Transient absorption measurements—kinetics at 690 nm
Figure 3a presents transient absorption kinetics at 690 nm of monomeric and trimeric PSI in solution and immobilized onto the conductive layer of FTO glass (Fig. 1a). Acceleration of the overall excitation decay in the immobilized PSI versus PSI in solution is well noticeable and consistent with previous, time-resolved fluorescence results (Szewczyk et al. 2017). On the other hand, there are no major differences in kinetic traces between monomeric and trimeric forms, in respective systems. In the following, we focus on trimeric PSI. Similar results for monomeric PSI obtained under identical experimental conditions are shown in supplementary information. Figure 3b presents kinetic traces of the trimeric PSI immobilized onto different substrates/in different systems: PSI deposited and dried on bare FTO (conditions with possible electron injection from PSI to FTO), PSI deposited and dried on silanized FTO (conditions with blocked electron injection by insulating layer of silane), and PSI deposited on bare FTO and being in contact with aqueous buffer. One can see that the traces are almost identical, except for the longest component, the lifetime and amplitude of which slightly vary for different samples (see below). A common feature of these three systems is the formation of a film of crowded PSI complexes. On the other hand, they differ either in electrical contact between PSI and FTO or in degree of hydration. Thus, we hypothesize that the major factor responsible for the acceleration of excitation decay in immobilized PSI complexes is their crowding on the substrate, and not drying or electron injection into the substrate. As shown in the inset of Fig. 3b, the experiments with PSI on silanized FTO and on silanized glass brought also almost identical results. This observation is interpreted in terms of the lack of electron transfer from PSI to the FTO conductive layer through the insulating silane layer by hypothetical tunneling effects or by the silane layer discontinuities.
Transient absorption measurements—PSI in solution—global analysis
Figure 4c shows the results of the global analysis performed for trimeric PSI in solution. The dynamics of the PSI complex can be described by three major decay-associated spectral (DAS) components of 0.3, 3.3, and 26 ps. The non-conservative shape of the fastest component with a smaller negative band at about 670 nm and a larger positive band at 690 nm indicates two different processes occurring on the same time scale: (1) relaxation from the Soret to Qy band of Chls (causing the appearance of stimulated emission in the Qy region—manifested as the positive band) and (2) excitation equilibration within the bulk antenna (energy transfer from “blue bulk Qy” to “red bulk Qy” Chl states, seen as the negative band at ~ 670 nm and a small contribution to the positive band at ~ 690 nm) (Fig. 5). The second component (3.3 ps) was also characterized with two bands, the negative one at about 685 nm and the positive one at about 710 nm. The existence of both positive and negative bands, with similar amplitudes (and integrated areas), indicates energy transfer, which was assigned to equilibration of bulk red Chls. The third component (26 ps) with one negative band at about 690 nm and a bump at about 705 nm was ascribed to photochemical quenching in the RC of the excitation equilibrated over bulk (690 nm) and red (705 nm) Chls. The additional, forth component with a small amplitude was assigned to uncoupled or loosely attached chlorophylls (Uchls), because of their slow excitation decay and blue-shifted spectrum. Similar DAS shapes and lifetimes for the closed RC were reported previously (Savikhin et al. 2000). The slight differences in the spectra reported in our study and those by Savikhin et al. may come from the model used (four- vs. five-component fit), different excitation wavelengths, and different ways of controlling the RC state (chemical vs. strong illumination).
Transient absorption measurements—PSI in solution—target analysis
In order to obtain more physical description of the excitation energy dynamics in PSI in solution and next in immobilized PSI, the target analysis was performed. Within the given signal-to-noise ratio, the four-compartment model was sufficient to characterize basic transitions for all the systems under study. This model contained the following compartments: “Soret”, “Bulk”, “Red”, and uncoupled (“Uchls”; Fig. 4a). It is essentially the same model as was used previously for analysis of time-resolved fluorescence data (Szewczyk et al. 2017) but with an extra “Soret” compartment which was possible to identify due to better temporal resolution of the transient absorption experiment. As a result, molecular lifetimes of each transition (reciprocals of molecular rate constants; Fig. 4a; Table 1) and spectral distributions of the states within each of the compartments (species-associated spectra (SAS)—Fig. 4b) were obtained. Excitation quenching by closed RC occurs with lifetime t
1 = 18.3 ps (Fig. 4a). Excitation energy transfer from bulk to red Chls with lifetime t
2 = 13.6 ps is coupled with backward transfer which is nearly three times faster than forward reaction: t
3 = 4.7 ps. The SAS band minima for bulk and red Chls are at 685.5 and 706 nm, respectively (Fig. 4b). The negative band of the “Red” SAS is of bigger amplitude than that of the “Bulk” SAS. Similar effect was observed also for monomers in solution (see Fig. S1 in Supplementary information). The blue-shifted spectrum with a maximum at about 680 nm and lifetime t
4 = 5 ns was assigned to uncoupled Chls. The last molecular lifetime, t
5 = 0.3 ps, was ascribed to “Soret” → “Bulk” transition as discussed above (Fig. 5).
Transient absorption measurements—PSI immobilized on substrates—global analysis
Figure 4f, i, l shows the results of the global analysis obtained for trimeric PSI immobilized in three different systems—onto conductive surface of FTO glass, onto conductive surface of FTO glass covered with insulating silane layer, and onto conductive surface of FTO glass in the cell system, respectively (compare to Fig. 1a, b, d). After PSI immobilization, the three-exponential character of the excitation dynamics is well preserved in all cases. The overall dynamics in all these systems is similar, in line with the results shown in Fig. 3b, and can be described by three major DAS components of 0.4–0.7, 3.6–5.1, and 20–23.7 ps. The most noticeable effects of immobilization are changes in relative amplitudes between the second and the third component (~ 3–5 and ~ 20–24 ps, respectively; compare Fig. 2c with Fig. 2f, i, l). In general, greater contribution of the faster phase over the slower one together with acceleration of the third component after immobilization implies the acceleration of the overall excitation energy decay. Although this effect is partly compensated by increasing lifetime of the second component after immobilization (from 3.3 ps in solution to up to 5.1 ps on bare FTO), direct comparison of the kinetics (Fig. 3) and average lifetimes (Table 1, t
av) demonstrates the overall acceleration effect. The same effect was observed for PSI monomers (Fig. S1 and Table S1 in supplementary information). The non-conservative shape of the second DAS component (3.6–5.1 ps) in the case of immobilized PSI (Fig. 4f, i, l) suggests mixing of two processes: (1) energy transfer assigned to equilibration of bulk red Chls, the same as that for PSI in solution (Fig. 4c) and (2) photochemical quenching of the excitation in the closed RC, absent in solution on this time scale.
The fourth, slowest component is for each PSI-substrate sample characterized by much shorter lifetime (~ 300–500 ps) than in solution (5 ns). These results are in line with those obtained previously with time-resolved fluorescence (Szewczyk et al. 2017) and are discussed below.
Transient absorption measurements—PSI immobilized on substrates—target analysis
After immobilization of trimeric PSI onto substrates, few main changes relative to PSI in solution can be observed in target analysis results (Fig. 4). The first difference is shortening of the lifetime t
1—from 18.3 ps in solution to 13–14 ps in immobilized PSI. The second one is the weaker coupling between bulk and red Chls reflected by increased values of t
2 and t
3 lifetimes: from 13.6/4.7 ps for PSI in solution to 22/7.9 ps, 17.9 /6.4 ps, and 15.5/5.2 ps for PSI on bare FTO, silanized FTO, and in the cell system, respectively. This effect is quite large for PSI-bare FTO sample, intermediate for PSI-silanized FTO, and weak for PSI in the cell system. The third effect is related to the positions of “Bulk” and “Red” SAS band maxima after PSI immobilization. In solution, the respective bands are at 685.5 and 706 nm. They are red-shifted to 689 and 710 nm for bare FTO and similarly to 688.5 and 709 nm for silanized FTO, respectively. Oppositely, the positions of SAS band maxima for the PSI in the cell system almost did not change in comparison to solution—they are at 686.5 and 706 nm. Also, the differences in amplitudes of SAS are firmly visible. Under “dry” conditions (bare and silanized FTO), the “Red” SAS shows strongly reduced amplitude (compare Fig. 4e, h–b). In immobilized PSI complexes in the cell system (“wet” conditions), the “Red” SAS is also of reduced amplitude albeit to a lower extent (compare Fig. 4k–b). This effect may indicate that the oscillator strength of the red Chls decreases if PSI is immobilized and densely packed. Similar effect was observed in the case of monomeric PSI (Fig. S1) and previously in aggregated (densely packed) LHCII particles in solution (Gruszecki et al. 2006).
Immobilization of PSI in all systems causes similar changes in the lifetime and shape of the uncoupled Chls’ SAS (Fig. 4e, h, k and Fig. S2C). The lifetime is shortened by one order of magnitude as noticed above (global analysis). Apart from main blue-shifted band with maximum at about 680 nm (present also in PSI in solution; Fig. 4b) being a fingerprint of unconnected Chls (Melkozernov et al. 2000), an additional small band at about 705–710 nm can be distinguished. This observation suggests that after immobilization some of the uncoupled Chls undergo transition from “blue” to “red” form. All the described modifications of excitation dynamics induced by immobilization of PSI on the solid substrate are very much consistent with previously published data obtained using time-resolved fluorescence method (Szewczyk et al. 2017).
To sum up, immobilization of PSI causes similar effects in all the systems under study although in the case of the cell system some of the features of SAS (Fig. 4 and Fig. S1) and also DAS (Fig. 4) are intermediate between those for PSI in solution and under the “dry” conditions (bare and silanized FTO).
Estimation of the effective numbers of red Chls
As shown previously (Szewczyk et al. 2017) and in the supplementary information, results of target analysis (molecular lifetimes and spectral positions of SAS; Fig. S3) may be used to estimate energetic parameters of bulk and red Chls (standard enthalpy difference, ΔH
0, and standard free energy difference, ΔG
0, between bulk and red Chls), and from those, effective number of bulk and red Chl states may be extracted. The results of these calculations as well as input data taken from the target analysis are shown in Table 1 for PSI trimers and in Table S1 for PSI monomers. The estimated effective number of red Chls per monomer in PSI trimers (4.3–4.6, Table 1) is independent of the system (PSI in solution and different systems with immobilized PSI), which is the same as that reported in the previous fluorescence studies (Szewczyk et al. 2017), from which also a similar number of 3.9–4.2 red Chls per monomer in trimeric PSI was estimated. The results shown in Table 1 consistently demonstrate that despite little spectral red-shift of red (and also bulk) Chls (see also Fig. S1) and the reduction of oscillator strength of red Chls (see above and Fig. 4), immobilization of PSI trimers influences neither the energetic parameters nor the numbers of red Chls.
In the case of the monomeric PSI, the results of global and target analyses are generally similar to those for trimers (Fig. S1), except for some differences regarding the red Chls. The estimated effective number of red Chls was increased from 3.1 per monomer in solution to 4.3–5.3 for systems with immobilized PSI (Table S1). Similar tendency was observed in fluorescence studies where immobilization caused an increase in the effective number of red Chls from 3–3.4 in solution to 6.3. In both absorption and fluorescence studies, these changes are related to modifications of energetic parameters, in particular standard free energy difference between bulk and red Chls, ΔG
0, whose absolute value decreases after immobilization. This observation confirms the previous hypothesis, that Chl–Chl interactions being most likely the origin of the extra red Chl states are more susceptible to modifications for PSI monomers than for trimers after immobilization/dense packing on the substrate (Szewczyk et al. 2017). Appearance of additional low-energy Chls, as a result of aggregation, was also observed previously in LHCII particles (Vasil’ev et al. 1997; Gruszecki et al. 2006; Andreeva et al. 2009; see also below).
Origin of the acceleration of the excitation energy decay in immobilized PSI
We have performed careful comparative analysis of PSI trimers and monomers immobilized onto different surfaces, conductive and non-conductive, dried, or being in contact with aqueous solution. In all cases, immobilization of PSI complexes caused similar acceleration of excitation decay within the protein. This result suggests that the acceleration of the PSI excitation decay is caused neither by electron injection into the substrate nor by drastically changed hydration state of the proteins, but most likely it is due to dense packing of PSI on the substrate. This conclusion is supported by earlier studies on a different pigment–protein photosynthetic complex, LHCII. For that system, accelerated excitation decay as well as the formation of a few red-shifted chlorophyll species as a result of aggregation, caused by low detergent concentration, was reported (Vasil’ev et al. 1997; Gruszecki et al. 2006; Andreeva et al. 2009). Furthermore, it was proposed that the origin of the new electronic low-energy levels is related to exciton coupling of protein-bound photosynthetic pigments (Gruszecki et al. 2006). Similar effects were observed by us: acceleration of the overall decay—for monomeric and trimeric PSI, and formation of additional red Chls—for monomeric PSI. Moreover, apart from the acceleration of overall excitation decay of Chls well coupled to RC occurring on 10–20-ps time scale (see t
av values in Table 1 and S1), we also observed ~ 10-fold acceleration of excitation decay within a very minor pool of Chls uncoupled to RC: from ~ 5 ns in solution to ~ 500 ps after immobilization on the substrate. For these reasons, we propose that dense packing of PSI on the substrate resembles the “dense packing” of LHCII complexes within the aggregates, although the exact mechanism of the observed acceleration remains to be discovered. The above consideration was performed for hypothetical homogenous packing, although scenario in which different “clusters” of packed proteins are formed cannot be excluded. The parameters retrieved from the analysis would then represent average/mean values. Finally, we conclude that despite the described spectral and dynamic modifications of PSI complexes immobilized in different systems, these proteins remain fully functional in terms of excitation energy transfer.