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.

Photosystem II (PSII) uses solar energy to oxidize water and delivers electrons to fix CO₂. Although the atomic resolution structure and basic photophysical and photochemical functions of PSII are well understood, many important questions remain. The activity of PSII in vitro and in vivo is routinely monitored by recording chlorophyll-a fluorescence induction kinetics (ChlF). According to the ‘mainstream’ model, the rise from the minimum level (F_o) to the maximum (F_m) of ChlF of dark-adapted PSII reflects the closure of all functionally active reaction centers, and the F_v/F_m ratio is equated with the maximum photochemical quantum yield of PSII (F_v=F_m–F_o). However, this model has never been free of controversies. A range of recent experimental data confirmed that the first single-turnover saturating flash (STSF), generating closed state (PSII_C), produces F₁<F_m; and uncovered rate-limiting steps – Δτ_1/2 half-waiting times – in the multi-STSF induced F₁-to-F_m increments, originating from the gradual formation of light-adapted charge-separated state (PSII_L) with significantly increased stability of charges compared to PSII_C elicited by a single STSF. All data show that the interpretation of ChlF must be laid on new grounds. Here we discuss the underlying physical mechanisms and the significance of structural/functional dynamics of PSII – as reflected by ChlF and variations of the novel parameter Δτ_1/2.

Photosystem II (PSII) catalyses the photoinduced oxygen evolution and, by producing reducing equivalents drives, in concert with PSI, the conversion of carbon dioxide to sugars. Our knowledge about the architecture of the reaction centre (RC) complex and the mechanisms of charge separation and stabilisation is well advanced. However, our understanding of the processes associated with the functioning of RC is incomplete: the photochemical activity of PSII is routinely monitored by chlorophyll-a fluorescence induction but the presently available data are not free of controversy. In this work, we examined the nature of gradual fluorescence rise of PSII elicited by trains of single-turnover saturating flashes (STSFs) in the presence of a PSII inhibitor, permitting only one stable charge separation. We show that a substantial part of the fluorescence rise originates from light-induced processes that occur after the stabilisation of charge separation, induced by the first STSF; the temperature-dependent relaxation characteristics suggest the involvement of conformational changes in the additional rise. In experiments using double flashes with variable waiting times (∆τ) between them, we found that no rise could be induced with zero or short ∆τ, the value of which depended on the temperature - revealing a previously unknown rate-limiting step in PSII.

Photosynthetic reaction centres are membrane-spanning proteins, found in several classes of autotroph organisms, where a photoinduced charge separation and stabilization takes place with a quantum efficiency close to unity. The protein remains stable and fully functional also when extracted and purified in detergents thereby biotechnological applications are possible, for example, assembling it in nano-structures or in optoelectronic systems. Several types of bionanocomposite materials have been assembled by using reaction centres and different carrier matrices for different purposes in the field of light energy conversion (e.g., photovoltaics) or biosensing (e.g., for specific detection of pesticides). In this review we will summarize the current status of knowledge, the kinds of applications available and the difficulties to be overcome in the different applications. We will also show possible research directions for the close future in this specific field.

Recent chlorophyll‐a fluorescence yield measurements, using single‐turnover saturating flashes (STSFs), have revealed the involvement of a rate‐limiting step in the reactions following the charge separation induced by the first flash. As also shown here, in diuron‐inhibited PSII core complexes isolated from Thermosynechococcus vulcanus the fluorescence maximum could only be reached by a train of STSFs. In order to elucidate the origin of the fluorescence yield increments in STSF series, we performed transient absorption measurements at 819 nm, reflecting the photooxidation and re‐reduction kinetics of the primary electron donor P680. Upon single flash excitation of the dark‐adapted sample, the decay kinetics could be described with lifetimes of 17 ns (∼50%) and 167 ns (∼30%), and a longer‐lived component (∼20%). This kinetics are attributed to re‐reduction of P680^•+ by the donor side of PSII. In contrast, upon second‐flash (with Δt between 5 μs and 100 ms) or repetitive excitation, the 819 nm absorption changes decayed with lifetimes of about 2 ns (∼60%) and 10 ns (∼30%), attributed to recombination of the primary radical pair P680^•+Pheo^•–, and a small longer‐lived component (∼10%). These data confirm that only the first STSF is capable of generating stable charge separation – leading to the reduction of Q_A; and thus, the fluorescence yield increments elicited by the consecutive flashes must have a different physical origin. Our double‐flash experiments indicate that the rate‐limiting steps, detected by chlorophyll‐a fluorescence, are not correlated with the turnover of P680.