Photoproduction of hydrogen peroxide in Photosystem II membrane fragments: A comparison of four signals

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Abstract
The present study describes the formation of different forms of peroxide in Photosystem II (PS II) by using a chemiluminescence detection technique. Four chemiluminescence signals (A, B, C and D) of the luminolperoxidase (Lu-Per) system, which detects peroxide, are found in illuminated O2-evolving Photosystem II (PS II) membrane fragments isolated from spinach. Signal A (‘free peroxide’) peaking around 0.2–0.3 s after mixing PS II membrane fragments with Lu-Per is eliminated by catalase or removal of oxygen from the suspension and ascribed to O2 interaction with reduced PS II electron acceptors. In contrast, signal B peaking around 1.5 min remains largely unaffected under anaerobic conditions, as well as in the presence of catalase (20 μg/ml). Under flash illumination the extent of this signal exhibits a weak period four oscillation (maximum at third and 7th flash). Its yield increases up to the third flash, but is close to zero in the fourth flash. An analogous behaviour is observed in flashes 5 to 8. Signal B is ascribed to Lu-Per interaction with the water-oxidizing system being in S2 and/or S3-state. Signal C (‘bound peroxide’) detected as free peroxide after acid 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 S2 or formed at S2 upon acid shock treatment. Signal D (‘slowly released peroxide’) peaking around 2–3 s after mixing is observed in samples after various 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 average amplitude of this signal corresponds with a yield of about 0.2 H2O2 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 oxidative pathway of the water oxidase in the S2 or the S3-state or give rise to peroxide formation due to higher accessibility of the Mn-cluster to water molecules.