SINGLET OXYGEN AND FREE-RADICAL PRODUCTION DURING ACCEPTOR-INDUCED AND DONOR-SIDE-INDUCED PHOTOINHIBITION - STUDIES WITH SPIN-TRAPPING EPR SPECTROSCOPY

High-intensity illumination of thylakoids results in the well-characterized impairment of Photosystem II electron transport (photoinhibition), followed by the degradation of the D1 reaction centre protein. The time-course and features of photodamage are different in fully functional thylakoid membranes, when photoinhibition is invoked by impairment of Photosystem II acceptor side electron transport, and in thylakoids which are unable to oxidize water, when the damage is a consequence of inactivation of Photosystem II donor side (reviewed by Aro, E.-M., Virgin, I. and Andersson, B. (1993) Biochim. Biophys. Acta 1134, 113-134). In the present study we followed the production of singlet oxygen and free radicals during both types of photoinhibition by EPR spectroscopy. Singlet oxygen was detected by following the formation of 2,2,6,6-tetramethylpiperidine-1-oxyl, a stable nitroxide radical yielded in the reaction of singlet oxygen with the sterically hindered amine 2,2,6,6-tetramethylpiperidine. Free radicals were detected as spin adducts of the spin trap 5,5-dimethyl-1-pyrroline N-oxide, and identified on the basis of hyperfine splitting constants of the EPR spectra. We found the following. (i) Singlet oxygen, a non-radical form of active oxygen, was detectable only in samples undergoing acceptor-side-induced photodamage. (ii) The acceptor-side-induced process was accompanied by the oxygen dependent production of carbon centred (alkyl or hydroxyalkyl) radicals, probably from the reaction of singlet oxygen with histidine residues. (iii) Donor-side-induced photoinhibition was dominated by hydroxyl radicals, which were produced in anaerobic samples, too. The production rate of these radicals, as well as D1 protein degradation, was dependent on the possibility of electron donation from manganese ions to Photosystem II. The marked distinction between the active oxygen forms produced in acceptor- and donor-side-induced photoinhibition are in agreement with earlier reports on the different mechanism of these processes.