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-characte rized 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 functio nal 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 o f inactivation of Photosystem II donor side (reviewed by Aro, E.-M., V irgin, I. and Andersson, B. (1993) Biochim. Biophys. Acta 1134, 113-13 4). In the present study we followed the production of singlet oxygen and free radicals during both types of photoinhibition by EPR spectros copy. 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 foun d the following. (i) Singlet oxygen, a non-radical form of active oxyg en, was detectable only in samples undergoing acceptor-side-induced ph otodamage. (ii) The acceptor-side-induced process was accompanied by t he oxygen dependent production of carbon centred (alkyl or hydroxyalky l) radicals, probably from the reaction of singlet oxygen with histidi ne 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 oxyg en forms produced in acceptor- and donor-side-induced photoinhibition are in agreement with earlier reports on the different mechanism of th ese processes.