Photoinhibition of photosynthesis 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 (F(oi)) with typical decay kinetics (fast, semistable, stable, and non-decaying). The fast-decaying state has no bound Q(B) but possesses a single reduced Q(A) Species with a 30-s decay half-time in the dark (Q(B), second quinone acceptor; Q(A), first quinone acceptor). In the semistable state, Q(A)- is stabilized for 2-3 min, most likely by protonation, and gives rise to the Q(A)- Fe2+ EPR signal in the dark. In the stable state, Q(A) 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 Q(A) H-2 has left its binding site. The first three photoinhibitory states are reversible in the dark through reestablishment Of Q(A) to Q(B) electron transfer. Significantly, illumination at 4 K of anaerobically photoinhibited centers trapped in all but the fast state gives rise to a spin-polarized triplet EPR signal from chlorophyll P680 (primary electron donor). When oxygen is introduced during anaerobic illumination, the light-inducible chlorophyll triplet is lost concomitant with induction of DI protein degradation. The results are integrated into a model for the photoinhibitory process involving initial loss of bound Q(B) followed by stable reduction and subsequent loss Of Q(A) facilitating chlorophyll P680 triplet formation. This in turn mediates light-induced formation of highly reactive and damaging singlet oxygen.
