Pressure-dependency of steady-state fluorescence spectra
As already mentioned in Introduction, spectral variations in ChlF of PSII CC of T. vulcanus occur at cryogenic temperatures during the induction, with most prominent changes observed at 80 K along the PSIIC→PSIIL transition phase (Sipka et al. 2021). For this reason, but also for improved spectral resolution, main measurements in this study are performed at 78 K. The light-induced reorganizations mostly affected relative amplitudes of the emissions belonging to the two inner antenna complexes of PSII CC named as CP43 and CP47. Distinct features of the exciton energy landscape were also identified in the core complex by fluorescence lifetime measurements reflecting different stages of Fv at 278 K. In this study, our focus centers on examining the spectral and temporal responses of Chl-a fluorescence from PSII CC of T. vulcanus at 78 K, induced by high-pressure compression of the protein complex at either RT or 78 K.
As seen in Fig. 1, the emission spectrum of PSII CC measured at 78 K and 1 bar is dominated by two bands peaking at around 685 and 694 nm. Responsible for these bands are Chl-a molecules bound within the inner antenna complexes of CP43 and CP47, respectively (Andrizhiyevskaya et al. 2005). At elevated pressures applied at 78 K, the maxima of both bands universally, albeit with slightly different rate, shift towards longer wavelengths (bathochromic or red shift). The shifts observed were approximately proportional with the pressure; the linear peak shift rates (slope) being about − 7.4 ± 1.0 cm–1 / kbar for CP43 and − 9.1 ± 2.4 cm–1 / kbar for CP47 (Fig. 1C). In wavelength scale, the peak positions of the emission bands gradually shifted from about 685 to 688 nm and from about 692 to 696 nm, respectively. As common (see, e.g., Timpmann et al. (2020)), the peak shifts are accompanied by broadening of the bands, but we refrain from discussing this complex phenomenon in this work. Most noticeable, however, is the pressure induced switch of the CP43 and CP47 emission bands intensity (Figs. 1A and 1B), whereby higher pressures seem to favour the CP47 emission over the CP43 emission, which dominates the PSII CC spectrum measured at normal pressure (Andrizhiyevskaya et al. 2005).
Below a protein glass temperature, the properties of the proteins generally depend on the handling history, i.e., on the order in which the sample is pressurized and cooled (Frauenfelder et al. 1990). So, drastic changes of spectral, kinetic and dynamic properties of the FMO bacteriochlorophyll a protein were observed under 6 kbar hydrostatic pressure at the temperatures below about 180 K for the samples compressed at RT and then cooled down, as compared to the samples first cooled and then pressurized (Freiberg et al. 1997). PSII CC exposes qualitatively similar behaviour. When the pressure was applied at RT, before cooling, then the emission observed at 78 K reveals not only almost twice increased slopes for CP43 and CP47 (− 12.7 ± 0.9 cm–1 / kbar and − 19.6 ± 2.7 cm–1 / kbar, respectively), but also greater difference between these slopes. Hence, the application of hydrostatic pressure induces a significantly greater red shift in both the CP43 and CP47 bands, which are more distinctly separated from each other when pressure is applied at RT compared to 78 K.
Figure 3 depicts a significant alteration in the relative intensities of the CP43 and CP47 bands observed during high-pressure compression. This change is especially remarkable in Panel B, characterized by a large decrease in CP43 emission alongside a big increase in CP47 emission under pressure. We tentatively attribute this effect to the thermal redistribution of the equilibrium CP43 and CP47 antenna populations toward lower-energy CP47 transitions. Notable also is that the transition point where CP43 and CP47 intensities roughly equalize seems to occur at considerably lower pressures when the sample is compressed at RT compared to compression at 78 K.
The application of high-pressure compression at ambient temperature has been demonstrated to induce structural, energetic, and dynamic changes within the system (Schreiber and Vidaver 1973; Frauenfelder et al. 1990; Foguel et al. 1992; Jalviste et al. 2020; Timpmann et al. 2020), which may or may not be permanent. Hence, it is crucial to assess the reversibility of these pressure effects. Figure 4 depicts the overall reversibility of both pressure- and temperature-induced changes evident in the fluorescence spectra of PSII CC. Notably, the initial (red) and final (green) spectra recorded under ambient conditions exhibit a considerable overlap, despite the great intermediate modifications in shape that the final spectrum underwent. This observation underscores the remarkable flexibility of the PSII CC protein structure in accommodating relatively large variations in pressure (up to 12 kbar) and temperature (approximately 220 K difference). Furthermore, the observed reversibility suggests that these parameter variations do not significantly disturb the pigment binding pockets within the PSII CC complex.
Upon cooling PSII CC to 78 K, a slight increase in fluorescence yield was observed, attributed to a decrease in the efficiency of energy transfer from the core antenna to the RC (Andrizhiyevskaya et al. 2004). In contrast, as outlined below, the application of high pressures at ambient temperature leads to fluorescence quenching and a reduction in its lifetime. Importantly, compression at 78 K did not induce fluorescence quenching in our samples. It is noteworthy that in PSII CC the primary source of fluorescence is the CP43 and CP47 antennas, comprising strongly coupled pigments supporting excitons. The inter-pigment exciton couplings are highly sensitive to pigment-pigment distances, relative orientations of the pigments, individual pigment conformations, and local dielectric constants in the binding pockets of cofactors. All of these factors can be modulated under externally applied hydrostatic high pressure, thereby influencing the antenna fluorescence properties.
The spectral shape changes depicted in Figs. 1–3, particularly the positions and shifts of the bands, offer valuable insights into the electronic structure of the involved pigments and the nature of the couplings between them (Warshel and Parson 1987; Freiberg et al. 1993; Renger 2004)(Chang et al. (1995))(Gall et al. 1997). In cases involving strongly coupled pigments, such as the primary donor of bRC, the optical spectra not only display excitonic couplings, which facilitate excitation energy transport between adjacent molecules or parts of a molecule but also couplings through charge transfer states, aiding in charge transfer between pigments. Due to the exponential distance dependence of charge transfer effects, significant spectral and dynamical responses to pressure have been both expected (Warshel and Parson 1987; Renger 2004) as well as observed (Clayton 1972; Clayton and Devault 1972). For instance, at 300 K, the 870-nm absorption band of the primary donor in the RC of Rhodobacter sphaeroides was found to redshift at a rate of -68 cm⁻¹ / kbar (Clayton and Devault 1972). The observed weakening of the special pair fluorescence in compressed bRCs at room temperature (Timpmann et al. 2020) was attributed to an increased free energy gap between the RC excited state and the charge-separated state, reducing the contribution of charge recombination to the overall emission and modifying the fluorescence kinetics. Similar large slopes have been noted in cyclic bacterial light-harvesting complexes LH2 and LH1 (Freiberg et al. 1993) (Tars et al. 1994), where bacteriochlorophyll pigments are arranged in strongly excitonically coupled dimers. Conversely, the extent of band shifts for weakly coupled pigments in B800 rings of LH2 (Timpmann et al. 2001) or FMO (Freiberg et al. 1997) complexes is several times smaller. Additionally, it has been demonstrated that pressure shifts diminish with cooling, a phenomenon associated with the temperature dependence of sample compressibility (Freiberg et al. 1993; Timpmann et al. 2003). For instance, the pressure shift rate of the B850 absorption band of LH2 decreases from − 60 to -40 cm⁻¹ / kbar over the temperature range from 300 K to 78 K. These previous works thus indicate that pressure-induced shifts of spectra in complexes containing excitonically coupled pigments within protein surroundings can broadly be attributed to the strength of inter-pigment couplings and the compressibility of the samples.
The slopes of the PSII CC emission band shift related to excitons in the CP43 and CP47 antenna complexes, recorded at 78 K (− 7 to − 9 cm–1 / kbar), are notably smaller than those observed in purple bacteria. However, they are up to twice greater compared to the main Chl-a absorption in PSII RC at 78 K (− 4 to − 8 cm–1 / kbar) (Chang et al. (1995)). These relatively modest shift rates observed for all major components of PSII CC suggest the absence of strongly coupled Chl-a molecules. This finding may seem surprising given structural data that reveal close contacts between the PSII CC chromophores (Umena et al. 2011), although it appears to align with the relatively simple circular dichroism spectra of the same samples in the Qy region of Chl-a (Magyar et al. 2022) (see also Årkshöld et al. 2005).
Pressure-dependencies of time resolved spectra and fluorescence kinetics
To gain further insight about the nature of PSIIC-to-PSIIL transition, the change of time-resolved fluorescence spectra of PSII CC from T. vulcanus was analysed at 78 K upon applying high hydrostatic pressures either at RT (Fig. 5) or at 78 K (Fig. 6).
The PSII CC fluorescence spectrum at 78 K in Fig. 3 exhibited a significant peak shift when pressure was applied at RT. This shift is clearly reflected in the spectral dependencies of the decay-associated spectra (DAS) shown in Fig. 5, which extend towards longer wavelengths. Pressure also impacts the relative amplitudes of DAS and the average lifetime, notably reducing the amplitude of the second (red) decay component and shortening the decay times of the remaining DAS components.
However, the most striking observation is the qualitative change in the 40-ps DAS component with a negative peak at around 693 nm, as depicted in Fig. 5A. This component was previously associated with energy transfer between domain 4 and Chl29 in the CP47 antenna of PSII CC (Shibata et al. (2013). The DAS exhibit notable alterations in Fig. 5B, especially the absence of a negative feature and the significantly reduced fluorescence lifetime. These effects indicate a modified energy transfer dynamic, partially attributed to the pressure-induced decrease in transition energy of the terminal emitter states in CP47 due to the high pressures applied at RT.
Conversely, when high-pressure conditions were established at 78 K (Fig. 6), apart from a slight redshift, qualitatively very similar DAS spectra were obtained, akin to those observed at 1 bar (compare Figs. 6A and 6B). Additionally, the distributions of average lifetimes (Fig. 6C) were strikingly similar. This outcome is expected, given the lower compressibility (greater rigidity) of glassy protein samples at 78 K compared to RT (Ivancich et al. (1997) (Timpmann et al. 2003) and an impediment the light-induced structural changes associated with the Fv process by cryogenic temperatures (Sipka et al. 2021).
In this context, it was intriguing to compare ChlF induction at 78 K in the presence and absence of high pressure, which could be monitored, at least in part, by examining the fluorescence lifetime of the emission exhibited by CP43. The variations in fluorescence decay kinetics (component lifetimes and average lifetime) were induced by high repetition rate picosecond pulses of the fluorescence excitation light, enabling, as depicted in Fig. 7, the detection of a segment of Fv at 1 bar pressure. However, in the presence of 12 kbar pressure, no variable fluorescence could be induced during the 900-second-long excitation period. Noteworthy is the significant shortening of the average lifetime under 12 kbar pressure compared to that at 1 bar. This is consistent with the observations of Timpmann et al. (2020) on Rba. sphaeroides RC, which demonstrated over twice shortening of a lifetime by increasing the pressure from 1 bar to 10 kbar.