Quantification of the number of spins in the S-2- and S-3-states of Ca2+-depleted photosystem II by pulsed-EPR spectroscopy

Ca2+-depletion of the photosystem II enzyme by a NaCl-washing in the light inhibits oxygen evolution. In Ca2+-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-II 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.