (RG660 filter) and with the photomultiplier shielded by interconversion of the two redox states has formed the basis a complementary blue filter (Coming 4-96). Light-minus-dark of functional models proposed to explain the water-splitting difference spectra were obtained by automatic scanning after reaction (31) or in relation to a redox link between PSII and giving a particular exposure to illumination. PSI (32). Within the isolated reaction center, cyt b559 exists mainly Quantification of the level of cyt b559 in reaction center in its low potential form. Because the ambient redox potential samples was carried out by absorption spectroscopy after the is usually in the region of 100-150 mV, the cytochrome addition of 1-2 mM ferricyanide in the dark, followed by the normally exists in its oxidized state. However, in the pres- addition ofa few grains of sodium dithionite (-2 mg/ml). The ence of added quinones and under aerobic conditions, it was dithionite-minus-ferricyanide difference spectrum, from 600 shown that it could be photoreduced (33, 34). In this paper we to 500 nm, was obtained, and the AA was measured at 559 nm. show that cyt bs59Lp within the isolated reaction center can be directly reduced by Pheo and present a model for the role of RESULTS cyt b559 as a protectant for both acceptor and donor side photoinhibition. Fig. 1 shows the absorption spectra of isolated reaction centers that had been preilluminated with white light minus the dark control. The light-minus-dark difference spectra MATERIALS AND METHODS were recorded at various times during the illumination period The reaction center of PSII was isolated from pea (Pisum and are characterized by two main positive bands at 430 and sativum) according to the method given in Chapman et al. 559 nm that are typical of cyt b559 reduction. The differences (33). The chlorophyll concentration of the reaction center in the spectra shown in Fig. 1 are due to the presence of solution used was 4 pg/ml, calculated by the method of Mn2+, which, in particular, distorted the AA at about 410 nm. Arnon (35). The samples were suspended in 50 mM Mes- The kinetics of the photoreduction of cyt b559 followed a NaOH (pH 6.0) containing 2 mM dodecyl maltoside and 0.2 monoexponential curve with a rate constant estimated to be M sucrose. For preillumination treatments, the samples were 0.08 sec-1. It was also found that there was no significant maintained at 10°C with a thermally controlled cuvette difference between the reduction rates in the presence or holder. Anaerobic conditions in the samples were obtained absence of Mn2+ when strong intensity illumination was by adding glucose oxidase (0.1 mg/ml) and catalase (0.1 used. This indicates that under anaerobic conditions the mg/ml) plus 5 mM glucose and incubating for 5 min before reaction center can provide an electron to reduce the cy- measurements or by extensive degassing of all the solutions tochrome without being damaged (note that in Fig. 1 no with oxygen-free nitrogen and sealing of the sample cuvette photobleaching of pigments was observed, suggesting that in a stream of nitrogen. the electron donor was neither chlorophyll nor 1-carotene). ,4min -2 min --1 min

-0.5 min 5

x

0

l0

03

400 500 600 400 500 600 Wavelength, nm FIG. 1. Absorption difference spectra of the illuminated reaction centers minus the dark control. Illumination was with white light [800 ,uE*m-2.s-1; 1 einstein (E) = 1 mol of photons] for 0.5, 1, 2, and 4 min, in the presence of 0.5 mM MnCl2 as electron donor (A) and in absence of MnCl2 (B). The experiments were carried out under anaerobic conditions using a glucose/glucose oxidase trap (see Materials and Methods). The chlorophyll concentration was 4 Ag/ml. Downloaded by guest on October 2, 2021 10944 Biochemistry: Barber and De Las Rivas Proc. Natl. Acad Sci. USA 90 (1993) and thus decreasing the probability ofdamage by radical pair recombination (19). The photochemical activity of samples in which reduced cyt b559 had been photoaccumulated by exposure to actinic light under anaerobic conditions was measured. First, in dark pretreated samples, the actinic light induced the reduction of cytochrome, indicated by an increase of AA at 559 nm (Fig. x 3A). In these samples no chromatic change was detected when monitored at 450 nm. After a 5-min illumination, all the .0 cyt b559 was reduced, and then, in these light-treated samples, 0 we observed the reversible photoinduced reduction of Pheo (Fig. 3B). The anion radical of Pheo (Pheo-) is spectrally characterized in Fig. 3B by the reversible light-induced positive signals at 450 nm and negative at 422 nm (3, 43) and -1 4 min,," by the light-minus-dark difference spectrum presented in Fig. 3C. This difference spectrum is similar to that reported F 1 680 previously (14) and is not distorted by irreversible bleaching 500 600 700 of chlorophyll since the intensity of the actinic light was Wavelength, nm significantly lower than that used to obtain the data of Fig. 2 ~~~~~~~~ and also because only the reversible difference spectrum has FIG. 2. Absorption difference spectra of5minKthe illuminated reaction been recorded. This result indicates that when all the cyt b559 centers minus the dark control. Illumination was with white light is reduced in the reaction center, a reversible photoaccumu- (1500 ,E.m-2 s-1) for 2, 3, 4, and 5 min in the presence of 0.5 mM lation of reduced Pheo can occur. This photoinduced reduc- MnCl2 as electron donor and in anaerobic conditions. The chloro- tion of Pheo was maximum in the presence of Mn2+ but did phyll concentration of the light-treated samples was 4 pg/ml. occur to a lesser extent without added electron donor. The A possible donor could be an amino acid residue such as quantification of this optical change observed at 450 nm and tyrosine or (36-39). measured with excess Mn2+ present indicated that the pho- A comparison of the maximum extent of the light-induced toaccumulation of reduced Pheo occurs in 80% of the reac- reduction of cyt b559 with its chemically induced reduction in tion centers [considering that one Pheo is reduced in each reaction center and using the e at 450 nm of 8.3 mM-1'cm-1, the dark (dithionite-minus-ferricyanide) gave practically the calculated from Nanba and Satoh (3)]. same AA559. The absorbance change corresponded to the The data presented so far imply that the photoreduction of reduction of 1 mol ofcyt b559 for every 6.5 mol ofchlorophyll cyt b559 is irreversible, since the difference spectra shown in when the e at 559 nm was taken as 17.5 mM-1 cm-1 (40). Fig. 1 were stable in the dark, at least over a period longer Considering that every reaction center contains one cy- than 10 min. If, however, 100 ,M oxidized 2,5-dibromo-3- tochrome and six chlorophyll (41, 42), the results methyl-6-isopropyl-p-benzoquinone (DBMIB) was intro- above indicate that the photoaccumulation of reduced cyt duced into the anaerobic suspension, there was rapid reox- b559 occurs in nearly all the reaction centers (i.e., 93% of the idation of the photoaccumulated reduced cytochrome (see centers). Fig. 4). Indeed, when 100 ,uM DBMIB is present during the Fig. 2 shows the irreversible pigment bleaching that occurs preillumination period, no accumulation of reduced cyt b559 when the isolated reaction centers are subjected to strong is observed. illumination for long periods of time (2-5 min). After 2 min, all the cyt b559 was reduced and only then did further illumination provoke bleaching in the red region, peaking at DISCUSSION 680 nm, indicating irreversible damage of P680 and other The discovery by Knaffand Arnon (1) that cyt b559Hp could be chlorophylls (19). This therefore indicates a possible protec- photooxidized at low temperatures when the water-splitting tive role of cyt b559 acting as an electron acceptor from Pheo reactions were inactivated was the first direct evidence that 422 nm 450 nnr

559nm FIG. 3. Kinetics of the light-induced x absorbance changes at 450 and 559 nm of the isolated reaction centers before preil- U.0 ~ ~ ~ ~ ~ J~~A A lumination (A) and after S min of illumi- nation with 800 uE-m-2.s- white light (B). (C) Automatically recorded light- AA 450 nm ( 545 nm minus-dark difference spectrum of the 450nmm reversible changes due to the photore- duction of Pheo. In all cases MnCl2 (0.5 mM) was present, and anaerobic condi- tions were achieved with a glucose/ glucose oxidase trap. The chlorophyll C wasconcentrationwith 100 tEwasm-2s-14 ,Ag/ml.red light.ExcitationLight I on, upward open arrowhead; light off, 500 600 downward filled arrowhead. The bar in- Time, min Wave-length, am dicates an absorbance change of 10-3. Downloaded by guest on October 2, 2021 , Biochemistry: Barber and De Las Rivas Proc. Natl. Acad. Sci. USA 90 (1993) 10945 now seems clear that both donor and acceptor side photoin- hibition can occur (8). Recognition of this fact therefore opens up the question of how cyt b559 could protect either route of photoinduced damage. Thompson and Brudvig (7) have presented a detailed scheme of how cyt b559 could serve x as a protectant against donor side photoinhibition. In this a) scheme, cyt b559Hp is redox poised so as to supply an electron directly or indirectly to P680+ when electron flow from water co 0 oxidation is inadequate. More recently, Nedbal et al. (44) .0 have, from indirect measurements, implied that cyt b559Lp can act as a direct oxidant of reduced Pheo and in so doing, protect PSII against acceptor side photoinhibition. The experiments presented in this paper clearly demon- strate that the light-driven photoreduction of cyt b559 can occur as a consequence of direct electron transfer from 500 600 700 reduced Pheo. Both the photoaccumulation of Pheo-, when Wavelength, nm all the cytochrome is reduced, and the lack of any quinone in FIG. 4. The effect of introducing 100 ,uM DBMIB into an anaer- the isolated reaction center complex support the conclusion obic sample ofPSII reaction centers that had been preilluminated for that the electron donor to cyt b559 is Pheo. Quantification of 5 min with 800 j,E'm-2.s-' white light so as to fully reduce cyt b559. the signal at 559 nm indicates that the cytochrome can be Spectrum a, light-minus-dark difference spectrum before adding reduced in all the reaction centers. We estimate the quantum DBMIB; spectrum b, spectrum after the addition of quinone. yield of this light-induced reduction to be about 0.025 (2.5%) based on 5% absorption of incident light and 100%o efficiency this component is closely associated with the reaction center for primary charge separation. of PSII. Their work was confirmed by others (2) and formed Our results give direct experimental support to the hypoth- the basis of a host of different postulates to describe the esis of Nedbal et al. (44)-that is, the cyt b559Lp can accept functional role of this in PSII activity. Isolation of the electrons from reduced Pheo reasonably efficiently and thus PSII reaction center (3, 14) confirmed that the cytochrome is reduce the risk of damage caused by radical pair recombi- intimately associated with the D1/D2 heterodimer and em- nation (8). The other important result of our work is that the phasized a significant difference in the composition of the reduced cytochrome is not efficiently reoxidized by the donor higher plant system as compared with the reaction center of side of PSII as would be expected if an effective cyclic purple photosynthetic bacteria. Another striking difference process were operative. Nevertheless, such a cycle has between the reaction centers ofPSII and purple bacteria is the frequently been suggested. To explain our observations we high vulnerability ofthe former to photoinhibitory damage and wish to propose a model that recognizes that cyt b559 can exist the remarkably high turnover rate for the Dl protein (8). Thus, in a high and low potential form and that both donor and of the many suggestions as to the function of cyt b559 within acceptor side photoinhibitory processes occur. We also as- PSII, the most popular has implicated a protective role against sume that there is only one cyt b559 per functional PSII in the photoinhibition (e.g., ref. 7). thylakaid membrane for which there is now very strong Our understanding of the molecular processes of photoin- experimental evidence (41, 45). Our model is schematically hibition have advanced considerably in recent years. Much of shown in Fig. 5 in which we propose a reversible change this advancement has come from studies with in vitro systems between high and low potential forms of cyt b559 with the ranging from isolated thylakoids (9, 10, 27) and PSII-enriched purpose of protecting against either donor or acceptor side membrane fragments (11, 12) to isolated oxygen-evolving photoinhibition. In its high potential form (Em +400 mV), PSII complexes (24) and PSII reaction centers (20, 22, 23). It cyt b559 is maintained in its reduced state and therefore ideally

v Acceptor side - 0.5V- XImpalrment (A \QB ov . Cytb5Cyt 59LP Ambient T I e FIG. 5. A scheme to emphasize the possible role of pAotentat molecular switch cyt b559 as a one-electron protectant against acceptor or donor side photoinhibition. The model postulates that Cytb559HP in order for the cyt b559 to act either as an electron +O.5V- acceptor (for acceptor side protection) or as an electron donor (for donor side protection) it shifts reversibly between its high and low potential forms, triggered by an unknown molecular switch. The reoxidation of Water reduced cyt b559Lp and the reduction of oxidized cyt b559Hp is suggested to involve electron exchange with P6880 the ambient redox system governed by factors such as + l.OV' Donor side the redox state of the plastoquinone pool and the Impairment presence of molecular oxygen. Downloaded by guest on October 2, 2021 10946 Biochemistry: Barber and De Las Rivas Proc. Natl. Acad Sci. USA 90 (1993) poised to give an electron to long lived, and potentially & Andersson, B. (1992) Proc. Natl. Acad. Sci. USA 89, dangerous, oxidized species on the donor side of PSII. Once 1408-1412. oxidized, the cytochrome can be rereduced by the ambient 13. Vass, I. & Styring, S. (1992) Biochemistry 31, 5957-5963. potential of its surroundings governed, for example, by the 14. Barber, J., Chapman, D. J. & Telfer, A. (1987) FEBS Lett. 220, redox state of the plastoquinone pool. The slowness of the 67-73. rereduction of photooxidized cyt b559 has been noted previ- 15. Takahashi, Y., Hansson, O., Mathis, P. & Satoh, K. (1987) Biochim. Biophys. Acta 893, 49-59. ously (46). However, under conditions when acceptor side 16. Wasielewski, M. R., Johnson, D. G., Seibert, M. & Govindjee, photoinhibition might occur due to complete reduction ofQA (1989) Proc. Natl. Acad. Sci. USA 86, 524-528. and QB (12, 13), cyt b559 could shift its potential to the low 17. Hastings, G., Durrant, J. R., Barber, J., Porter, G. & Klug, potential form. Such a shift in response to strong illumination D. R. (1992) Biochemistry 31, 7638-7647. has already been observed (27) and was faster than the 18. Durrant, J. R., Giorgi, L. B., Barber, J., Klug, D. R. & Porter, subsequent appearance ofphotoinhibitory damage. In its low G. (1990) Biochim. Biophys. Acta 1017, 167-175. potential form, cyt b559 exists in an oxidized state and 19. Telfer, A., De Las Rivas, J. & Barber, J. (1991) Biochim. therefore is ideally poised to act as an electron acceptor. The Biophys. Acta 1060, 106-114. reoxidation of the reduced cyt b559Lp would occur due to the 20. De Las Rivas, J., Shipton, C. A., Ponticos, M. & Barber, J. ambient potential of the system, which again could be gov- (1993) Biochemistry 32, 6944-6950. erned by the redox state of the plastoquinone pool. Indeed 21. Greenberg, B. M., Gaba, V., Mattoo, A. K. & Edelman, M. the experiment presented in Fig. 4 indicates the effectiveness (1987) EMBO J. 6, 2865-2869. 22. Shipton, C. A. & Barber, J. (1991) Proc. Natl. Acad. Sci. USA of DBMIB in oxidizing the photoreduced heme within the 88, 6691-6695. isolated PSII reaction center. 23. Barbato, R., Shipton, C. A., Giacometti, G. M. & Barber, J. The scheme shown in Fig. 5 suggests that the conversion (1991) FEBS Lett. 290, 162-166. between cyt b559Hp and cyt b559Lp is reversible. Although the 24. De Las Rivas, J., Andersson, B. & Barber, J. (1992) FEBS Lett. conversion from high to low potential is well documented, the 301, 246-252. reverse is less commonly observed. However, such a con- 25. Cramer, W. A. & Whitmarsh, J. (1977) Annu. Rev. Plant version does occur in vivo when non-QB PSII complexes, Physiol. 28, 133-172. located in stromal lamellae, are "activated" to become 26. Lam, E., Baltimore, B., Ortiz, W., Chollar, S., Melis, A. & oxygen-evolving QB active PSII complexes in the grana (47). Malkin, R. (1983) Biochim. Biophys. Acta 724, 201-211. The nature of the "molecular switch" that allows this inter- 27. Styring, S., Virgin, I., Ehrenberg, A. & Andersson, B. (1990) Biochim. Biophys. Acta 1015, 269-278. conversion to occur is unknown but is likely to involve a 28. Thompson, L. K., Miller, A.-F., Bruser, C. A., de Paula, J. C. change in the environment of the heme or a minor confor- & Brudvig, G. W. (1989) Biochemistry 28, 8048-8056. mational change of the PSII complex as indicated from in 29. Matsuda, H. & Butler, W. L. (1983) Biochim. Biophys. Acta vitro studies (25-28). One obvious mechanism could involve 725, 320-324. protonation/deprotonation events associated with changes in 30. Larsson, C., Jansson, C., Ljunberg, U., Akerlund, H.-E. & local pH. Although the proposed model needs further exper- Andersson, B. (1983) in Advances in Photosynthesis Research, imental support, we hope that it will give a new framework ed. Sybesma, C. (Nijhoff, Amsterdam) Vol. 1, pp. 363-366. on which to discuss the functional role of cyt b559 in photo- 31. Butler, W. L. (1979) FEBS Lett. 95, 19-25. synthesis. In its final form it will need to reconcile the 32. Ortega, J. M., Hervas, M. & Losada, M. (1989) Biochim. Biophys. Acta 975, 244-251. existence of data that, at first sight, do not easily fit into this 33. Chapman, D. J., Gounaris, K. & Barber, J. (1988) Biochim. model, including the work of Buser et al. (48, 49), which Biophys. Acta 933, 423-431. implicates the QB site in the photoreduction of cyt b559. 34. Satoh, K., Hansson, 0. & Mathis, P. (1988) Biochim. Biophys. Acta 1016, 121-126. We acknowledge the technical support of Caroline Woollin. We 35. Arnon, D. I. (1949) Plant Physiol. 24, 1-15. wish to thank the Agricultural and Food Research Council, The 36. Debus, R. (1992) Biochim. Biophys. Acta 1102, 269-352. Federation of Biochemical Societies, and the Spanish government 37. Vermaas, W. F. J., Rutherford, A. W. & Hansson, 0. (1988) for financial support. Proc. Natl. Acad. Sci. USA 85, 8477-8481. 38. Boussac, A., Zimmermann, J.-L., Rutherford, A. W. & La- 1. Knaff, D. B. & Arnon, D. I. (1969) Proc. Natl. Acad. Sci. USA vergne, J. (1990) Nature (London) 347, 303-306. 63, 956-962. 39. Ono, T.-A. & Inoue, Y. (1992) Biochim. Biophys. Acta 1099, 2. Vermeglio, A. & Mathis, P. (1974) Biochim. Biophys. Acta 368, 185-192. 9-17. 40. Cramer, W. A., Theg, S. M. & Widger, W. R. (1986) Photo- 3. Nanba, 0. & Satoh, K. (1987) Proc. Natl. Acad. Sci. USA 84, synth. Res. 10, 393-403. 109-112. 41. Miyazaki, A., Shina, T., Toyoshima, Y., Gounaris, K. & 4. Barber, J., Gounaris, K. & Chapman, D. J. (1987) in Cy- Barber, J. (1989) Biochim. Biophys. Acta 975, 142-147. tochrome Systems: Molecular Biology and Bioenergetics, eds. 42. Gounaris, K., Chapman, D. J., Booth, P., Crystall, B., Giorgi, Papa, S., Chance, B. & Ernster, L. (Plenum, New York), pp. L. B., Klug, D. R., Porter, G. & Barber, J. (1990) FEBS Lett. 657-666. 265, 88-92. 5. Tang, X.-S., Fushimi, K. & Satoh, K. (1990) FEBS Lett. 273, 43. Klimov, V. V., Allakhverdiev, S. I., Demeter, S. & Kras- 257-260. novskii, A. A. (1989) Dokl. Akad. Nauk. SSSR 249, 227-230. 6. Pakrasi, H. B., Nyhus, K. J. & Granok, H. (1990) Z. Natur- 44. Nedbal, J., Samson, G. & Whitmarsh, J. (1992) Proc. Natl. forsch. C Biosci. 45, 423-429. Acad. Sci. USA 89, 7929-7933. 7. Thompson, L. K. & Brudvig, G. W. (1988) Biochemistry 27, 45. Buser, C. A., Diner, B. A. & Brudvig, G. W. (1992) Biochem- 6653-6658. istry 31, 11441-11448. 8. Barber, J. & Andersson, B. (1992) Trends Biochem. Sci. 17, 46. Yerkes, C. T. & Crofts, A. R. (1984) in Advances in Photo- 61-66. synthesis Research, ed. Sybesma, C. (Nijhoff Junk, The 9. Blubaugh, D. J., Atamian, M., Babcock, G. T., Golbeck, J. H. Hague), Vol. 1, pp. 489-492. & Cheniae, G. M. (1991) Biochemistry 30, 7586-7597. 47. Cox, R. P. & Andersson, B. (1981) Biochem. Biophys. Res. 10. Demeter, S., Neale, P. J. & Melis, A. (1987) FEBS Lett. 241, Commun. 103, 1336-1342. 370-374. 48. Buser, C. A., Diner, B. A. & Brudvig, G. W. (1992) Biochem- 11. Jegerschold, C., Virgin, I. & Styring, S. (1990) Biochemistry 29, istry 31, 11449-11459. 6179-6186. 49. Buser, C. A., Thompson, L. K., Diner, B. A. & Brudvig, 12. Vass, I., Styring, S., Hundal, T., Koivuniemi, A., Aro, E.-M. G. W. (1990) Biochemistry 29, 8977-8985. Downloaded by guest on October 2, 2021