Electron paramagnetic resonance Signal II in spinach chloroplasts. I. Kinetic analysis for untreated chloroplasts

An analysis of electron paramagnetic resonance Signal II in spinach chloroplasts has been made using both continuous and flashing light techniques. In order to perform the experiments we developed a method which allows us to obtain fresh, untreated chloroplasts with low dark levels of Signal II. Under these conditions a single 10-μs flash is sufficient to generate greater than 80% of the possible light-induced increase in Signal II spin concentration. The risetime for this flash-induced increase in Signal II is approx. 1 s. The close association of Signal II with Photo-system II is confirmed by the observations that red light is more effective than is far red light in generating Signal II, and that 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) does not inhibit the formation of the radical. Single flash saturation curves for the flash-induced increase in Signal I and Signal II indicate that the quantum efficiency for Signal II formation is close to that for Signal I. While one or two flashes (spaced 10 ms apart) are quite efficient in generating Signal II, three or four flashes are much less effective. However, if this spacing is decreased to 100 μs, three or four flashes become as efficient as one or two flashes. From observations of a deficiency of O₂ evolved during the initial flashes of dark-adapted chloroplasts, we conclude that the species which gives rise to Signal II is able to compete with water for oxidizing equivalents generated by Photosystem II. On the basis of these results we postulate a model in which Signal II arises from an oxidized radical which is produced by a slow electron transfer to the specific states S₂ and S₃ on the water side of Photo-system II.