Although fluorescence is widely used to study photosynthetic systems,
the mechanisms that affect the fluorescence in photosystem II (PSII) a
re not completely understood. The aim of this study is to define the l
ow-temperature steady-state fluorescence quenching of redox-active cen
ters that function on the electron donor side of PSII. The redox state
s of the electron donors and accepters were systematically varied by u
sing a combination of pretreatments and illumination to produce and tr
ap, at low temperature, a specific charge-separated state. Electron pa
ramagnetic resonance spectroscopy and fluorescence intensity measureme
nts were carried out on the same samples to obtain a correlation betwe
en the redox state and the fluorescence. It was found that illuminatio
n 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 s
ide of PSII. This pathway involves electron donation to the primary el
ectron donor in PSII, P680, from cytochrome b(559) 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) suff
icient to quench 70% of the fluorescence intensity. This implies that
neighboring PSII reaction centers are energetically connected, allowin
g 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 P
SII studied were the O-2-evolving complex, the redox-active tyrosines
(Y-Z and Y-D), and cytochrome b(559). No significant changes in fluore
scence intensity could be attributed to changes in the redox state of
these components. The fast phase of fluorescence quenching is attribut
ed to the rapid photooxidation of Chl(Z), and the slow phase is attrib
uted to multiple turnovers providing for further oxidation of Chl(Z) a
nd irreversible photoinhibition. Significant photoinhibition only occu
rred at Chl concentrations below 0.7 mg/mL and above 150 K. The revers
ible oxidation of Chl(Z) in intact systems may function as a photoprot
ection mechanism under high-light conditions and account for a portion
of the nonphotochemical fluorescence quenching.