An improved procedure for photoactivation of photosynthetic oxygen evolution: Effect of artificial electron acceptors on the photoactivation yield of NH2OH-treated wheat Photosystem II membranes

The conditions for photoactivation of oxygen evolution in Mn-depleted wheat Photosystem II (PS II) membranes reported by Tamura and Cheniae (Tamura, N. and Cheniae, G. (1987) Biochim. Biophys. Acta 890, 179–194) were examined in more detail. Treatment with 1.0 mM NH 2 OH of wheat PS II membranes lacking the extrinsic 23 kDa and 16 kDa proteins removed almost all the Mn atoms from the PS II complex and reduced the oxygen-evolving activity to less than 5% of that before the treatment. About a half of the lost activity was restored by a 60 min incubation of the treated membranes with supplements of 1.0 mM Mn 2+ /20 mM Ca 2+ /150 mM Cl − /100 μM 2,6-dichlorophenolindophenol (DCIP) under weak light illumination according to the protocol of Tamura and Cheniae. On lowering the DCIP concentration during illumination, the rate of photoactivation was accelerated and a much higher activity was restored: with 5–20 μM DCIP the activity restoration reached about 80% of the original level in 15 min. Dependence of photoactivation yield by repetitive flashes on flash interval indicated that a low concentration (5 μM) of DCIP increased the yield without affecting the rates of light-driven formation and decay of the intermediate state, while DCIP above 5 μM specifically accelerated its decay, resulting in a lower yield. Phenyl- p -benzoquinone at 0.4 mM also stimulated the flash photoactivation yield, but the maximum activity restoration under continuous illumination was at most 50% of the original. This limited restoration is mainly due to light-induced inactivation of photoactivated centers in the presence of the quinone. Characteristics of PS II electron acceptors required for photoactivation are discussed.