Investigation of the Inhibitory Effect of Nitrite on Photosystem II

Abstract

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 NO₂^–. However, the S₂-state <i>g</i> = 4.1 signal is favored over the <i>g</i> = 2 multiline signal with increasing NO₂^– concentrations. This could indicate competition of NO₂^– for the Cl^– site at higher NO₂^– concentrations. In addition to the donor-side chemistry, there is clear evidence of an acceptor-side effect of NO₂^–. The <i>g</i> = 1.9 Fe­(II)-Q_A^–• signal is replaced by a broad <i>g</i> = 1.6 signal in the presence of NO₂^–. Additionally, a <i>g</i> = 1.8 Fe­(II)-Q^–• signal is present in the dark, indicating the formation of a NO₂^–-bound Fe­(II)-Q_B^–• species in the dark. Electron-transfer assays suggest that the inhibitory effect of NO₂^– 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 NO₂^–