Detection of an EPR multiline signal for the S-0 state in photosystem II

The S-0* state was generated by incubation of dark-adapted (S-1 state) photosystem II membranes either with the exogenous two electron reductant hydrazine and subsequent 273 K illumination in the presence of DCMU or by dark incubation with low amounts of the one electron reductant hydroxylamine. In agreement with earlier reports, the S-1 and S-1 states were found to be electron paramagnetic resonance (EPR) silent. However, in the presence of 0.5-1.5% methanol, a weak EPR multiline signal centered around g = 2.0 was observed at 7 K for the S-0* states generated by both procedures. This signal has a similar average line splitting to the well-characterized S-2 state multiline EPR signal, but can be clearly distinguished from that and other modified S-2 multiline signals by differences in line position and intensities. In addition, at 4 K it can be seen that the S-0* multiline has a greater spectral breadth than the S-2 multilines and is composed of up to 26 peaks. The S-0* signal is not seen in the absence of methanol and is not affected by 1 mM EDTA in the buffer medium. We assign the S-0* multiline signal to the manganese cluster of the oxygen evolving complex in a mixed valence state of the form (MnMnMnMnIII)-Mn-II-Mn-III-Mn-III, (MnMnMnMnIV)-Mn-II-Mn-III-Mn-IV, or (MnMnMnMnIV)-Mn-III-Mn-III-Mn-III. Addition of methanol may be helpful in future to find an EPR signal originating from the natural S-0 state.