By measuring ms delayed fluorescence emission, we have investigated the interaction of protons in the reactions of the watersplitting enzyme of photosystem II. (1) In the presence of the electron transport mediators 2,3,5,6-tetramethyl-p-phenylene diamine (DAD) (in its reduced form) and methyl viologen and of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), the maximal intensity of the delayed fluorescence measured between 1 and 1.5m s after illumination was greater than the level reached in the

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... presence of DCMU alone. (2) The stimulation of the delayed fluorescence was greater when the suspension was preilluminated by 1 - 8 xenon (or laser) flashes prior to addition of DCMU, and the maximal intensity varied with the number of preilluminating flashes given. (3) The amplitude of the intensity of delayed fluorescence oscillated with a periodicity of 4, with a maximum after 2, and minimum after 4 flashes. A negligible oscillation wras observed in the absence of the electron transport mediators. (4) When the prompt fluorescence intensity was measured under these conditions, a weak oscillation of period 4 with superimposed periodicity of 2 was observed in F0, but of insufficient amplitude to account for the observed changes of delayed fluorescence intensity in terms of the bade reaction caused by addition of DCMU. (5) We therefore suggest that the enhancement of the delayed fluorescence is due to the release of protons accompanying transitions of the S-states, and that this release occurs not in synchrony with 02, but in all the transitions of the S-states with the exception of Si → S2 . We discuss the relative potentials of the transitions at the low internal pH which is generated as a result of cyclic electron transport around photosystem I in the conditions of our experiments.