The identity and rearrangements of substrate water molecules in photosystem II (PSII) water oxidation are of great mechanistic interest and addressed herein by comprehensive analysis of NH₄⁺/NH₃ binding. Time-resolved detection of O₂ formation and recombination fluorescence as well as Fourier transform infrared (FTIR) difference spectroscopy on plant PSII membrane particles reveals the following. (1) Partial inhibition in NH₄Cl buffer occurs with a pH-independent binding constant of ∼25 mM, which does not result from decelerated O₂ formation, but from complete blockage of a major PSII fraction (∼60%) after reaching the Mn(IV)₄ (S₃) state. (2) The non-inhibited PSII fraction advances through the reaction cycle, but modified nuclear rearrangements are suggested by FTIR difference spectroscopy. (3) Partial inhibition can be explained by anticooperative (mutually exclusive) NH₃ binding to one inhibitory and one non-inhibitory site; these two sites may correspond to two water molecules terminally bound to the "dangling" Mn ion. (4) Unexpectedly strong modifications of the FTIR difference spectra suggest that in the non-inhibited PSII, ammonia binding obliterates the need for some of the nuclear rearrangements occurring in the S₂-S₃ transition as well as their reversal in the O₂ formation transition, in line with the carousel mechanism [Askerka, M., et al. (2015) Biochemistry 54, 5783]. (5) We observe the same partial inhibition of PSII by NH₄Cl also for thylakoid membranes prepared from mesophilic and thermophilic cyanobacteria, suggesting that the results described above are valid for plant and cyanobacterial PSII.

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