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Photoinhibition ofphotosynthesis was studied in isolated photosystem II membranes by using chlorophyll fluorescence and electron paramagnetic resonance (EPR) spectroscopy combined with protein analysis. Under anaerobic conditions four sequentially intermediate steps in the photoinhibitory process were identified and characterized. These intermediates show high dark chlorophyll fluorescence (Fo) with typical decay kinetics (fast, semistable, stable, and nondecaying). The fast-decaying state has no bound Q. but possesses a single reduced QA species with a 30-s decay half-time in the dark (QB, second quinone acceptor; QA, first quinone acceptor). In the semistable state, Q-is stabilized for 2-3 min most likely by protonation, and gives rise to the Q-Fe2' EPR signal in the dark. In the stable state, QA has become double reduced and is stabilized for 0.5-2 hr by protonation and a protein conformational change. The final, nondecaying state is likely to represent centers where QA H2 has left its binding site. The first three photoinhibitory states are reversible in the dark through reestablishment of QA to QB electron transfer. Significantiy, illumination at 4 K of anaerobically photoinhibited centers trapped in all but the fast state gives rise to a spinpolarized triplet EPR signal from chlorophyll Pawl (primary electron donor). When oxygen is introduced during anaerobic illumination, the light-inducible chlorophyll triplet is lost concomitant with induction of D1 protein degradation. The results are integrated into a model for the photoinhibitory process involving initial loss of bound QuB followed by stable reduction and subsequent loss of QA facilitating chlorophyll P6w, triplet formation. This in turn mediates lght-induced formation of highly reactive and damaging singlet oxygen.Oxidative damage to proteins is a normal consequence of a variety ofbiochemical reactions (1). The repair often requires degradation and resynthesis of the damaged proteins. Such protein turnover is known to occur for one protein in plant chloroplasts under conditions of high light stress (2-4). This protein, designated D1, is one of the reaction center subunits of photosystem II (PSII) that catalyzes the important watersplitting reaction (4). The turnover of the D1 protein is an event subsequent to the photoinactivation of PSII electron transport that occurs at high light intensities. There is at present no consensus with respect to the molecular mechanism behind these photoinactivation and protein-damaging events in plants normally referred to as photoinhibition.The first inactivation of PSII electron transport has been proposed to be targeted to the primary (QA) (4,5) or the secondary (QB) quinone acceptor (2,3,6) or the primary charge separation (7). The photoinactivation is thought to result in damage to the D1 protein, which thereby becomes triggered for degradation. This damage has been suggested to include formation of toxic oxygen species (8, 9). Under certain conditions, D1 protein degradation may be triggered by lo...

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An immunological approach using a polyclonal phosphothreonine antibody is introduced for the analysis of thylakoid protein phosphorylation in vivo. Virtually the same photosystem II (PSII) core phosphoproteins (D1, D2, CP43, and the psbH gene product) and the lightharvesting chlorophyll a/b complex II (LHCII) phosphopolypeptides (LHCB1 and LHCB2), as earlier identified by radiolabeling experiments, were recognized in both pumpkin and spinach leaves. Notably, the PSII core proteins and LHCII polypeptides were found to have a different phosphorylation pattern in vivo with respect to increasing irradiance. Phosphorylation of the PSII core proteins in leaf discs attained the saturation level at the growth light intensity, and this level was also maintained at high irradiances. Maximal phosphorylation of LHCII polypeptides only occurred at low light intensities, far below the growth irradiance, and then drastically decreased at higher irradiances. These observations are at variance with traditional studies in vitro, where LHCII shows a light-dependent increase in phosphorylation, which is maintained even at high irradiances. Only a slow restoration of the phosphorylation capacity for LHCII polypeptides at the low light conditions occurred in vivo after the high light-induced inactivation. Furthermore, if thylakoid membranes were isolated from the high light-inactivated leaves, no restoration of LHCII phosphorylation took place in vitro. However, both the high light-induced inactivation and low light-induced restoration of LHCII phosphorylation seen in vivo could be mimicked in isolated thylakoid membranes by incubating with reduced and oxidized dithiothreitol, respectively. We propose that stromal components are involved in the regulation of LHCII phosphorylation in vivo, and inhibition of LHCII phosphorylation under increasing irradiance results from reduction of the thiol groups in the LHCII kinase. Photosystem (PS)1 II is a multiprotein complex of the thyla- (14) protein phosphatases, active in dephosphorylation of thylakoid phosphoproteins, are reported to be present in plant chloroplasts. Distinct physiological roles have been established for the phosphorylation of LHCII polypeptides, being implicated in the regulation of excitation energy distribution between PSII and PSI (1, 2), and in the protection of PSII against photoinhibition (15,16). Under conditions where PSII becomes more excited than PSI, the redox-controlled kinase is activated and phosphorylates LHCII polypeptides. The outer LHCII subcomplex, consisting of LHCB1 and LHCB2 proteins (see, e.g., Ref. 17), then detaches from the PSII complex and migrates out from the appressed region of the thylakoid membrane, thereby reducing the excitation of PSII (1). Dephosphorylation of this mobile LHCII subcomplex allows its subsequent migration back to the stacked grana region and reassociation with PSII (16).The only function so far established for reversible phosphorylation of PSII core proteins is the regulation of the degradation of photodamaged D1 protein ...

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The photosystem II reaction center D1 protein is known to turn over frequently. This protein is prone to irreversible damage caused by reactive oxygen species that are formed in the light; the damaged, nonfunctional D1 protein is degraded and replaced by a new copy. However, the proteases responsible for D1 protein degradation remain unknown. In this study, we investigate the possible role of the FtsH protease, an ATP-dependent zinc metalloprotease, during this process. The primary light-induced cleavage product of the D1 protein, a 23-kD fragment, was found to be degraded in isolated thylakoids in the dark during a process dependent on ATP hydrolysis and divalent metal ions, suggesting the involvement of FtsH. Purified FtsH degraded the 23-kD D1 fragment present in isolated photosystem II core complexes, as well as that in thylakoid membranes depleted of endogenous FtsH. In this study, we definitively identify the chloroplast protease acting on the D1 protein during its light-induced turnover. Unlike previously identified membrane-bound substrates for FtsH in bacteria and mitochondria, the 23-kD D1 fragment represents a novel class of FtsH substratefunctionally assembled proteins that have undergone irreversible photooxidative damage and cleavage. INTRODUCTIONProteins that are rendered nonfunctional due to interactions with reactive oxygen species or free radicals undergo proteolysis and are replaced by newly synthesized copies. This is particularly significant in the chloroplast thylakoid membrane. Here, enzymes operate in a highly oxidizing environment and therefore are susceptible to impairment of structure and function. Within the thylakoid membrane, photosystem II (PSII) is the component most sensitive to oxidative damage. This sensitivity is partially due to its function in water splitting, a reaction that requires an oxidizing potential of 1.1 V, but it is also due to the intrinsic formation by PSII of toxic oxygen species (Andersson and Barber, 1994). PSII is a large multisubunit protein complex integral to the thylakoid membrane (Andersson and Barber, 1994). Its reaction center contains the homologous D1 and D2 proteins, PsbI, PsbW (in which Psb stands for PSII, and I and W denote specific subunits), and cytochrome b 559 . The D1/D2 heterodimer binds all of the chlorophylls, quinones, and metal ligands necessary to perform primary PSII photochemistry and electron transport. The structure of the PSII reaction center recently has been determined at a resolution of 8 Å (Rhee et al., 1998), and the structure of the dimeric PSII core complex has been set at a resolution of ‫ف‬ 9 Å (Hankamer et al., 1999).Under conditions of high light intensity, electron transport within the complex is arrested, and consequently, the photosynthetic process is inactivated. This phenomenon is known as photoinhibition (Barber and Andersson, 1992;Prasil et al., 1992). The process is thought to occur via overreduction of the acceptor side of PSII, chlorophyll triplet formation, and production of toxic singlet oxygen (Vass et a...

Redox-controlled phosphorylation of thylakoid membrane proteins represents a unique system for the regulation of light energy utilization in photosynthesis. The molecular mechanisms for this process remain unknown, but current views suggest that the plastoquinone pool directly controls the activation of the kinase. On the basis of enzyme activation by a pH shift in the darkness combined with f lash photolysis, EPR, and optical spectroscopy we propose that activation occurs when plastoquinol occupies the quinoloxidation (Qo) site of the cytochrome bf complex, having its high-potential path components in a reduced state. A linear correlation between kinase activation and accessibility of the Qo site to plastoquinol was established by quantification of the shift in the g y EPR signal of the Rieske Fe-S center resulting from displacement of the Qo-site plastoquinol by a quinone analog. Activity persists as long as one plastoquinol per cytochrome bf is still available. Withdrawal of one electron from this plastoquinol after a single-turnover f lash exciting photosystem I leads to deactivation of the kinase parallel with a decrease in the g z EPR signal of the reduced Rieske Fe-S center. Cytochrome f, plastocyanin, and P 700 are rereduced after the f lash, indicating that the plastoquinol at the Qo site is limiting in maintaining the kinase activity. These results give direct evidence for a functional cytochrome bf-kinase interaction, analogous to a signal transduction system where the cytochrome bf is the receptor and the ligand is the plastoquinol at the Qo site.Light is not only the source for photosynthetic energy conversion but also an essential regulatory factor in plant signal transduction (1-5). The light-induced protein kinase activity of the chloroplast thylakoid membrane (1, 6, 7) connects light quality and intensity with the versatility of regulatory protein phosphorylation. This reversible post-translational modification regulates the balance of excitation energy between the two photosystems by means of phosphorylation of the lightharvesting chlorophyll (Chl) a͞b-protein complex (LHC)II (7, 8), optimizing photosynthetic efficiency under ever-changing irradiance. The ensuing regulation of the reducing power produced by photosynthetic electron transfer controls the translation of key chloroplast-encoded proteins (3, 4). Moreover, phosphorylation regulates light-stress-induced turnover of photosystem (PS)II reaction center proteins (9-11). The redox-controlled signaling system for regulation of nucleusencoded chloroplast cab gene expression was also proposed to be initiated by thylakoid protein phosphorylation (5).Despite numerous studies aimed at the elucidation of this unique light-induced protein phosphorylation process, our understanding of the mechanism connecting light with kinase activation is still very limited. The activation͞deactivation of the enzyme has been correlated to the redox state of the plastoquinone pool (7, 12), which depends on the excitation and electron flow of PSII relative ...