Quantification of the number of spins in the S 2 - and S 3 -states of Ca 2+ -depleted photosystem II by pulsed-EPR spectroscopy

Ca 2+ -depletion of the photosystem II enzyme by a NaCl-washing in the light inhibits oxygen evolution. In Ca 2+ -depleted photosystem II the S 3 charge storage state exhibits a split EPR signal attributed to the magnetic interaction between a radical and the Mn-cluster. Further treatment of photosystem II by EGTA modifies the shape of the EPR signal of the Mn-cluster in the S 2 charge storage state. The percentage of centers in which the S 2 modified signal and the split S 3 signal can be observed has been estimated by using pulsed-EPR spectroscopy. On the basis of one tyrosine D radical per reaction center, the field-swept spin echo spectrum of the modified S 2 state in dark-adapted photosystem II was detected in a large majority of the reaction centers. The derivative of the S 2 field-swept spectrum with respect to the magnetic field resulted in a spectrum similar to that observed by cw-EPR. The additional light-induced split S 3 signal appeared on top of the envelope of the S 2 signal and was detected in the same proportion of centers as that which exhibited the S 2 signal prior to the illumination. In the formal S 3 state, the hyperfine lines of the Mn field-swept echo spectrum were no longer detectable. The storage of PS-11 at 77 K after formation of the S 3 Q A − state by freezing the membranes under continuous illumination resulted in a decrease of the S 3 signal but the pulsed-EPR S 2 manganese signal was conserved.