Reversible and irreversible intermediates during photoinhibition of ...

Proc. Natl. Acad. Sci. USA Vol. 89, pp. 1408-1412, February 1992 Biophysics

Reversible and irreversible intermediates during photoinhibition of photosystem II: Stable reduced QA species promote chlorophyll triplet formation (protein turnover/photosynthesis/D1 protein/electron paramanetic resonance)

IMRE VASS*, STENBJORN STYRING, TORILL HUNDAL, ANTTI KOIVUNIEMIt, EVA-MARi AROt, AND BERTIL ANDERSSON Department of Biochemistry, Stockholm University, S-106 91 Stockholm, Sweden

Communicated by Daniel Arnon, October 22, 1991

ABSTRACT 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.

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 long-lived oxidizing species such as P6N+ (primary electron donor) and/or Tyr-Z' (10, 11) and occur in the absence of oxygen (12, 13). The actual degradation is catalyzed by proteolytic activity in the PSII complex (4). Light-induced impairment of PSII electron transport in the absence of oxygen can be restored in the dark without D1 protein turnover (14, 15). This indicates the trapping of reversibly inhibited intermediates during photoinhibition under anaerobic conditions. Here we have investigated the mechanism of light-induced impairment of electron transport and its connection to D1 protein degradation by characterizing such reversible intermediates by fluorescence and electron paramagnetic resonance (EPR) spectroscopy combined with D1 protein analyses.

MATERIALS AND METHODS Spinach thylakoids and PSII membranes were isolated by standard procedures (4). Photoinhibition of photosynthesis under anaerobic conditions was performed in sealed 1-cm fluorescence cuvettes in which the suspension medium had been flushed with argon for 10 min before addition of sample [20 ,jg of chlorophyll (Chl) per ml]. The argon stream was maintained above the stirred solution during illumination with white light of 3000 microeinsteins (,uE) m-2-s-1. The oxygen level was