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A Functional Model for the Role of Cytochrome B559 in the Protection

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A Functional Model for the Role of Cytochrome B559 in the Protection Proc. Natl. Acad. Sci. USA Vol. 90, pp. 10942-10946, December 1993 Biochemistry A functional model for the role of cytochrome 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 water-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 redox 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 oxygen 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 proteins (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 chlorophylls that ible interconversion between the two redox forms so as to constitute P680 (ref. 19) and a breakdown of the Dl protein 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 chlorophyll 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.
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