Recent steady-state and time-resolved spectroscopy investigations have revealed that Photosystem II core complexes (PSII CCs) are capable of undergoing marked light-induced structural reorganizations even upon the formation of stable charge separation state PSII C . These reversible changes observed at physiological and cryogenic temperatures lead to the gradual formation of light adapted charge-separated state PSII L . It has been proposed that the underlying physical mechanisms involve complex dielectric relaxation processes due to the generation of stationary and transient electric fields, in which structural rigidity and flexibility of the related protein complexes play equally important roles. In order to gain further insights into the nature of structural dynamics of PSII, here, the response of the chlorophyll- a transient fluorescence in PSII CC prepared from Thermosthicus vulcanus was studied at 78 K under high hydrostatic pressures applied either at room temperature or at 78 K. PSII CC exhibits remarkable flexibility against high hydrostatic pressures up to 12 kbar and cryogenic temperatures down to 78 K, as evidenced by the fair shape overlap between the initial fluorescence spectrum at ambient conditions and the final fluorescence spectra recorded under various pressure-temperature treatments. This observed reversibility further implies that the variations in these parameters do not significantly disrupt the pigment binding pockets within PSII CC. However, as is typical of glassy protein samples, the pressure-induced spectral and kinetic effects were contingent on the sample's treatment history. These effects, such as bathochromic shifts and broadenings of the spectra, were not only quantitatively greater, but also qualitatively different, such as the disruption of antenna energy transfer pathways or inhibition of the induction of variable chlorophyll fluorescence when pressure was applied at ambient temperature compared to 78 K. The relatively modest spectral shift rates, not exceeding about − 20 cm⁻¹/kbar, further suggest the absence of strongly coupled chlorophyll units significantly contributing to PSII CC fluorescence.

Hydrogen bonding plays a crucial role in stabilizing proteins throughout their folding process. In photosynthetic light-harvesting chromoproteins, enriched with pigment chromophores, hydrogen bonds also fine-tune optical absorption to align with the solar irradiation spectrum. Despite its significance for photosynthesis, the precise mechanism of spectral tuning through hydrogen bonding remains inadequately understood. This study investigates wild-type and genetically engineered LH2 and LH1 light-harvesting complexes from Rhodobacter sphaeroides using a unique set of advanced spectroscopic techniques combined with simple exciton modeling. Our findings reveal an intricate interplay between exciton and site energy shift mechanisms, challenging the prevailing belief that spectral changes observed in these complexes upon the modification of tertiary structure hydrogen bonds almost directly follow shifting site energies. These deeper insights into natural adaptation processes hold great promise for advancing sustainable solar energy conversion technologies.

Photosynthesis is a vital process that converts sunlight into energy for the Earth’s ecosystems. Color adaptation is crucial for different photosynthetic organisms to thrive in their ecological niches. Although the presence of collective excitons in light-harvesting complexes is well known, the role of delocalized excited states in color tuning and excitation energy transfer remains unclear. This study evaluates the characteristics of photosynthetic excitons in sulfur and non-sulfur purple bacteria using advanced optical spectroscopic techniques at reduced temperatures. The exciton effects in these bacteriochlorophyll a-containing species are generally much stronger than in plant systems that rely on chlorophylls. Their exciton bandwidth varies based on multiple factors such as chromoprotein structure, surroundings of the pigments, carotenoid content, hydrogen bonding, and metal ion inclusion. The study nevertheless establishes a linear relationship between the exciton bandwidth and Qy singlet exciton absorption peak, which in case of LH1 core complexes from different species covers almost 130 nm. These findings provide important insights into bacterial color tuning and light-harvesting, which can inspire sustainable energy strategies and devices.

Much of the thermodynamic parameter values that support life are set by the properties of proteins. While the denaturing effects of pressure and temperature on proteins are well documented, their precise structural nature is rarely revealed. This work investigates the destabilization of multiple Ca2+ binding sites in the cyclic LH1 light-harvesting membrane chromoprotein complexes from two Ca-containing sulfur purple bacteria by hydrostatic high-pressure perturbation spectroscopy. The native (Ca-saturated) and denatured (Ca-depleted) phases of these complexes are well distinguishable by much-shifted bacteriochlorophyll a exciton absorption bands serving as innate optical probes in this study. The pressure-induced denaturation of the complexes related to the failure of the protein Ca-binding pockets and the concomitant breakage of hydrogen bonds between the pigment chromophores and protein environment were found cooperative, involving all or most of the Ca2+ binding sites, but irreversible. The strong hysteresis observed in the spectral and kinetic characteristics of phase transitions along the compression and decompression pathways implies asymmetry in the relevant free energy landscapes and activation free energy distributions. A phase transition pressure equal to about 1.9 kbar was evaluated for the complexes from Thiorhodovibrio strain 970 from the pressure dependence of biphasic kinetics observed in the minutes to 100 h time range.

Photosynthesis is a critical process that harnesses solar energy to sustain life across Earth's intricate ecosystems. Central to this phenomenon is nuanced adaptation to a spectrum spanning approximately from 300 nm to nearly 1100 nm of solar irradiation, a trait enabling plants, algae, and phototrophic bacteria to flourish in their respective ecological niches. While the Sun’s thermal radiance and the Earth’s atmospheric translucence naturally constrain the ultraviolet extent of this range, a comprehension of how to optimize the utilization of near-infrared light has remained an enduring pursuit. This study unveils the remarkable capacity of the bacteriochlorophyll b -containing purple photosynthetic bacterium Blastochloris viridis to harness solar energy at extreme long wavelengths, a property attributed to a synergistic interplay of exciton and site energy shift mechanisms. Understanding the unique native adaptation mechanisms offers promising prospects for advancing sustainable energy technologies of solar energy conversion.

Cooperative luminescence (CL) of Yb3+ pairs was investigated in Yb-doped Li6Y(BO3)(3) crystals. It was reliably distinguished from other types of up-conversion luminescence related to the energy transfer from Yb3+ ions to other rare-earth ions unintentionally introduced into the crystal. The CL was observed in the region of 19,000-21,000 cm(-1) (526-476 nm) under excitation at about 10,285 cm(-1) (972.3 nm) by continuous wave or pulsed lasers. The emission band is nearly featureless at room temperature but develops a pronounced line spectrum at low temperatures. The width of the absorption line corresponding to the F-2(7/2) -> F-2(5/2) transition of the Yb3+ ions at 10,285 cm(-1) demonstrates an extreme dependence on temperature, increasing by a factor of 250 from 6 to 300 K. The dependence of the CL intensity on laser irradiation power in the continuous wave mode is interpreted by an analytical model based on the intensity saturation effect strongly related to the peak amplitude of the absorption line. The model updates earlier descriptions of this dependence given in terms of power functions with "anomalous" exponents differing from the value of 2 expected for the luminescence of pair centres and accounts for the observed sharp temperature dependence of the saturation intensity. The decay kinetics of CL was shown to be approximately twice as fast as that of a single-ion luminescence, as expected. However, the decay times were found to be decreasing with increasing Yb concentration, which is explained by excitation escape from Yb3+ pairs due to energy transfer along Yb chains.

The denaturation of globular proteins by high pressure is frequently associated with the release of internal voids and/or the exposure of the hydrophobic protein interior to a polar aqueous solvent. Similar evidence with respect to membrane proteins is not available. Here, we investigate the impact of hydrostatic pressures reaching 12 kbar on light-harvesting 2 integral membrane complexes of purple photosynthetic bacteria using two types of innate chromophores in separate strategic locations: bacteriochlorophyll-a in the hydrophobic interior and tryptophan at both protein-solvent interfacial gateways to internal voids. The complexes from mutant Rhodobacter sphaeroides with low resilience against pressure were considered in parallel with the naturally robust complexes of Thermochromatium tepidum. In the former case, a firm correlation was established between the abrupt blue shift of the bacteriochlorophyll-a exciton absorption, a known indicator of the breakage of tertiary structure pigment-protein hydrogen bonds, and the quenching of tryptophan fluorescence, a supposed result of further protein solvation. No such effects were observed in the reference complex. While these data may be naively taken as supporting evidence of the governing role of hydration, the analysis of atomistic model structures of the complexes confirmed the critical part of the structure in the pressure-induced denaturation of the membrane proteins studied.

Anna A. Oleshkevich, Specific features of change in enzymate activity in

Flexible color adaptation to available ecological niches is vital for the photosynthetic organisms to thrive. Hence, most purple bacteria living in the shade of green plants and algae apply bacteriochlorophyll a pigments to harvest near infra-red light around 850–875 nm. Exceptions are some Ca2+-containing species fit to utilize much redder quanta. The physical basis of such anomalous absorbance shift equivalent to ~5.5 kT at ambient temperature remains unsettled so far. Here, by applying several sophisticated spectroscopic techniques, we show that the Ca2+ ions bound to the structure of LH1 core light-harvesting pigment–protein complex significantly increase the couplings between the bacteriochlorophyll pigments. We thus establish the Ca-facilitated enhancement of exciton couplings as the main mechanism of the record spectral red-shift. The changes in specific interactions such as pigment–protein hydrogen bonding, although present, turned out to be secondary in this regard. Apart from solving the two-decade-old conundrum, these results complement the list of physical principles applicable for efficient spectral tuning of photo-sensitive molecular nano-systems, native or synthetic.

While photosynthesis thrives at close to normal pressures and temperatures, it is presently well known that life is similarly commonplace in the hostile environments of the deep seas as well as around hydrothermal vents. It is thus imperative to understand how key biological processes perform under extreme conditions of high pressures and temperatures. Herein, comparative steady-state and picosecond time-resolved spectroscopic studies were performed on membrane-bound and detergent-purified forms of a YM210W mutant reaction center (RC) from Rhodobacter sphaeroides under modulating conditions of high hydrostatic pressure applied at ambient temperature. A previously established breakage of the lone hydrogen bond formed between the RC primary donor and the protein scaffold was shown to take place in the membrane-bound RC at an almost 3 kbar higher pressure than in the purified RC, confirming the stabilizing role of the lipid environment for membrane proteins. The main change in the multi-exponential decay of excited primary donor emission across the experimental 10 kbar pressure range involved an over two-fold continuous acceleration, the kinetics becoming increasingly mono-exponential. The fastest component of the emission decay, thought to be largely governed by the rate of primary charge separation, was distinctly slower in the membrane-bound RC than in the purified RC. The change in character of the emission decay with pressure was explained by the contribution of charge recombination to emission decreasing with pressure as a result of an increasing free energy gap between the charge-separated and excited primary donor states. Finally, it was demonstrated that, in contrast to a long-term experimental paradigm, adding a combination of sodium ascorbate and phenazine methosulfate to the protein solution potentially distorts natural photochemistry in bacterial RCs.

An increased robustness against high temperature and the much red-shifted near-infrared absorption spectrum of excitons in the LH1-RC core pigment-protein complex from the thermophilic photosynthetic purple sulfur bacterium Thermochromatium tepidum has recently attracted much interest. In the present work, thermal and hydrostatic pressure stability of the peripheral LH2 and core LH1-RC complexes from this bacterium were in parallel investigated by various optical spectroscopy techniques applied over a wide spectral range from farultraviolet to near-infrared. In contrast to expectations, very distinct robustness of the complexes was established, while the sturdiness of LH2 surpassed that of LH1-RC both with respect to temperatures between 288 and 360 K, and pressures between 1 bar and 14 kbar. Subtle structural variances related to the hydrogen bond network are likely responsible for the extra stability of LH2.

Photochemical charge separation is key to biological solar energy conversion. Although many features of this highly quantum-efficient process have been described, others remain poorly understood. Herein, ultrafast fluorescence barospectroscopy is used for the first time to obtain insights into the mechanism of primary charge separation in a YM210W mutant bacterial reaction center under novel surrounding modulating conditions. Over a range of applied hydrostatic pressures reaching 10 kbar, the rate of primary charge separation monotonously increased and that of the electron transfer to secondary acceptor decreased. While the inferred free energy gap for charge separation generally narrowed with increasing pressure, a pressure-induced break of a protein-cofactor hydrogen bond observed at similar to 2 kbar significantly (by 219 cm(-1) or 27 meV) increased this gap, resulting in a drop in fluorescence. The findings strongly favor a model for primary charge separation that incorporates charge recombination and restoration of the excited primary pair state, over a purely sequential model. We show that the main reason for the almost threefold acceleration of the primary electron transfer rate is the pressure-induced increase of the electronic coupling energy, rather than a change of activation energy. We also conclude that across all applied pressures, the primary electron transfer in the mutant reaction center studied can be considered nonadiabatic, normal region, and thermally activated.

As a basis of photosynthesis, photoinduced oxidation of (bacterio)chlorophyll molecules in the special reaction center complexes has been a subject of extensive research. In contrast, the generally harmful photooxidation of antenna chromoproteins has received much less attention. Here, we have established the permanent structural changes in the LH2 antenna bacteriochlorophyll-protein complex from a sulfur photosynthetic purple bacterium Ectothiorhodospira haloalkaliphila taking place at physiological conditions upon intense optical irradiation. To this end, a crystal structure of the LH2 complex from E. haloalkaliphila was first resolved by X-ray diffraction to 3.7 Å, verifying a great similarity with the earlier structure from Phaesporillum molischianum. Analysis of the various steady-state and picosecond time-resolved optical spectroscopy data and related model simulations then confirmed that the major spectral effects observed-bleaching and blue-shifting of the B850 exciton band and correlated emergence of a higher-energy C700 exciton band-are associated with photooxidation of increasing numbers of B850 bacteriochlorophylls into 3-acetyl-chlorophylls, with no noticeable damage to the pigment-binding protein scaffold. A prospective noninvasive method for an in situ optical control of excitons by selective photooxidation of pigment chromophores was thus revealed and demonstrated in a structurally well-defined native system.

The optical absorption and fluorescence response to external high pressure of the reaction center membrane chromoprotein complex from the wild-type non-sulfur photosynthetic bacterium Rhodobacter sphaeroides was investigated using the native pigment cofactors as local molecular probes of the reaction center structure at physiological (ambient) and cryogenic (79 K) temperatures. In detergent-purified complexes at ambient temperature, abrupt blue shift and accompanied broadening of the special pair band was observed at about 265 MPa. These reversible in pressure features were assigned to a pressure-induced rupture of a lone hydrogen bond that binds the photo-chemically active L-branch primary electron donor bacteriochlorophyll cofactor to the surrounding protein scaffold. In native membrane-protected complexes the hydrogen bond rupture appeared significantly restricted and occurred close to about 500 MPa. The free energy change associated with the rupture of the special pair hydrogen bond in isolate complexes was estimated to be equal to about 12 kJ mol(-1). In frozen samples at cryogenic temperatures the hydrogen bond remained apparently intact up to the maximum utilized pressure of 600 MPa. In this case, however, heterogeneous spectral response of the cofactors from the L-and M-branches was observed due to anisotropic build-up of the protein structure. While in solid phase, the special pair fluorescence as a function of pressure exactly followed the respective absorption spectrum at a constant Stokes shift, at ambient temperature, the two paths began to deviate strongly from one other at the hydrogen bond rupture pressure. This effect was tentatively interpreted by different emission properties of hydrogen-bound and hydrogen-unbound special pair exciton states.

The LH1-RC core complex from the thermophilic photosynthetic purple sulfur bacterium Thermochromatium tepidum has recently attracted interest of many researchers because of its several unique properties, such as increased robustness against environmental hardships and the much red-shifted near infrared absorption spectrum of the LH1 antenna exciton polarons. The known near-atomic-resolution crystal structure of the complex well supported this attention. Yet several mechanistic aspects of the complex prominence remained to be understood. In this work, samples of the native, Ca2+-containing core complexes were investigated along with those destabilized by Ba2+ substitution, using various spectrally selective steady-state and picosecond time-resolved spectroscopic techniques at physiological and cryogenic temperatures. As a result, the current interpretation of exciton spectra of the complex was significantly clarified. Specifically, by evaluating the homogeneous and inhomogeneous compositions of the spectra, we showed that there is little to no effect of cation substitution on the dynamic or kinetic properties of antenna excitons. Reasons of the extra red shift of absorption/fluorescence spectra observed in the Ca-LH1-RC and not in the Ba-LH1-RC complex should thus be searched in subtle structural differences following the inclusion of different cations into the core complex scaffold.

In the present work, spectral and kinetic changes accompanying the assembly of the light-harvesting 1 (LH1) complex with the reaction center (RC) complex into monomeric RC-LH1 and dimeric RC-LH1-PufX core complexes of the photosynthetic purple bacterium Rhodobacter sphaeroides are systematically studied over the temperature range of 4.5-300K. The samples were interrogated with a combination of optical absorption, hole burning, fluorescence excitation, steady state and picosecond time resolved fluorescence spectroscopy. Fair additivity of the LH1 and RC absorption spectra suggests rather weak electronic coupling between them. A low-energy tail revealed at cryogenic temperatures in the absorption spectra of both monomeric and dimeric core complexes is proved to be due to the special pair of the RC. At selected excitation intensity and temperature, the fluorescence decay time of core complexes is shown to be a function of multiple factors, most importantly of the presence/absence of RCs, the supramolecular architecture (monomeric or dimeric) of the complexes, and whether the complexes were studied in a native membrane environment or in a detergent - purified state.

In the purple phototrophic bacterium Rhodobacter sphaeroides, light harvesting LH2 complexes transfer absorbed solar energy to RC–LH1–PufX core complexes, which are mainly found in the dimeric state. Many other purple phototrophs have monomeric core complexes and the basis for requiring dimeric cores is not fully established, so we analysed strains of Rba. sphaeroides that contain either native dimeric core complexes or altered monomeric cores harbouring a deletion of the first 12 residues from the N-terminus of PufX, which retains the PufX polypeptide but removes the major determinant of core complex dimerization. Membranes were purified from strains with dimeric or monomeric cores, and with either high or low levels of the LH2 complex. Samples were interrogated with absorption, steady-state fluorescence, and picosecond time-resolved fluorescence kinetic spectroscopies to reveal their light-harvesting and energy trapping properties. We find that under saturating excitation light intensity the photosynthetic membranes containing LH2 and monomeric core complexes have fluorescence lifetimes nearly twice that of membranes with LH2 plus dimeric core complexes. This trend of increased lifetime is maintained with RCs in the open state as well, and for two different levels of LH2 content. Thus, energy trapping is more efficient when photosynthetic membranes of Rba. sphaeroides consist of RC–LH1–PufX dimers and LH2 complexes.

Bacterial light-harvesting pigment-protein complexes are very efficient at converting photons into excitons and transferring them to reaction centers, where the energy is stored in a chemical form. Optical properties of the complexes are known to change significantly in time and also vary from one complex to another; therefore, a detailed understanding of the variations on the level of single complexes and how they accumulate into effects that can be seen on the macroscopic scale is required. While experimental and theoretical methods exist to study the spectral properties of light-harvesting complexes on both individual complex and bulk ensemble levels, they have been developed largely independently of each other. To fill this gap, we simultaneously analyze experimental low-temperature single-complex and bulk ensemble optical spectra of the light-harvesting complex-2 (LH2) chromoproteins from the photosynthetic bacterium Rhodopseudomonas acidophila in order to find a unique theoretical model consistent with both experimental situations. The model, which satisfies most of the observations, combines strong exciton-phonon coupling with significant disorder, characteristic of the proteins. We establish a detailed disorder model that, in addition to containing a C_{2}-symmetrical modulation of the site energies, distinguishes between static intercomplex and slow conformational intracomplex disorders. The model evaluations also verify that, despite best efforts, the single-LH2-complex measurements performed so far may be biased toward complexes with higher Huang-Rhys factors.

We have recorded fluorescence-excitation and emission spectra from single LH2 complexes from Rhodopseudomonas (Rps.) acidophila. Both types of spectra show strong temporal spectral fluctuations that can be visualized as spectral diffusion plots. Comparison of the excitation and emission spectra reveals that for most of the complexes the lowest exciton transition is not observable in the excitation spectra due to the cutoff of the detection filter characteristics. However, from the spectral diffusion plots we have the full spectral and temporal information at hand and can select those complexes for which the excitation spectra are complete. Correlating the red most spectral feature of the excitation spectrum with the blue most spectral feature of the emission spectrum allows an unambiguous assignment of the lowest exciton state. Hence, application of fluorescence-excitation and emission spectroscopy on the same individual LH2 complex allows us to decipher spectral subtleties that are usually hidden in traditional ensemble spectroscopy.

In this study, we use the photosynthetic purple bacterium Rhodobacter sphaeroides to find out how the acclimation of photosynthetic apparatus to growth conditions influences the rates of energy migration toward the reaction center traps and the efficiency of charge separation at the reaction centers. To answer these questions we measured the spectral and picosecond kinetic fluorescence responses as a function of excitation intensity in membranes prepared from cells grown under different illumination conditions. A kinetic model analysis yielded the microscopic rate constants that characterize the energy transfer and trapping inside the photosynthetic unit as well as the dependence of exciton trapping efficiency on the ratio of the peripheral LH2 and core LH1 antenna complexes, and on the wavelength of the excitation light. A high quantum efficiency of trapping over 80% was observed in most cases, which decreased toward shorter excitation wavelengths within the near infrared absorption band. At a fixed excitation wavelength the efficiency declines with the LH2/LH1 ratio. From the perspective of the ecological habitat of the bacteria the higher population of peripheral antenna facilitates growth under dim light even though the energy trapping is slower in low light adapted membranes. The similar values for the trapping efficiencies in all samples imply a robust photosynthetic apparatus that functions effectively at a variety of light intensities.