PHOTOPRODUCTION OF HYDROGEN-PEROXIDE IN PHOTOSYSTEM-II MEMBRANE-FRAGMENTS - A COMPARISON OF 4 SIGNALS

The present study describes the formation of different forms of peroxi de in Photosystem II (PS II) by using a chemiluminescence detection te chnique. Four chemiluminescence signals (A, B, C and D) of the luminol -peroxidase (Lu-Per) system, which detects peroxide, are found in illu minated O-2-evolving Photosystem II (PS II) membrane fragments isolate d from spinach. Signal A ('free peroxide') peaking around 0.2-0.3 s af ter mixing PS II membrane fragments with Lu-Per is eliminated by catal ase or removal of oxygen from the suspension and ascribed to O-2 inter action with reduced PS II electron accepters. In contrast, signal B pe aking around 1.5 min remains largely unaffected under anaerobic condit ions, as well as in the presence of catalase (20 mu L/ml). Under flash illumination the extent of this signal exhibits a weak period four os cillation (maximum at third and 7th flash). Its yield increases up to the third flash, but is close to zero in the fourth flash. An analogou s behaviour is observed in flashes 5 to 8. Signal B is ascribed to Lu- Per interaction with the water-oxidizing system being in S-2 and/or S- 3-state. Signal C ('bound peroxide') detected as free peroxide after a cid decomposition of illuminated PS II particles is observed on the 1 st flash and oscillates with period 2 with superposition of period 4. It is evidently related to peroxide either released from S-2 or formed at S-2 upon acid shock treatment. Signal D ('slowly released peroxide ') peaking around 2-3 s after mixing is observed in samples after vari ous treatments (LCC-incubation, washing with 1 M NaCl at pH 8 or with 1 M CaCl2, Cl--depletion) that lead to at least partial removal of the extrinsic proteins of 18, 24 and 33 kDa without Mn extraction. The av erage amplitude of this signal corresponds with a yield of about 0.2 H 2O2 molecules per RC and flash. In a flash train, the extent of signal D exhibits an oscillation pattern with a minimum at the 3rd flash. We assume that these treatments increase the release of 'bound' peroxide (upon injection into the Lu-Per assay) either formed in the normal ox idative pathway of the water oxidase in the S-2 or the S-3-state or gi ve rise to peroxide formation due to higher accessibility of the Mn-cl uster to water molecules.