EPR SPECTROSCOPY DETECTION OF ACTIVE OXYGEN AND FREE-RADICALS IN THYLAKOIDS EXPOSED TO PHOTOINHIBITION

High intensity illumination of thylakoids results in the well-characte rized impairment of Photosystem Il 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. In the present study we f ollowed the production of singlet oxygen and free radicals during both types of photoinhibition by EPR spectroscopy. Singlet oxygen was dete cted by following the formation of 2,2,6,6-tetramethylpiperidine-1-oxy l, a stable nitroxide radical yielded in the reaction of singlet oxyge n with the sterically hindered amine 2,2,6,6-tetramethylpiperidine. Fr ee radicals were detected as spin adducts of the spin trap 5,5-dimethy l-1-pyrrolin-N-oxide, and identified on the basis of hyperfine splitti ng constants of the EPR spectre. We found that (i) singlet oxygen, a n on-radical form of active oxygen was detectable only in samples underg oing 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 o f singlet oxygen with histidine residues. (iii) Donor side induced pho toinhibition was dominated by hydroxyl radicals, which were produced i n anaerobic samples, too. The production rate of these radicals, as we ll as D1 protein degradation, was dependent on the possibility of elec tron donation From manganese ions to Photosystem II. The marked distin ction between the active oxygen forms produced in acceptor and donor s ide induced photoinhibition are in agreement with earlier reports on t he different mechanism of these processes.