A 230-G wide EPR signal is induced in acetate-treated photosystem II by 30 s of illumination at 277 K followed by freezing under illumination to 77 K [MacLachlan, D. J., and Nugent, J. H. A. (1993) Biochemistry 32, 9772-9780]. This signal, referred to as the S3 EPR signal, has been interpreted to arise from an S2X+ species where X+ is an amino acid radical. Investigation of the factors responsible for the formation and decay of the S3 EPR signal reveals that the yield of the S3 EPR signal is strongly temperature-dependent and depends on the rate of oxidation of Q(A)-. Quantitation of the number of centers contributing to the S3 EPR signal produced by the optimal continuous illumination times of 3 min at 250 K, 30 s at 273 K, and 5 s at 294 K gave values of 13, 38, and 49 ± 3%, respectively. By using 5 s of illumination at 294 K to induce the S3 EPR signal, and then illumination at 200 K to reduce Q(A), both the S3 and Q(A)-Fe EPR signals were induced in high yield. This result indicates that the S3 EPR signal does not arise from an acceptor-side species. When saturating laser flashes were used to induce the S3 EPR signal in u dark-prepared, dark-adapted, acetate-treated sample, the yield was small after one flash and close to maximal after two flashes. An EPR signal at g = 4.1 was observed to be formed at intermediate times during the decay of the S3 EPR signal in the dark; the rates of decay of the S3 EPR signal at 273 and 294 K corresponded to the rates of formation of the g = 4.1 EPR signal. These results, together with the flush results, indicate that two steps are involved in both the generation and decay of the S3 EPR signal. The rates of formation and decay of both the S3 and Q(A)-Fe EPR signals were measured at 250, 273, and 294 K. A kinetic model is presented that accounts for these kinetic data and the yield of the S3 EPR signal.
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