Although fluorescence is widely used to study photosynthetic systems, the mechanisms that affect the fluorescence in photosystem II (PSII) are not completely understood. The aim of this study is to define the low-temperature steady-state fluorescence quenching of redox-active centers that function on the electron donor side of PSII. The redox states of the electron donors and acceptors were systematically varied by using a combination of pretreatments and illumination to produce and trap, at low temperature, a specific charge- separated state. Electron parmagnetic resonance spectroscopy and fluorescence intensity measurements were carried out on the same samples to obtain a correlation between the redox state and the fluorescence. It was found that illumination of PSII at temperatures between 85 and 260 K induced a fluorescence quenching state in two phases. At 85 K, where the fast phase was most prominent, only one electron-transfer pathway is active on the donor side of PSII. This pathway involves electron donation to the primary electron donor in PSII, P680, from cytochrome b559 and a redox-active chlorophyll molecule, Chl(Z). Oxidized Chl(Z) was found to be a potent quencher of chlorophyll fluorescence with 15%of oxidized Chl(Z) sufficient to quench 70% of the fluorescence intensity. This implies that neighboring PSII reaction centers are energetically connected, allowing oxidized Chl(Z) in a few centers to quench most of the fluorescence. The presence of a well-defined quencher in PSII may make it possible to study the connectivity between antenna systems in different sample preparations. The other redox-active components on the donor side of PSII studied were the O2-evolving complex, the redox-active tyrosines (Y(Z) and Y(D)), and cytochrome b559. No significant changes in fluorescence intensity could be attributed to changes in the redox state of these components. The fast phase of fluorescence quenching is attributed to the rapid photooxidation of Chl(Z), and the slow phase is attributed to multiple turnovers providing for further oxidation of Chl(Z) and irreversible photoinhibition. Significant photoinhibition only occurred at Chl concentrations below 0.7 mg/mL and above 150 K. The reversible oxidation of Chl(Z) in intact systems may function as a photoprotection mechanism under high-light conditions and account for a portion of the nonphotochemical fluorescence quenching.
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