The resilience of biological systems to fluctuating environmental conditions is a crucial evolutionary advantage. In this study, we examine the thermo- and piezo-stability of the LH1-RC pigment-protein complex, the simplest photosynthetic unit, in three species of phototropic purple bacteria, each containing only this core complex. Among these species, Blastochloris viridis and Blastochloris tepida utilize bacteriochlorophyll b as the main lightharvesting pigment, while Rhodospirillum rubrum relies on bacteriochlorophyll a. Through spectroscopic analyses, we observed limited reversibility in the effects of temperature and pressure, likely due to the malleability of pigment binding sites within the light-harvesting LH1 complex. In terms of thermal robustness, LH1 complexes in a detergent environment progressively dissociate into dimeric (B820) and monomeric (B777) subunits. However, in the native membrane, degradation primarily occurs directly into B777 without the intermediate formation of B820. Interestingly, while high-pressure compression of core complexes from Blastochloris viridis and Blastochloris tepida caused significant changes in compressibility around 1.3 kbar and the formation of B777 and B820 subunits upon decompression, no such compressibility changes or pressure-induced dissociation were observed in Rhodospirillum rubrum complexes, even at pressures as high as 11 kbar. This study reveals significant differences in the piezo- and thermal properties of phototrophs containing either BChl a or BChl b, underscoring the critical role of structural factors in understanding the temperature- and pressure-induced denaturation phenomena in photosynthetic complexes. Rhodospirillum rubrum, in particular, stands out as one of the most thermodynamically stable systems among phototrophic microorganisms, capable of withstanding temperatures up to 70 degrees C and pressures exceeding 11 kbar.

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.

Irc3 is one of the six mitochondrial helicases described in Saccharomyces cerevisiae. Physiological functions of Irc3 are not completely understood as both DNA metabolic processes and mRNA translation have been suggested to be direct targets of the helicase. In vitro analysis of Irc3 has been hampered by the modest thermostability of the S. cerevisiae protein. Here, we purified a homologous helicase (Irc3op) of the thermotolerant yeast Ogataea pol-ymorpha that retains structural integrity and catalytic activity at temperatures above 40 degrees C. Irc3op can com-plement the respiratory deficiency phenotype of a S. cerevisiae irc3 Delta mutant, indicating conservation of biochemical functions. The ATPase activity of Irc3op is best stimulated by branched and double-stranded DNA cofactors. Single-stranded DNA binds Irc3op tightly but is a weak activator of the ATPase activity. We could also detect a lower level stimulation with RNA, especially with molecules possessing a compact three-dimensional structure. These results support the idea that that Irc3 might have dual specificity and remodel both DNA and RNA molecules in vivo. Furthermore, our analysis of kinetic parameters predicts that Irc3 could have a regulatory function via sensing changes of the mitochondrial ATP pool or respond to the accumulation of single-stranded DNA.

Color-tuning is a critical survival mechanism for photosynthetic organisms. Calcium ions are believed to enhance both spectral tuning and thermostability in obligatory calcium-containing sulfur purple bacteria. This study examined the thermo- and piezo stability of the LH1-RC complexes from two calcium-containing sulfur purple bacteria notable for their extreme red-shifted spectra. The results generally show limited reversibility of both temperature and pressure effects related to the malleability of calcium-binding sites. While the pressure-induced decomposition product closely resembles the calcium-depleted form of the chromoproteins, the thermally induced products reveal monomeric B777 and dimeric B820 forms of bacteriochlorophyll a, similar to those seen in non-sulfur purple bacteria treated with detergent. The study further found nearly unison melting of the protein tertiary and secondary structures. Overall, our findings do not support a direct link between color adjustment and thermodynamic stability in light-harvesting chromoproteins.

The conceptual formalism to understand the properties and function of chlorophylls in the gas and solution phases as well as in protein matrices is reviewed.This formalism is then applied to interpret modern high-resolution spectroscopic data, resulting from methods such as differential fluorescence line-narrowing spectroscopy and selective fluorescence excitation spectroscopy, which resolve individual vibrational transitions within the inhomogeneously broadened emission and absorption spectra of chlorophyll-a, bacteriochlorophyll-a, and pheophytin-a.Density functional theory and ab initio quantum chemical calculations are applied to interpret this data and fill in missing information needed to understand photosynthetic processes.The focus is placed on recognizing environmental and thermal effects, as well as the roles of Duschinsky rotation and non-adiabatic coupling in controlling the spectra.A critical feature of chlorophyll spectroscopy is determined to be absorption-emission asymmetry.Its ramifications for chlorophyll's function in photosystems are expected to be significant, as most current models for understanding their function assume that absorption and emission are symmetric, i.e. in the absence of relaxation processes, molecules coherently re-emit the light that they absorbed to enact exciton transport.The effect of the Duschinsky rotation is that after vibrational excitation during the electronic transition chlorophylls mostly emit light at different energies to what they absorb, while the effect of non-adiabatic coupling is that the polarization of the light is changed.

SummaryThe Saccharomyces cerevisiae Irc3 protein is a mitochondrial Superfamily II DNA helicase that, according to genomic data, is conserved in different yeast species. Here we characterize Irc3 helicase from the thermotolerant yeast Ogataea polymorpha (Irc3op) that throughout its helicase motor domain has approximately 82% similarity with the S. cerevisiae protein. Irc3op retains enzymatic activity at considerably higher temperatures than Irc3sc, displaying the fastest rate of ATP hydrolysis at 41 °C and a Tm of 45.3 °C for its secondary structure melting. We demonstrate that Irc3op is a structure-specific DNA helicase translocating on both single- and double-stranded DNA molecules. Like the homolog of S. cerevisiae, Irc3op can unwind only DNA molecules that contain branched structures. Different DNA molecules containing three- and four-way branches are utilized by Irc3op as unwinding substrates. Importantly, the preferred unwinding substrate of Irc3op is a DNA fork containing the nascent lagging strand, suggesting a possible role in replication restart following a block in leading strand polymerization. A lower unwinding efficiency of four-way branched DNA molecules could explain why Irc3op only partially complements the irc3Δ phenotype in S. cerevisiae.

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.

Significant asymmetry found between the high-resolution Qy emission and absorption spectra of chlorophyll-a is herein explained, providing basic information needed to understand photosynthetic exciton transport and photochemical reactions. The Qy spectral asymmetry in chlorophyll has previously been masked by interference in absorption from the nearby Qx transition, but this effect has recently been removed using extensive quantum spectral simulations or else by analytical inversion of absorption and magnetic circular dichroism data, allowing high-resolution absorption information to be accurately determined from fluorescence-excitation spectra. To compliment this, here, we measure and thoroughly analyze the high-resolution differential fluorescence line narrowing spectra of chlorophyll-a in trimethylamine and in 1-propanol. The results show that vibrational frequencies often change little between absorption and emission, yet large changes in line intensities are found, this effect also being strongly solvent dependent. Among other effects, the analysis in terms of four basic patterns of Duschinsky-rotation matrix elements, obtained using CAM-B3LYP calculations, predicts that a chlorophyll-a molecule excited into a specific vibrational level, may, without phase loss or energy relaxation, reemit the light over a spectral bandwidth exceeding 1,000 cm−1 (0.13 eV) to influence exciton-transport dynamics.

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.

A weak absorption tail related to the Qy singlet electronic transition of solvated chlorophyll a is discovered using sensitive anti-Stokes fluorescence excitation spectroscopy. The quasi-exponentially decreasing tail was, at ambient temperature, readily observable as far as −2400 cm−1 from the absorption peak and at relative intensity of 10−7. The tail also weakened rapidly upon cooling the sample, implying its basic thermally activated nature. The shape of the spectrum as well as its temperature dependence were qualitatively well reproduced by quantum chemical calculations involving the pigment intramolecular vibrational modes, their overtones, and pairwise combination modes, but no intermolecular/solvent modes. A similar tail was observed earlier in the case of bacteriochlorophyll a, suggesting generality of this phenomenon. Long vibronic red tails are, thus, expected to exist in all pigments of light-harvesting relevance at physiological temperatures.

Optical absorption and fluorescence spectra of molecules in condensed phases often show extensive sidebands. Originating from electron-vibrational and electron-phonon couplings, these spectral tails bear important information on the dynamics of the electronic states and processes the molecules are involved in. The sidebands observed in conjugate Qy absorption and fluorescence spectra of chlorophyll a and bacteriochlorophyll a are relatively weak, characterized by the Huang-Rhys factor much less than one. Therefore, it is widely considered that only fundamental intramolecular modes are responsible for their formation. Here we provide evidence for extra-long and structured fluorescence tails extending to as far as ~960 nm in chlorophyll a and ~1140 nm in bacteriochlorophyll a into the infrared part of the optical spectrum. According to quantum chemical simulations, these sidebands are mainly contributed to by vibrational overtones of the fundamental modes. Because energy transfer and relaxation processes generally depend on vibronic overlap integrals, these findings potentially contribute into better understanding of many vital photoinduced phenomena including photosynthetic light harvesting.

In photosynthesis, collective pigment excitations – excitons – facilitate chemical reactions for sustainable biological function. Here, the effect of hydrostatic pressure – an important thermodynamic stress factor – on optical spectral properties of excitons in the cyclic light-harvesting 2 pigment-protein complex from photosynthetic bacterium Rhodoblastus acidophilus was first studied. The high pressure-induced modifications of absorption, fluorescence and polarized fluorescence excitation spectra were theoretically analyzed in terms of the disordered molecular exciton model. We uniquely show that the observed shift of the spectra under pressure is largely governed by the pressure-induced rise of two factors: the exciton displacement energy and the exciton coupling energy, which increases the spread of the exciton state manifold. A significant increase of static energy disorder revealed by model calculations suggests that high-pressure compressing of the complex is actually accompanied by altered spatial orientations and conformations of the protein-embedded pigment molecules.