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 NH₂OH 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 Mn²⁺/20 mM Ca²⁺/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.