Photosystem II (PSII) uses solar energy to oxidize water and delivers electrons for life on Earth. The photochemical reaction center of PSII is known to possess two stationary states. In the open state (PSII_O), the absorption of a single photon triggers electron-transfer steps, which convert PSII into the charge-separated closed state (PSII_C). Here, by using steady-state and time-resolved spectroscopic techniques on Spinacia oleracea and Thermosynechococcus vulcanus preparations, we show that additional illumination gradually transforms PSII_C into a light-adapted charge-separated state (PSII_L). The PSII_C-to-PSII_L transition, observed at all temperatures between 80 and 308 K, is responsible for a large part of the variable chlorophyll-a fluorescence (F_v) and is associated with subtle, dark-reversible reorganizations in the core complexes, protein conformational changes at noncryogenic temperatures, and marked variations in the rates of photochemical and photophysical reactions. The build-up of PSII_L requires a series of light-induced events generating rapidly recombining primary radical pairs, spaced by sufficient waiting times between these events—pointing to the roles of local electric-field transients and dielectric relaxation processes. We show that the maximum fluorescence level, F_m, is associated with PSII_L rather than with PSII_C, and thus the F_v/F_m parameter cannot be equated with the quantum efficiency of PSII photochemistry. Our findings resolve the controversies and explain the peculiar features of chlorophyll-a fluorescence kinetics, a tool to monitor the functional activity and the structural-functional plasticity of PSII in different wild-types and mutant organisms and under stress conditions.

Extraction of plant light-harvesting complex II (LHCII) from the native thylakoid membrane or from aggregates by the use of surfactants brings about significant changes in the excitonic circular dichroism (CD) spectrum and fluorescence quantum yield. To elucidate the cause of these changes, e.g. trimer-trimer contacts or surfactant-induced structural perturbations, we compared the CD spectra and fluorescence kinetics of LHCII aggregates, artificial and native LHCII-lipid membranes, and LHCII solubilized in different detergents or trapped in polymer gel. By this means we were able to identify CD spectral changes specific to LHCII-LHCII interactions - at (-)437 nm and (+)484 nm, and changes specific to the interaction with the detergent n-dodecyl-β-maltoside (β-DM) or membrane lipids - at (+)447 nm and ( )494 nm. The latter change is attributed to the conformational change of the LHCII-bound carotenoid neoxanthin, by analyzing the CD spectra of neoxanthin-deficient plant thylakoid membranes. The neoxanthin-specific band at ( )494 nm was not pronounced in LHCII in detergent-free gels or solubilized in the α isomer of DM but was present when LHCII was reconstituted in membranes composed of phosphatidylcholine or plant thylakoid lipids, indicating that the conformation of neoxanthin is sensitive to the molecular environment. Neither the aggrega-tion-specific CD bands, nor the surfactant-specific bands were positively associated with the onset of fluorescence quenching, which could be triggered without invoking such spec-tral changes. Significant quenching was not active in reconstituted LHCII proteoliposomes, while a high degree of energetic connectivity, depending on the lipid:protein ratio, in these membranes allows for efficient light harvesting.