Photosynthetic O2 evolution takes place at the Mn cluster in photosystem II (PSII) by oxidation of water. It has been proposed that ammonia, one of water analogues, functions as an inhibitor of O2 evolution at alkaline pH. However, the detailed mechanism of inhibition has not been understood yet. In this study, we investigated the mechanism of ammonia inhibition by examining the NH4Cl-induced inhibition of O2 evolution in a wide pH range (pH 5.0-8.0) and by detecting the interaction site using Fourier transform infrared (FTIR) spectroscopy. In addition to intact PSII membranes from spinach, PSII membranes depleted of the PsbP and PsbQ extrinsic proteins were used as samples to avoid the effect of the release of these proteins by salt treatments. In both types of samples, oxygen evolution activity decreased by approximately 40% by addition of 100 mM NH4Cl in the range of pH 5.0-8.0. The presence of inhibition at acidic pH without significant pH dependence strongly suggests that NH4+ cation functions as a major inhibitor in the acidic pH region, where neutral NH3 scarcely exists in the buffer. The NH4Cl treatment at pH 6.5 and 5.5 induced prominent changes in the COO- stretching regions in FTIR difference spectra upon the S1 → S2 transition measured at 283 K. The NH4Cl concentration dependence of the amplitude of the spectral changes showed a good correlation with that of the inhibition of O2 evolution. From this observation, it is proposed that NH4+ cation interacts with carboxylate groups coupled to the Mn cluster as direct ligands or proton transfer mediators, causing inhibition of the O2 evolving reaction.
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