Unique properties of light-induced changes of variable chlorophyll fluorescence in purified PSII core complexes of...

A new type of kinetic chlorophyll fluorometer (Klughammer et al. 2024, Photosynth Res 161:151-176) which enables measuring the changes of rel. fluorescence yield during application of saturating single-turnover 15µs flashes (ST-kinetics, STK) simultaneously with the PAM relaxation kinetics induced by the same flash, was applied on purified PSII core complexes (PSII CC) isolated from Thermosynechococcus vestitus (Lambertz et al. 2023, Biochim Biophys Acta 1864:148953). While the main text concentrates on purified PSII dimers, under Supplementary Materials also comparative measurements with purified monomers and heterodimers are presented. Freshly solubilized, non-preilluminated samples display extremely high ratios of maximal/minimal fluorescence yields up to 26, corresponding to F_v/F_m = 0.96. Both PAM and STK techniques reveal period-4 oscillations in presence of 2 µM dichlorobenzoquinone (DCBQ). Period-2 oscillations are observed in the PAM relaxation kinetics after relaxation of donor-side dependent quenching (DQ). Maximal fluorescence yields in STK responses are lowered with respect to the corresponding PAM responses by High Intensity Quenching (HIQ), consisting of DQ and carotenoid triplet quenching (TQ). The DQ observed in presence of 20µM DCMU lowers flash-induced F_v by about 30%, relaxing with a half-time of about 40µs, as revealed by double flash STK measurements. When a train of ST with 5s dark intervals is applied in the absence of artificial acceptors, up to ST#3 both STK and PAM measurements show about 30% quenching (DQ) with respect to maximal fluorescence yield (F_m). Suppression of DQ occurs in a train of additional ST, reflected in a multi-step further rise to F_m. This "terminal rise" is inhibited by 20 µM DCMU and by 2µM DCBQ, suggesting that redox-reactions involving reduced Q_B are involved in this phenomenon. It is proposed that double reduced Q_B is either protonated, so that after release of PQH₂ the PSII acceptor side is inactivated, or reoxidized by the donor side (via cyclic PSII, PSII-CEF). Based on this rationale a simple scheme is presented to explain the phenomenon. A similar "terminal rise" is observed using the PAM technique upon illumination by strong continuous light. It is suggested that the "terminal rise" in PSII CC and the "thermal" I₁-I₂ phase in vivo may involve the same pathways of DQ suppression, the molecular steps of which remain to be clarified by further research.