Dependence of the rate-limiting steps in the dark-to-light transition of photosystem II on the lipidic environment of the reaction center

Abstract

In our earlier works, we have identified rate-limiting steps in the dark-to-light transition of PSII. By measuring chlorophyll a fluorescence transients elicited by single-turnover saturating flashes (STSFs) we have shown that in diuron-treated samples an STSF generates only F₁ (< F_m) fluorescence level, and to produce the maximum (F_m) level, additional excitations are required, which, however, can only be effective if sufficiently long Δτ waiting times are allowed between the excitations. Biological variations in the half-rise time (Δτ _1/2) of the fluorescence increment suggest that it may be sensitive to the physicochemical environment of PSII. Here, we investigated the influence of the lipidic environment on Δτ _1/2 of PSII core complexes of Thermosynechococcus vulcanus. We found that while non-native lipids had no noticeable effects, thylakoid membrane lipids considerably shortened the Δτ _1/2, from ~ 1 ms to ~ 0.2 ms. The importance of the presence of native lipids was confirmed by obtaining similarly short Δτ _1/2 values in the whole T. vulcanus cells and isolated pea thylakoid membranes. Minor, lipid-dependent reorganizations were also observed by steady-state and time-resolved spectroscopic measurements. These data show that the processes beyond the dark-to-light transition of PSII depend significantly on the lipid matrix of the reaction center.

Keywords: closed state of PSII; conformational changes; dielectric relaxation; light-adapted state of PSII, light-induced changes; proteoliposomes..

Fig. 1. Dependence of the F₁-to-F₂ increment on the waiting time (Δτ), between the first and second STSFs, of PSII CC of Thermosynechococcus vulcanus in solution (blue) (A,B), and in buffer containing 0.03% β-DDM (red) (A), and embedded into PC liposomes (yellow) (B) – at 5°C in the presence of 40 μM DCMU. Continuous lines represent logistic-function fits of the data points, which represent mean values ± SD (n = 3–5). The magnitude of the increment at each Δτ was determined by measuring the F₂ fluorescence level elicited by the second STSF delivered after Δτ waiting time for the first flash.

Fig. 2. Dependence of the F₁-to-F₂ increment on the waiting time (Δτ), between the first and second STSFs, of Thermosynechococcus vulcanus PSII CC in solution (blue) and embedded into thylakoid lipids (TM lip, red) at 5°C (A), and of T. vulcanus cells (yellow) at RT (B) – in the presence of 40 μM DCMU. Continuous lines represent logistic-function fits of the data points, which represent mean values ± SD (n = 3–5). The magnitude of the increment at each Δτ was determined by measuring the F₂ fluorescence level elicited by the second STSF delivered after Δτ waiting time for the first flash.

Fig. 3. Absorption (A) and CD (B) spectra of Thermosynechococcus vulcanus PSII CC in solution (PSII) and embedded into TM-lipid membranes. The spectra are normalized to the unity absorbance of each sample at 675 nm.

Fig. 4. 77 K steady-state fluorescence emission spectra of Thermosynechococcus vulcanus PSII CCs in solution (PSII) and embedded into different lipid membranes (TM lipid and PC-PE); excitation wavelength, 440 nm. The spectra are normalized to the maximum fluorescence intensities and are corrected for the detector spectral sensitivity.

Fig. 5. Decay-associated fluorescence emission spectra of Thermosynechococcus vulcanus PSII CC in solution (A) and embedded into TM lipid (B) and PC-PE membranes (C) – in F₀ state; the spectra are obtained by global lifetime analysis of the fluorescence decays recorded at RT with 632-nm excitation.