The photogeneration of hydroxyl radicals (OH(*)) in photosystem II (PSII) membranes was studied using EPR spin-trapping spectroscopy. Two kinetically distinguishable phases in the formation of the spin trap-hydroxyl (POBN-OH) adduct EPR signal were observed: the first phase (t(1/2) = 7.5 min) and the second phase (t(1/2) = 30 min). The generation of OH(*) was found to be suppressed in the absence of the Mn-complex, but it was restored after readdition of an artificial electron donor (DPC). Hydroxyl radical generation was also lost in the absence of oxygen, whereas it was stimulated when the oxygen concentration was increased. The production of OH(*) during the first kinetic phase was sensitive to the presence of SOD, whereas catalase and EDTA diminished the production of OH(*) during the second kinetic phase. The POBN-OH adduct EPR signal during the first phase exhibits a similar pH-dependence as the ability to oxidize the non-heme iron, as monitored by the Fe(3+) (g = 8) EPR signal: both EPR signals gradually decreased as the pH value was lowered below pH 6.5 and were absent at pH 5. Sodium formate decreases the production of OH(*) in intact and Mn-deleted PSII membranes. Upon illumination of PSII membranes, both superoxide, as measured by EPR signal from the spin trap-superoxide (EMPO-OOH) adduct, and H(2)O(2), measured colormetrically, were generated. These results indicated that OH(*) is produced on the electron acceptor side of PSII by two different routes, (1) O(2)(*)(-), which is generated by oxygen reduction on the acceptor side of PSII, interacts with a PSII metal center, probably the non-heme iron, to form an iron-peroxide species that is further reduced to OH(*) by an electron from PSII, presumably via Q(A)(-), and (2) O(2)(*)(-) dismutates to form free H(2)O(2) that is then reduced to OH(*) via the Fenton reaction in the presence of metal ions, the most likely being Mn(2+) and Fe(2+) released from photodamaged PSII. The two different routes of OH(*) generation are discussed in the context of photoinhibition.

Mixed photosystem II (PSII) samples consisting of Cl(-)-depleted and active, or Ca(2+)-depleted and active PSII enriched membrane fragments, respectively, were investigated with respect to their susceptibility to light. In the presence of Cl(-)-depleted PSII, active centers were damaged more severely, most likely caused by a higher amount of reactive oxygen species formed in the nonfunctional centers. Cl(-) depletion led to an increased H(2)O(2) production, which seemed to be responsible for the stimulation of PSII activity loss. To distinguish between direct H(2)O(2) formation by partial water oxidation and indirect H(2)O(2) formation by oxygen reduction involving the prior formation of O(2)(-?), the production of reactive oxygen species was followed by spin trapping EPR spectroscopy. All samples investigated, i.e. PSII with a functional water splitting complex, Ca(2+)- and Cl(-)-depleted PSII, produced upon illumination O(2)(-?) and OH(?) radicals on the acceptor side, while Cl(-)-depleted PSII produced additionally OH(?) radicals originating from H(2)O(2) formed on the donor side of PSII.