Trehalose as a tool for studying the molecular mechanisms of electron transfer in photosynthetic systems

1 Introduction

Elucidating how the local structural environment governs electron transfer (ET) in photosynthetic protein complexes remains a central challenge in biophysics of photosynthesis. In recent years, increasing attention has been directed toward the role of external matrices and confinement effects in modulating protein function. In this context, trehalose (ɑ-D-glucopyranosyl-ɑ-D-glucopyranoside), a natural disaccharide, has emerged as a valuable compound due to its exceptional ability to stabilize biomolecular structures under conditions of dehydration, thermal stress, and osmotic imbalance. Some organisms survive desiccation by overproducing trehalose, which serves to preserve internal structure by vitrification (Argüelles, 2000). Besides an important role of trehalose in maintaining desert plants, bacteria, yeasts, some invertebrate animals in reversible anhydrobiotic state, it is a useful and unique tool for studying isolated proteins. Trehalose is classified as a kosmotrope (Kilburn et al., 2006): it is capable of ordering surrounding water molecules, and this property is extremely important for understanding the mechanism of trehalose effects on the protein function.

Trehalose is widely recognized for its ability to stabilize proteins against dehydration, denaturation, or loss of function. It can suppress the thermal denaturation of bovine pancreatic RNase A (Lin and Timasheff, 1996; Kaushik and Bhat, 2003) and the mitochondrial enzyme rhodanese (Singer and Lindquist, 1998), counteract urea-induced denaturation of α-chymotrypsin (Kumar et al., 2010), and protect β-galactosidase during freeze-thaw processes (Mitchell et al., 2019). Furthermore, trehalose inhibits the dissociation of tetrameric rabbit lactate dehydrogenase under acidic pH conditions (Simongini et al., 2023). Notably, this disaccharide also enhances the catalytic activities of bovine pancreatic DNase I and the NcoI restriction endonuclease (Carninci et al., 1998). Trehalose is thought to modulate protein dynamics through its impact on the surrounding hydration water. This view has been supported by recent neutron diffraction studies of myoglobin in trehalose solutions, which provide direct experimental evidence for trehalose-induced alterations in hydration structure (Ahlgren et al., 2026).

Despite these well-documented effects on soluble proteins, the influence of trehalose on non-photosynthetic membrane protein complexes remains comparatively underexplored. Notably, trehalose has been shown to protect the plasma membrane H⁺-ATPase from Kluyveromyces lactis against denaturation and aggregation during dehydration and freezing (Sampedro et al., 1998), as well as against thermal denaturation (Felix et al., 1999). These protective effects are attributed to the ability of trehalose to preserve native protein conformation and prevent irreversible structural alterations.

Trehalose forms an amorphous, glass-like matrix that preserves native protein conformations while significantly reducing conformational mobility. This property provides a unique platform for dissecting the interplay between protein dynamics and function. In ET systems, incorporation into a trehalose matrix can significantly affect both the kinetics and efficiency of charge transport by modulating conformational flexibility, restructuring hydration shells, and altering the hydrogen bond network of the proteins (reviewed in Gorka et al., 2020; Möbius et al., 2020).

Photosynthetic pigment-protein complexes are among the most appropriate objects to investigate the effects of trehalose on the photosynthetic ET processes (Mamedov et al., 2021; Venturoli et al., 2024). Their functioning is coupled to multistep charge transfer reactions in a wide time range–from subpicoseconds to seconds, including both forward and backward ET as well as proton uptake and release.

Previous studies of bacterial reaction centers (bRCs) embedded in dry trehalose matrices were largely performed by G. Venturoli and co-authors (Palazzo et al., 2002; Francia et al., 2003; 2004; 2008). In general, the data indicated that this disaccharide has a profound effect on ET rates and protein dynamics in bRC. The progressive dehydration of trehalose matrices lead to an increased rates of charge recombination of P⁺Q_A– state (with mean reaction rate increasing from 8.7 s^-1 to 26.6 s^-1), as well as almost complete inhibition of forward electron transfer from Q_A to Q_B. These observations supported a model in which the protein was trapped in various states during the experiment.

These states are reflected in the highly distributed kinetics of charge recombination, with lifetimes that resemble those of the dark-adapted state. The origin of this inhibition does not appear to arise from significant alterations in protein structure, relative cofactor orientation (Savitsky et al., 2010; Malferrari, Savitsky, Lubitz, et al., 2016), or water mobility (Nalepa et al., 2017), but rather in the simple inability to adopt different conformations over short time scales. The effect of trehalose was also investigated in purified PS II complexes in solution (Mamedov et al., 2015; 2018; Yanykin et al., 2016) and PS I complexes incorporated in dehydrated trehalose glasses (Malferrari, Savitsky, Mamedov, et al., 2016).

In this review, we focus primarily on recent advances that have not been covered in earlier reviews (Gorka et al., 2020; Möbius et al., 2020). The main focus is on the effects of trehalose on modulation of charge transfer processes in photosynthetic systems (in solution, in a trehalose matrix, and immobilized on a nitrocellulose membrane filter).

2 Flash-induced electron transfer in photosystem II embedded into trehalose glassy matrix

Photosystem II (PS II) is a multisubunit protein complex embedded in the thylakoid membrane of oxygenic photosynthetic organisms that catalyzes light-driven water oxidation and plastoquinone (PQ) reduction. As a photo-oxidoreductase, it represents one of the most sophisticated biological energy transducers; the plant PS II monomer comprises ∼20 protein subunits, ∼25 integral lipids, 35 chlorophyll a molecules, 2 pheophytins, 12 carotenoids, 3 PQ, the Mn₄Ca cluster, and a chloride ion (Shevela and Messinger, 2012; Nickelsen and Rengstl, 2013; Shevela et al., 2023). The minimal functional unit, the PS II core complex, supports rapid forward electron transfer from the primary donor P₆₈₀ to the primary quinone acceptor Q_A, followed by reduction of photo-oxidized P₆₈₀ ⁺ by the redox-active tyrosine Y_Z. In turn, Y_Z is re-reduced by the water-oxidizing complex (WOC), which contains the Mn₄Ca cluster. Mn-depleted PS II samples allow direct observation of P₆₈₀ ⁺ reduction pathways without continuous electron input from the WOC. Under these conditions, the redox-active tyrosine Y_Z can still donate an electron to P₆₈₀ ⁺, but is not efficiently re-reduced. Thus, the system is functionally modified while remaining structurally relevant, making Mn-depleted PS II an ideal model for probing how hydration and conformational dynamics regulate ET.

Impact of trehalose on ET within PS II from spinach was studied using time-resolved optical spectroscopy at 820 nm (Mamedov et al., 2021). These transient changes reflect the redox state of the primary electron donor P₆₈₀, and can be interpreted within the established structural and functional organization of PS II cofactors (Figure 1).

Upon photoexcitation, fast charge separation occurs between the primary electron donor P₆₈₀ and the primary quinone acceptor Q_A (Kern and Renger, 2007; Shevela and Messinger, 2012; Shen, 2015). Under physiological conditions, the oxidized P₆₈₀ ⁺ is efficiently reduced by the redox-active tyrosine Y_Z, thereby suppressing charge recombination. When donor-side electron transfer is impaired, backwards electron transfer from Q_A ⁻ to P₆₈₀ ⁺ becomes a competitive pathway. Accordingly, P₆₈₀ ⁺ reduction can proceed via two pathways: fast, physiologically relevant ET from Y_Z and a slower, nonproductive recombination with Q_A ⁻. The kinetics of ET from Y_Z to P₆₈₀ ⁺ varies significantly depending on the functional state of the PS II complex. In intact complexes, it occurs within 20–250 ns; upon removal of the Mn₄CaO₅ cluster at pH 6.5 this reaction slows down to 1–10 µs (Conjeaud and Mathis, 1980; Hays et al., 1999; Schlodder et al., 2015). It is believed that this slowdown is mediated by the changes of the protonation states of amino acids between Y_Z and P₆₈₀, which modulate the local dielectric environment of the protein medium and hydrogen-bonding network, thereby affecting the ET rate (Tommos and Babcock, 2000; Chamorovsky et al., 2007; Schlodder et al., 2015).

Kinetic analysis of P₆₈₀ ⁺ decay in Mn-depleted PS II at pH 6.5 in solution (trace a, Figure 2A) shows that its reduction is dominated (>90%) by a fast component (τ < 10 µs) attributed to the Y_Z-dependent pathway (Figure 2B). A minor fraction (∼10%) exhibits slower decay (>30 µs), consistent with charge recombination with Q_A ⁻. The complexes were subsequently embedded in a trehalose glass and equilibrated in controlled environment with fixed relative humidity level (r = 6–74%). The residual water content, determined by FTIR spectroscopy, corresponds to trehalose/water molar ratio decreasing from ∼9.2 at r = 74% to ∼1 at r = 6%.

Representative kinetics at high and low hydration (r = 74% and 11%, Figure 2A) show multiphasic P₆₈₀ ⁺ decay spanning over three orders of magnitude in time. Embedding PS II in trehalose and dehydration to r = 11% significantly slow the decay, increasing the overall lifetime by more than an order of magnitude relative to the solution. Rehydration to r = 74% partially restores faster kinetics, although the decay remains slower than in the solution.

Dehydration alters both the rates and amplitudes of decay components (Figure 2B): the contribution of charge recombination with Q_A ⁻ increases at the expense of the Y_Z-mediated electron transfer. When the residual water content is sufficiently reduced, but still relatively high (∼4 H₂O per trehalose molecule), the two pathways contribute equally, indicating that Y_Z → P₆₈₀ ⁺ electron transfer is virtually suppressed in about half of the PS II population. Further dehydration produces a negligible additional change, suggesting an establishment of PS II subpopulation trapped in a nonfunctional conformational state. The dehydration is accompanied by a significant slowdown of all kinetic components by ∼3–5 times, reflecting conformational “freezing” of the complex. Notably, dissolving the trehalose matrix returned the kinetics to its original lifetimes, demonstrating that PS II complex interaction with trehalose is fully reversible (trace d in Figure 2A).

These observations suggest that ET from Y_Z to P₆₈₀ ⁺ is strongly dependent on protein conformational dynamics. The progressive immobilization of the protein matrix induced by trehalose dehydration restricts these dynamics, thereby inhibiting the donor-side electron transfer in a fraction of the complexes. In contrast, charge recombination between Q_A ⁻ and P₆₈₀ ⁺ is less sensitive to such conformational constraints and becomes increasingly dominant as functional electron donation from Y_Z is suppressed. These results are comparable to low temperature studies, which describe forward electron transfer both from Y_Z to P₆₈₀ and from Q_A ⁻ to Q_B as inhibited below ∼240 K (Reinman and Mathis, 1981).

Time-resolved structural studies demonstrate that oxidation of tyrosine Y_Z by P₆₈₀ ⁺ is a dynamically coupled process involving subtle repositioning of Y_Z toward the oxidized chlorophyll pair, rearrangement of the Y_Z-His190 hydrogen bond, and reorganization of the surrounding water network to enable efficient proton-coupled electron transfer (Li et al., 2024). Embedding PS II in a dehydrated trehalose matrix interferes with these processes by strongly restricting protein conformational mobility and perturbing hydration-dependent hydrogen-bond networks. As a result, both the geometric and energetic requirements for Y_Z oxidation are compromised: necessary conformational substates become inaccessible, proton transfer pathways are disrupted, and nuclear reorganization is suppressed.

3 Trehalose-mediated stabilization of voltage generation by immobilized photosystem I under continuous illumination

The integration of light-driven thylakoid membrane-embedded protein complexes from oxygenic photosynthetic organisms into hybrid (semi-synthetic) systems represents a promising strategy for converting solar energy with high quantum efficiency and molecular-level selectivity.

Direct measurements of light-induced photovoltage (ΔV) have previously been reported for spinach oxygen-evolving PS II core complexes immobilized on a Millipore membrane filter (MF) and interfaced with an indium tin oxide (ITO) electrode under continuous illumination (Zaspa et al., 2020). The highest and most stable photoresponse was observed in the presence of trehalose and 2,6-dichloro-1,4-benzoquinone, which acts as a redox mediator facilitating electron transfer from the primary quinone acceptor Q_A to the electrode. The sustained steady-state photoactivity at room temperature in this ITO|PSII-MF|ITO system is likely due to trehalose-mediated retention of hydration water at the PSII surface, which stabilizes protein structure and preserves functional integrity under ambient conditions.

An analogous experiment was also performed using intact bacterial photosynthetic membrane vesicles (chromatophores) from the nonsulfur purple bacterium C. sphaeroides (Vitukhnovskaya et al., 2023). The maximal ΔV was observed in the presence of an ascorbate/N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) redox couple, coenzyme Q₀, the disaccharide trehalose, and antimycin A, an inhibitor of the cytochrome bc _*1* complex. Importantly, ∼70% of the initial photovoltage was retained after 30 days of storage at 23 °C in the dark under aerobic conditions, following reinjection of a small volume of fresh buffer containing the redox mediators into the ITO|Chr–MF|ITO system.

The protein–pigment complex of photosystem I (PS I) that mediates light-driven ET in particular have attracted considerable attention owing to their high stability, long-lived charge-separated states, and well-defined redox arrangement of cofactors within the reaction center (RC) (Golbeck, 2006; Hippler and Nelson, 2021; Bruce and Allakhverdiev, 2025). Despite these advantages, the practical implementation of PS I complexes in electrode-immobilized architectures remains limited by structural instability, interfacial denaturation, and loss of ET activity under non-physiological conditions.

Immobilization of PS I onto various matrices: TiO₂, FTO–fluorine-doped tin oxide, ITO–indium-doped tin oxide, platinum, gold, silicon, graphene, gallium (III) arsenide (GaAs), carbon nanotubes or other nanotubes, and redox polymers (see (Nguyen and Bruce, 2014) for review) often disrupts the delicate protein hydration shell, leading to altered orientation and diminished long-term photocurrent output. To address these limitations, various matrix-forming and stabilizing agents have been explored to emulate the native aqueous environment and mitigate interfacial stress at the bio–solid interface. Among these additives, disaccharide trehalose has emerged as a particularly effective bioprotectant.

We employed direct photovoltage (ΔV) technique developed in our laboratory (Zaspa et al., 2022; Mamedov et al., 2025) (Figure 3) to assess the effect of trehalose on the structural integrity and photoelectrochemical performance of PS I from cyanobacerium Synechocystis sp. PCC 6803 (Mamedov et al., 2025). In brief, PS I samples (∼30 μL, 2 mg Chl mL^-1) were deposited onto a nitrocellulose membrane filter (MF) (0.22 μm pore size, 150 μm thickness) placed on an indium tin oxide (ITO)-coated semiconductor glass slide and incubated in the dark for 10–15 min. A second ITO electrode was assembled to form an ITO|PS I–MF|ITO sandwich. Photovoltage was recorded using copper wires connected to an operational amplifier (Burr Brown 3554BM, United States) and ITO|PS I–MF|ITO. Signals were digitized using a CS8012 Gage system. Saturating continuous illumination was provided by an LXHL-LW3C white light LED (∼2.0 mW cm^-2).

In the presence of the ascorbate (Asc)/2,6-dichlorophenolindophenol (DCPIP) redox couple and methyl viologen–conditions optimal for PS I functioning in a buffer–detergent solution, as established by light-induced absorbance changes at 703 nm, continuous illumination of the ITO|PS I–MF|ITO system induced a rapid rise in ΔV amplitude during the first 20 s, followed by a decay to approximately one-third of the maximum amplitude over ∼400 s (Zaspa et al., 2022).

Bacterial RC (Francia et al., 2008), cyanobacterial PS I (Malferrari, Savitsky, Mamedov, et al., 2016) as well as spinach PS II core complexes (Mamedov et al., 2021) embedded into dry trehalose glass matrices have been shown to remain stable for months at room temperature. Consistently, in the system described above, addition of this disaccharide to the PS I suspension prior to adsorption onto the MF-coated ITO markedly altered the ΔV kinetics, resulting in rapid ΔV generation followed by a stable signal over 200 s of illumination (Zaspa et al., 2022; Mamedov et al., 2025). Figure 4 (bars 1 and 4) depicts the amplitude of ΔV generation by the ITO|PS I–MF|ITO assembly under continuous illumination in the presence of 10 mM Asc, 200 µM DCPIP, and 0.75 M trehalose.

This effect likely reflects the unique physicochemical properties of trehalose, as sucrose does not exhibit comparable behavior under conditions of similar viscosity. This finding was in line with previously reported protective effects of this disaccharide on the ΔV signal of PS II core complexes adsorbed onto an MF coated on an ITO (Zaspa et al., 2020).

After 24 h of storage at 22 °C in the dark in laboratory room, the ΔV amplitude decreases dramatically (by ∼9-fold), as shown in Figure 4 (bar 2). The latter effect is likely due to desiccation of the PS I microenvironment. Indeed, re-injection of ∼50 μL of fresh buffer containing exogenous mediators directly into the gap between the electrodes in the ITO|PS I-MF|ITO system restored the initial amplitude of photoresponse by ∼80% (bar 3). Similar results were also observed in spinach PS II core complexes (Zaspa et al., 2020) and bacterial intracytoplasmic membrane vesicles (chromatophores) (Vitukhnovskaya et al., 2023).

Considering these observations, we recently studied the effect of relative humidity (r) on ΔV generation in the ITO|PS I–MF|ITO system. A relative air humidity of ∼63% was maintained in a desiccator using a saturated NH₄NO₃ salt solution at 22 °C ± 1 °C during long term experiments. In the presence of the Asc/DCPIP redox couple and disaccharide trehalose, continuous illumination produced a maximal ΔV (bar 4). It was observed that the ΔV amplitude decreased by ∼15% after 7 days of storage at r = 65% (bar 5), followed by a substantial decline in the signal by day 14 (bar 6). Signal recovery to near-initial levels was achieved upon re-injection of fresh buffer without added redox mediators (bar 7), albeit with slower kinetics (see (Mamedov et al., 2025)). These results indicate that the storage of ITO|PS I–MF|ITO system at elevated controlled humidity in the presence of trehalose significantly enhances PS I stability, in contrast to almost complete signal loss within 24 h under ambient laboratory conditions. Notably, under these conditions, the redox mediators also retain functional activity over extended incubation periods.

Thus, trehalose, as a natural osmolyte, when dehydrated at r = 65% almost fully preserves the long-term functional and, presumably, structural integrity of the protein complex by suppressing conformational fluctuations and maintaining the hydration shell. Taken together, these effects indicate that the storage of the system at the constant relative humidity of 65% significantly prolongs the ability of PS I to generate the light-induced ΔV at room temperature (Mamedov et al., 2025). At the same time, for the ITO|PS I–MF|ITO system stored at room temperature over extended periods, the key limiting factor for maintaining photoactivity is the effective electronic coupling between PS I and the electrodes. In this context, maintaining water balance becomes critically important, as it determines the mobility of redox mediators and, consequently, the efficiency of their diffusion and trans-electrode ET. Therefore, a systematic investigation of the effect of relative humidity in the wide range (33%–85%) on the formation of a steady-state photoresponse appears to be a promising approach to reveal the most optimal conditions for the longest preservation of PS I activity in the ITO|PS I–MF|ITO system at room temperature.

These findings underscore the importance of hybrid photosynthetic systems as a platform for probing fundamental energy conversion processes while advancing the development of next-generation devices in solar energy, bioelectronics, and sensing.

4 Effect of trehalose on the flash-induced charge transfer in bacterial chromatopores in solution

Trehalose at high concentration in buffer solutions does not affect electron transfer in isolated PS I complexes, but significantly influences reactions in PS II. In PS II core complexes, trehalose stimulates the steady-state rate of oxygen evolution by the WOC (Mamedov et al., 2015) and enhances O₂ photoconsumption in Mn-depleted PS II complexes (Yanykin et al., 2016). Furthermore, the addition of trehalose to suspension of proteoliposomes containing PS II core-complexes accelerates the kinetics of electrogenic proton transport during the S₂ → S₃ and S₄ → S₀ transitions of the WOC (Mamedov et al., 2018). These observations suggest that high concentrations of trehalose stabilize the WOC in the conformation optimal for efficient O₂ and proton release. However, to the best of our knowledge, until recently the effect of trehalose in buffer solution had not been studied in bacterial chromatophores.

In this work, we used two different methods–a direct electrometrical method (Drachev et al., 1981) and electrochromic carotenoid shift (CS) (Crofts et al., 1983), to test the influence of trehalose on electrogenic reactions accompanying flash-induced charge transfer in chromatophores of the purple sulfur bacterium Cereibacter sphaeroides.

The direct electrometric method was used to monitor the flash-induced electrogenic reactions in bRC of chromatophores (Drachev et al., 1989). The results demonstrated that under conditions of experiment the 1^st flash induced fast unresolvable generation of transmembrane electric potential (Δψ) due to the charge separation between the primary electron donor, bacteriochlorophyll molecule P₈₇₀ (or P), and the secondary ubiquinone acceptor Q_B with the formation of P₈₇₀ ⁺Q_B ^–state. Besides, there was a further increase in the submillisecond time range due to electrogenic reduction of P⁺ by endogenous cytochrome (cyt) c ₂.

The second flash resulted in an additional increase in Δψ due to the electrogenic protonation of the secondary quinone acceptor Q_B ^–to Q_BH₂. The addition of trehalose induced a decrease and deceleration of the P⁺Q_B ^– → P⁺Q_BH₂ electrogenic reaction by ∼25%, but did not affect the cyt c ₂ ²⁺P₈₇₀ ⁺ → c ₂ ³⁺P₈₇₀ ET. We propose that the effect of trehalose on the protonation of Q_B ⁻ to Q_BH₂ arises from conformational changes of both the protein and Q_B ⁻ associated with this step, as previously demonstrated by Okamura et al. (Okamura et al., 2000), whereas reduction of P₈₇₀ ⁺ by cyt c ₂ does not require significant conformational rearrangement.

The measurements of the flash-induced CS changes were performed mostly to monitor electrogenic reactions in the cyt bc ₁-complex (Figure 5). In accordance with previous results (Drachev et al., 1989), addition of antimycin (Ant), which inhibits the reduction of ubiquinone-reducing Q_i redox center and prevents further turnovers of the cyt bc ₁-complex, results in a significant decrease in the slow phase of the CS. Subsequent addition of myxothiazol (Myx), which fully inhibits the oxidation of QH₂ at the ubiquinol-oxidizing center Q_o, leads to complete elimination of the slow phase of the CS.

The half-time of the slow Ant-sensitive kinetic phase is ∼2.8 ms for the control, and slows down to 12.6 ms in the presence of 0.8 M trehalose. This effect points to a significant slowing of electron transfer along the high-potential branch of cyt bc ₁-complex chain upon the oxidation of QH₂ at the Q_o site. The half-time of Myx-sensitive kinetic phase (in the presence of Ant) is 1.1 ms for the control, and slows down to 4.5 ms in the presence of trehalose.

The effect of Ant, both in the absence and in the presence of trehalose, is more pronounced than the effect of Myx in the presence of Ant. Addition of Myx without Ant, also completely inhibits the slow phase of the CS.

It is safe to suggest that trehalose, which affects the conformational dynamics and the global network of hydrogen bonds in the proteins, should primarily influence the charge transfer reactions associated with conformational mobility. Consequently, since charge transfer in the bc ₁-complex is accompanied by a significant displacement of the Rieske Iron Sulphur Protein (ISP) from the site in the vicinity of Q_o oxidizing center towards cyt c ₁, trehalose slows down electron transfer in the high-potential branch of the bc ₁. This reaction includes the sequential electron transfer Q_o → ISP → cyt c ₁ and the accompanying release of two protons from QH₂ in the Q_o-site inside chromatophore. The second electron transfers from Q_o ⁻ to the low-potential branch of the bc ₁ via b _L, b _H and Q_i. The ET reactions in the high and low potential branches proceed in a concerted manner. The rate-limiting step of the overall process is the movement of ISP from Q_o to cyt c ₁, which is affected by a high concentration of trehalose.

It is worth mentioning that, since the Q_o site and particularly ISP are located close to the inner surface of the chromatophore membrane, the effect of trehalose implies that this disaccharide is capable of penetration into these vesicles. The trehalose content inside chromatophores was determined by a colorimetric method with anthrone in a highly acidic environment (Dreywood, 1946). Thus, unexpectedly, the chromatophore membrane appeared to be permeable to trehalose.

5 Discussion

Desiccation of PS II in trehalose glassy matrices strongly restricts internal protein mobility, leading to (i) a pronounced slowdown of both forward ET (P₆₈₀ ⁺ reduction by Y_Z) and back-reaction (P₆₈₀ ⁺Q_A ^–- recombination), and (ii) heterogeneous inhibition of Y_Z oxidation in approximately half of the PS II population, in which charge recombination becomes more prominent. Comparable coupling between restricted protein dynamics and electron-transfer kinetics is observed in all three photosynthetic RC types—PS II, PS I, and bRC; the effects of trehalose considered in this review are summarized in Table 1. However, PS II is particularly sensitive to the effect of trehalose matrix, which becomes evident at a higher water/trehalose molar ratios than in PS I and bRC.

TABLE 1. Comparison of trehalose effects on different photosynthetic complexes.

Water retention capacity of trehalose matrix differs substantially depending on the incorporated photosynthetic complex. In PS I–trehalose systems equilibrated at 11% relative humidity, the water/trehalose molar ratio drops down to ∼0.5 (Malferrari, Savitsky, Mamedov, et al., 2016), similar to that of pure trehalose glass (Malferrari et al., 2014). In contrast, intact PS II–trehalose systems exhibit significantly higher water/trehalose ratio (∼1.3), while removal of Mn cluster lowers the ratio only slightly to ∼1.1 (Mamedov et al., 2021). Given that the trehalose/photosystem molar ratio in these preparations is on the order of tens of thousands, these data imply that PS II retains several thousand additional water molecules relative to PS I, likely corresponding at minimum to a complete first hydration shell surrounding the pigment–protein complex. This interpretation is consistent with structural studies demonstrating that PS II contains a substantially larger population of tightly bound water molecules than either PS I or bRC.

The persistence of a considerable pool of PS II-associated water even within highly dehydrated trehalose matrices provides important insight into the structural organization and dynamic properties of trehalose–protein glasses. These observations are inconsistent with the classical water-replacement hypothesis and instead support models based on water entrapment and anchorage, in which residual protein-bound water is retained and structurally stabilized within the glassy matrix.

Comparison with bRC further illustrates the complex relationship between hydration, conformational dynamics, and electron transfer. In bRCs, the addition of viscous agents such as glycerol, sucrose, or ethylene glycol slows recombination of the P⁺Q_A– charge-separated state, seemingly in accordance with modified Kramers equation, which implies that viscosity of external medium can dampen bRC protein vibrational modes coupled to electron transfer (Shopes and Wraight, 1987; Beece et al., 2002). In contrast, severe dehydration of bRCs in LDAO films (at the same low relative humidity of r = 11%) accelerates P⁺Q_A– recombination (Malferrari et al., 2011; Malferrari et al., 2013). In bRCs, photoinduced electron transfer from Q_A to Q_B is accompanied by conformational rearrangements that stabilize the charge-separated state; under extreme dehydration these motions are hindered, leading to destabilization of charge separation and faster recombination. No analogous stabilization-associated conformational changes have been identified in PS II, which likely explains why dehydration in trehalose matrices produces the opposite kinetic effect in bRC and PSII.

Studies of ITO|PS I-MF|ITO systems further demonstrate that trehalose not only restricts conformational dynamics and diffusion of soluble redox mediators, but also efficiently stabilizes the structure and function of photoactive systems during long-term storage. This stabilization is likely associated with preservation of hydrogen-bond networks at the protein–matrix interface, including the PS I hydration shell and surface-exposed residues. Consequently, trehalose protects biohybrid photoelectrical systems from dehydration-induced degradation while maintaining photoactivity under continuous illumination and elevated relative humidity for period of more than a week.

Trehalose has also been shown to permeate the membrane of bacterial chromatophores, indicating that its effects are not limited to external matrix interactions but also involve direct modulation of protein-complexes inside chromatophores. It is possible that chromatopore membrane permeability to trehalose is stipulated not by lipid properties but by so far unidentified nonspecific sugar transporter of protein nature.

Taken together, studies of trehalose matrix effects on ET in photosynthetic pigment-protein complexes–including PS II, PS I, and bRCs–provide insights extending well beyond fundamental structure-function relationships. By modulating protein dynamics, hydration, and dielectric properties, trehalose offers a versatile platform for stabilizing functional states of complex biomolecular assemblies. These findings deepen our understanding of how environmental constraints regulate biological ET and may serve as a foundation for the development of robust biohybrid and bioinspired systems for solar energy conversion, as well as new approaches for stabilization, storage, and deployment of functional protein complexes in biotechnology and medicine.

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Footnotes

Edited by: Alberto Mezzetti, Sorbonne Universités, France

Reviewed by: Mohammad Yusuf Zamal, University of Hyderabad, India

Huating Kong, Chinese Academy of Sciences (CAS), China