The role of chloride in photosystem II (PSII) is unclear. Several monovalent anions compete for the Cl- site(s) in PSII, and some even support activity. NO2- has been reported to be an activator in Cl--depleted PSII membranes. In this paper, we report a detailed investigation of the chemistry of NO2- with PSII. NO2- is shown to inhibit PSII activity, and the effects on the donor side as well as the acceptor side are characterized using steady-state O2-evolution assays, electron paramagnetic resonance (EPR) spectroscopy, electron-transfer assays, and flash-induced polarographic O2 yield measurements. Enzyme kinetics analysis shows multiple sites of NO2- inhibition in PSII with significant inhibition of oxygen evolution at <5 mM NO2-. By EPR spectroscopy, the yield of the S2 state remains unchanged up to 15 mM NO 2-. However, the S2-state g = 4.1 signal is favored over the g = 2 multiline signal with increasing NO2 - concentrations. This could indicate competition of NO 2- for the Cl- site at higher NO 2- concentrations. In addition to the donor-side chemistry, there is clear evidence of an acceptor-side effect of NO 2-. The g = 1.9 Fe(II)-QA-• signal is replaced by a broad g = 1.6 signal in the presence of NO 2-. Additionally, a g = 1.8 Fe(II)-Q-• signal is present in the dark, indicating the formation of a NO2 --bound Fe(II)-QB-• species in the dark. Electron-transfer assays suggest that the inhibitory effect of NO 2- on the activity of PSII is largely due to the donor-side chemistry of NO2-. UV-visible spectroscopy and flash-induced polarographic O2 yield measurements indicate that NO2- is oxidized by the oxygen-evolving complex in the higher S states, contributing to the donor-side inhibition by NO 2-.
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