The S-0 state was generated by incubation of dark-adapted (S-1 state)
photosystem II membranes either with the exogenous two electron reduc
tant hydrazine and subsequent 273 K illumination in the presence of DC
MU or by dark incubation with low amounts of the one electron reductan
t hydroxylamine. In agreement with earlier reports, the S-1 and S-1 st
ates were found to be electron paramagnetic resonance (EPR) silent. Ho
wever, in the presence of 0.5-1.5% methanol, a weak EPR multiline sign
al centered around g = 2.0 was observed at 7 K for the S-0 states gen
erated by both procedures. This signal has a similar average line spli
tting to the well-characterized S-2 state multiline EPR signal, but ca
n 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 b
readth 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 signa
l to the manganese cluster of the oxygen evolving complex in a mixed v
alence state of the form (MnMnMnMnIII)-Mn-II-Mn-III-Mn-III, (MnMnMnMnI
V)-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.