Effect of Hydroxylamine on Photosystem II: Reinvestigation of Electron Paramagnetic Resonance Characteristics Reveals Possible S State Intermediates

Previous work in many laboratories has established that hydroxylamine reduces the S(1) state of the water oxidizing complex (WOC) in one-electron steps. Significant levels of what can now be defined as the S(-1)* state are achieved by specific (concentration and incubation length) hydroxylamine treatments. This state has already been studied by electron paramagnetic resonance spectrometry (EPR), and unusual EPR signals were noted (for example, see Sivaraja, M., and Dismukes, G. C. (1988) Biochemistry 27, 3467-3475). We have now reinvestigated these initial experiments and confirmed many of the original observations. We then utilized more recent EPR markers for the S(0) and S(1) states to further explore the S(-1)* state. The broad radical "split" type EPR signal, produced by 200 K illumination of samples prepared to give a high yield of the S(-1)* state, is shown to most likely reflect a trapped intermediate state between S(-1)* and S(0)*, since samples where this signal is present can be warmed in the dark to produce S(0)*. The threshold for advancement from S(-1)* to S(0)* is near 200 K, as the yield of broad radical decreases and S(0)* multiline EPR signal increases with length of 200 K illumination. Advancement of S(0)* to S(1) is limited at 200 K, but S(1) can be restored by 273 K illumination. Illumination of these hydroxylamine-treated samples at temperatures below 77 K gives a second broad radical EPR signal. The line shape, decay, and other properties of this new radical signal suggest that it may arise from an interaction in the S(-2)* or lower S states, which are probably present in low yield in these samples. Illumination below 20 K of S(0)* state samples containing methanol, and therefore exhibiting the S(0) multiline signal, gives rise to a third broad radical with distinctive line shape. The characteristics of the three broad radicals are similar to those found from interactions between Y(Z)(*) and other S states. The evidence is presented that they do represent intermediate states in S state turnover. Further work is now needed to identify these radicals.