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We present a nonlinear spectroelectrochemical technique to investigate photosynthetic protein complexes. The PEC2DES setup combines photoelectrochemical detection (PEC) that selectively probes the protein photogenerated charges output with two-dimensional electronic spectroscopy (2DES) excitation that spreads the nonlinear optical response of the system in an excitation-detection map. PEC allows us to distinguish the contribution of charge separation (CS) from other de-excitation pathways, whereas 2DES allows us to disentangle congested spectral bands and evaluate the exciton dynamics (decays and coherences) of the photosystem complex. We have developed in operando phase-modulated 2DES by measuring the photoelectrochemical reaction rate in a biohybrid electrode functionalized with a plant photosystem complex I-light harvesting complex I (PSI-LHCI) layer. Optimizing the photoelectrochemical current signal yields reliable linear spectra unequivocally associated with PSI-LHCI. The 2DES signal is validated by nonlinear features like the characteristic vibrational coherence at 750 cm-1. However, no energy transfer dynamics is observed within the 450 fs experimental window. These intriguing results are discussed in the context of incoherent mixing resulting in reduced nonlinear contrast for multichromophoric complexes, such as the 160 chlorophyll PSI. The presented PEC2DES method identifies generated charges unlike purely optical 2DES and opens the way to probe the CS channel in multichromophoric complexes.
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Cyanobacteria were the first microorganisms that released oxygen into the atmosphere billions of years ago. To do it safely under intense sunlight, they developed strategies that prevent photooxidation in the photosynthetic membrane, by regulating the light-harvesting activity of their antenna complexes-the phycobilisomes-via the orange-carotenoid protein (OCP). This water-soluble protein interacts with the phycobilisomes and triggers nonphotochemical quenching (NPQ), a mechanism that safely dissipates overexcitation in the membrane. To date, the mechanism of action of OCP in performing NPQ is unknown. In this work, we performed ultrafast spectroscopy on a minimal NPQ system composed of the active domain of OCP bound to the phycobilisome core. The use of this system allowed us to disentangle the signal of the carotenoid from that of the bilins. Our results demonstrate that the binding to the phycobilisomes modifies the structure of the ketocarotenoid associated with OCP. We show that this molecular switch activates NPQ, by enabling excitation-energy transfer from the antenna pigments to the ketocarotenoid.
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Proteínas Bacterianas , Carotenoides , Cianobacterias , Ficobilisomas , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Carotenoides/química , Carotenoides/metabolismo , Cianobacterias/metabolismo , Cianobacterias/química , Ficobilisomas/química , Ficobilisomas/metabolismo , Pigmentos Biliares/química , Pigmentos Biliares/metabolismo , Procesos FotoquímicosRESUMEN
Improving photosynthesis, the fundamental process by which plants convert light energy into chemical energy, is a key area of research with great potential for enhancing sustainable agricultural productivity and addressing global food security challenges. This perspective delves into the latest advancements and approaches aimed at optimizing photosynthetic efficiency. Our discussion encompasses the entire process, beginning with light harvesting and its regulation and progressing through the bottleneck of electron transfer. We then delve into the carbon reactions of photosynthesis, focusing on strategies targeting the enzymes of the Calvin-Benson-Bassham (CBB) cycle. Additionally, we explore methods to increase CO2 concentration near the Rubisco, the enzyme responsible for the first step of CBB cycle, drawing inspiration from various photosynthetic organisms, and conclude this section by examining ways to enhance CO2 delivery into leaves. Moving beyond individual processes, we discuss two approaches to identifying key targets for photosynthesis improvement: systems modeling and the study of natural variation. Finally, we revisit some of the strategies mentioned above to provide a holistic view of the improvements, analyzing their impact on nitrogen use efficiency and on canopy photosynthesis.
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Oxygenic photosynthesis, the process that converts light energy into chemical energy, is traditionally associated with the absorption of visible light by chlorophyll molecules. However, recent studies have revealed a growing number of organisms capable of using far-red light (700-800 nm) to drive oxygenic photosynthesis. This phenomenon challenges the conventional understanding of the limits of this process. In this review, we briefly introduce the organisms that exhibit far-red photosynthesis and explore the different strategies they employ to harvest far-red light. We discuss the modifications of photosynthetic complexes and their impact on the delivery of excitation energy to photochemical centers and on overall photochemical efficiency. Finally, we examine the solutions employed to drive electron transport and water oxidation using relatively low-energy photons. The findings discussed here not only expand our knowledge of the remarkable adaptation capacities of photosynthetic organisms but also offer insights into the potential for enhancing light capture in crops.
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Oxígeno , Fotosíntesis , Luz Roja , Clorofila/metabolismo , Clorofila/química , Transporte de Electrón , Oxidación-Reducción , Oxígeno/metabolismo , Agua/metabolismo , Agua/químicaRESUMEN
For many applications that involve measuring ultrafast optical phenomena, the streak camera is the device of choice because of its excellent time resolution, its high sensitivity, the possibility to simultaneously measure lifetimes and spectra, and because it can capture the temporal dynamics in a single shot. Nevertheless, to obtain a good time resolution, often narrow slits have to be employed that restrict the image source area and, therefore, limit the light collection efficiency in the experiment. For some applications, it is therefore challenging to find an acceptable balance between the time resolution and signal-to-noise ratio. To overcome this limitation, we have devised the propagation synchronous integration principle for the streak camera, in which an effective spatio-dependent time-shift in the excitation of a sample is introduced and counteracted by the streak sweep, thereby effectively allowing for an increased image source area while maintaining the optimal time resolution. Using the Optronis streak camera with tunable streak sweep and large (1 mm) photocathode width, we could achieve a sevenfold increase in light collection efficiency without affecting the time resolution. Furthermore, we were also able to achieve an 11-fold increase in light collection at the cost of a 26% decrease in the time resolution.
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Plants are designed to utilize visible light for photosynthesis. Expanding this light absorption toward the far-red could boost growth in low-light conditions and potentially increase crop productivity in dense canopies. A promising strategy is broadening the absorption of antenna complexes to the far-red. In this study, we investigated the capacity of the photosystem I antenna protein Lhca4 to incorporate far-red absorbing chlorophylls d and f and optimize their spectra. We demonstrate that these pigments can successfully bind to Lhca4, with the protein environment further red-shifting the chlorophyll d absorption, markedly extending the absorption range of this complex above 750 nm. Notably, chlorophyll d substitutes the canonical chlorophyll a red-forms, resulting in the most red-shifted emission observed in a plant light-harvesting complex. Using ultrafast spectroscopy, we show that the introduction of these novel chlorophylls does not interfere with the excited state decay or the energy equilibration processes within the complex. The results demonstrate the feasibility of engineering plant antennae to absorb deeper into the far-red region while preserving their functional and structural integrity, paving the way for innovative strategies to enhance photosynthesis.
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Clorofila , Complejos de Proteína Captadores de Luz , Clorofila A , Complejos de Proteína Captadores de Luz/química , Clorofila/metabolismo , Fotosíntesis , Análisis Espectral , Complejo de Proteína del Fotosistema I/química , PlantasRESUMEN
Nonphotochemical quenching (NPQ) is the process that protects photosynthetic organisms from photodamage by dissipating the energy absorbed in excess as heat. In the model green alga Chlamydomonas reinhardtii, NPQ is abolished in the knock-out mutants of the pigment-protein complexes LHCSR3 and LHCBM1. However, while LHCSR3 is a pH sensor and switches to a quenched conformation at low pH, the role of LHCBM1 in NPQ has not been elucidated yet. In this work, we combined biochemical and physiological measurements to study short-term high-light acclimation of npq5, the mutant lacking LHCBM1. In low light in the absence of this complex, the antenna size of PSII was smaller than in its presence; this effect was marginal in high light (HL), implying that a reduction of the antenna was not responsible for the low NPQ. The mutant expressed LHCSR3 at the wild-type level in HL, indicating that the absence of this complex is also not the reason. Finally, NPQ remained low in the mutant even when the pH was artificially lowered to values that can switch LHCSR3 to the quenched conformation. We concluded that both LHCSR3 and LHCBM1 are required for the induction of NPQ and that LHCBM1 is the interacting partner of LHCSR3. This interaction can either enhance the quenching capacity of LHCSR3 or connect this complex with the PSII supercomplex.
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Chlamydomonas reinhardtii , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Luz , Complejo de Proteína del Fotosistema II/metabolismo , Fotosíntesis/fisiología , Calor , Complejos de Proteína Captadores de Luz/genética , Complejos de Proteína Captadores de Luz/metabolismoRESUMEN
The antenna complexes of Photosystem I present low-lying states visible as red-shifted and broadened absorption and fluorescence bands. Among these, Lhca4 has the most evident features of these "red" states, with a fluorescence band shifted by more than 25 nm from typical LHC emission. This signal arises from a mixing of exciton and charge-transfer (CT) states within the excitonically coupled a603-a609 chlorophyll (Chl) dimer. Here we combine molecular dynamics, multiscale quantum chemical calculations, and spectral simulations to uncover the molecular mechanism for the formation and tuning of exciton-CT interactions in Lhca4. We show that the coupling between exciton and CT states is extremely sensitive to tiny variations in the Chl dimer arrangement, explaining both the red-shifted bands and the switch between conformations with blue and red emission observed in single-molecule spectroscopy. Finally, we show that mutating the axial ligand of a603 diminishes the exciton-CT coupling, removing any red-state fingerprint.
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Allophycocyanins are phycobiliproteins that absorb red light and transfer the energy to the reaction centers of oxygenic photosynthesis in cyanobacteria and red algae. Recently, it was shown that some allophycocyanins absorb far-red light and that one subset of these allophycocyanins, comprising subunits from the ApcD4 and ApcB3 subfamilies (FRL-AP), form helical nanotubes. The lowest energy absorbance maximum of the oligomeric ApcD4-ApcB3 complexes occurs at 709 nm, which is unlike allophycocyanin (AP; ApcA-ApcB) and allophycocyanin B (AP-B; ApcD-ApcB) trimers that absorb maximally at ~ 650 nm and ~ 670 nm, respectively. The molecular bases of the different spectra of AP variants are presently unclear. To address this, we structurally compared FRL-AP with AP and AP-B, performed spectroscopic analyses on FRL-AP, and leveraged computational approaches. We show that among AP variants, the α-subunit constrains pyrrole ring A of its phycocyanobilin chromophore to different extents, and the coplanarity of ring A with rings B and C sets a baseline for the absorbance maximum of the chromophore. Upon oligomerization, the α-chromophores of all AP variants exhibit a red shift of the absorbance maximum of ~ 25 to 30 nm and band narrowing. We exclude excitonic coupling in FRL-AP as the basis for this red shift and extend the results to discuss AP and AP-B. Instead, we attribute these spectral changes to a conformational alteration of pyrrole ring D, which becomes more coplanar with rings B and C upon oligomerization. This study expands the molecular understanding of light-harvesting attributes of phycobiliproteins and will aid in designing phycobiliproteins for biotechnological applications.
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Drought is a major abiotic stress that impairs plant growth and development. Despite this, a comprehensive understanding of drought effects on the photosynthetic apparatus is lacking. In this work, we studied the consequences of 14-d drought treatment on Arabidopsis thaliana. We used biochemical and spectroscopic methods to examine photosynthetic membrane composition and functionality. Drought led to the disassembly of PSII supercomplexes and the degradation of PSII core. The light-harvesting complexes (LHCII) instead remain in the membrane but cannot act as an antenna for active PSII, thus representing a potential source of photodamage. This effect can also be observed during nonphotochemical quenching (NPQ) induction when even short pulses of saturating light can lead to photoinhibition. At a later stage, under severe drought stress, the PSI antenna size is also reduced and the PSI-LHCI supercomplexes disassemble. Surprisingly, although we did not observe changes in the PSI core protein content, the functionality of PSI is severely affected, suggesting the accumulation of nonfunctional PSI complexes. We conclude that drought affects both photosystems, although at a different stage, and that the operative quantum efficiency of PSII (ΦPSII ) is very sensitive to drought and can thus be used as a parameter for early detection of drought stress.
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Arabidopsis , Arabidopsis/metabolismo , Complejo de Proteína del Fotosistema I/metabolismo , Sequías , Complejo de Proteína del Fotosistema II/metabolismo , Fotosíntesis/fisiología , Complejos de Proteína Captadores de Luz/metabolismo , Clorofila/metabolismoRESUMEN
While photosynthesis transforms sunlight energy into sugar, aerobic and anaerobic respiration (fermentation) catabolizes sugars to fuel cellular activities. These processes take place within one cell across several compartments, however it remains largely unexplored how they interact with one another. Here we report that the weak acids produced during fermentation down-regulate both photosynthesis and aerobic respiration. This effect is mechanistically explained with an "ion trapping" model, in which the lipid bilayer selectively traps protons that effectively acidify subcellular compartments with smaller buffer capacities - such as the thylakoid lumen. Physiologically, we propose that under certain conditions, e.g., dim light at dawn, tuning down the photosynthetic light reaction could mitigate the pressure on its electron transport chains, while suppression of respiration could accelerate the net oxygen evolution, thus speeding up the recovery from hypoxia. Since we show that this effect is conserved across photosynthetic phyla, these results indicate that fermentation metabolites exert widespread feedback control over photosynthesis and aerobic respiration. This likely allows algae to better cope with changing environmental conditions.
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Respiración de la Célula , Fotosíntesis , Anaerobiosis , Fermentación , RespiraciónRESUMEN
The photosynthetic light-harvesting complexes (LHCs) are responsible for light absorption due to their pigment-binding properties. These pigments are primarily Chlorophyll (Chl) molecules of type a and b, which ensure an excellent coverage of the visible light spectrum. To date, it is unclear which factors drive the selective binding of different Chl types in the LHC binding pockets. To gain insights into this, we employed molecular dynamics simulations on LHCII binding different Chl types. From the resulting trajectories, we have calculated the binding affinities per each Chl-binding pocket using the Molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) model. To further examine the importance of the nature of the axial ligand in tuning the Chl selectivity of the binding sites, we used Density Functional Theory (DFT) calculations. The results indicate that some binding pockets have a clear Chl selectivity, and the factors governing these selectivities are identified. Other binding pockets are promiscuous, which is consistent with previous in vitro reconstitution studies. DFT calculations show that the nature of the axial ligand is not a major factor in determining the Chl binding pocket selectivity, which is instead probably controlled by the folding process.
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Clorofila , Complejos de Proteína Captadores de Luz , Clorofila/química , Complejos de Proteína Captadores de Luz/química , Complejos de Proteína Captadores de Luz/metabolismo , Ligandos , Sitios de UniónRESUMEN
To compete in certain low-light environments, some cyanobacteria express a paralog of the light-harvesting phycobiliprotein, allophycocyanin (AP), that strongly absorbs far-red light (FRL). Using cryo-electron microscopy and time-resolved absorption spectroscopy, we reveal the structure-function relationship of this FRL-absorbing AP complex (FRL-AP) that is expressed during acclimation to low light and that likely associates with chlorophyll a-containing photosystem I. FRL-AP assembles as helical nanotubes rather than typical toroids due to alterations of the domain geometry within each subunit. Spectroscopic characterization suggests that FRL-AP nanotubes are somewhat inefficient antenna; however, the enhanced ability to harvest FRL when visible light is severely attenuated represents a beneficial trade-off. The results expand the known diversity of light-harvesting proteins in nature and exemplify how biological plasticity is achieved by balancing resource accessibility with efficiency.
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Clorofila , Cianobacterias , Clorofila/metabolismo , Microscopía por Crioelectrón , Clorofila A/metabolismo , Cianobacterias/metabolismo , Luz , FotosíntesisRESUMEN
CAM plants are superior to C3 plants in drought resistance because of their peculiar photosynthesis pathway and morphological features. While those aspects have been studied for decades, little is known about the photosynthetic machinery of CAM plants. Here, we used a combination of biochemical and biophysical methods to study the photosynthetic apparatus of Tillandsia flabellate, an obligatory CAM plant. Most of the Photosystems super- and sub-complexes have properties very similar to those of Arabidopsis, with the main difference that in Tillandsia PSI-LHCI complexes bind extra LHCI. Functional measurements show that the PSI/PSII ratio is rather low compared to other plants and that the antenna size of both PSI and PSII is small. Upon 30-day water deficiency, the composition of the photosystems does not change significantly, PSII efficiency remains high and no Photosystem II photoinhibition was detected despite a reduction of non-photochemical quenching (NPQ).
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Arabidopsis , Tillandsia , Tillandsia/metabolismo , Clorofila/metabolismo , Agua/metabolismo , Complejo de Proteína del Fotosistema I/metabolismo , Fotosíntesis/fisiología , Complejo de Proteína del Fotosistema II/metabolismo , Arabidopsis/metabolismo , LuzRESUMEN
The first step of photosynthesis in plants is performed by the light-harvesting complexes (LHC), a large family of pigment-binding proteins embedded in the photosynthetic membranes. These complexes are conserved across species, suggesting that each has a distinct role. However, they display a high degree of sequence homology and their static structures are almost identical. What are then the structural features that determine their different properties? In this work, we compared the two best-characterized LHCs of plants: LHCII and CP29. Using molecular dynamics simulations, we could rationalize the difference between them in terms of pigment-binding properties. The data also show that while the loops between the helices are very flexible, the structure of the transmembrane regions remains very similar in the crystal and the membranes. However, the small structural differences significantly affect the excitonic coupling between some pigment pairs. Finally, we analyzed in detail the structure of the long N-terminus of CP29, showing that it is structurally stable and it remains on top of the membrane even in the absence of other proteins. Although the structural changes upon phosphorylation are minor, they can explain the differences in the absorption properties of the pigments observed experimentally.
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Complejos de Proteína Captadores de Luz , Complejo de Proteína del Fotosistema II , Complejos de Proteína Captadores de Luz/química , Complejo de Proteína del Fotosistema II/metabolismo , Tilacoides/metabolismo , Fotosíntesis , Proteínas de Plantas/química , Plantas/metabolismo , Clorofila/metabolismoRESUMEN
Lhca1 is one of the four pigment-protein complexes composing the outer antenna of plant Photosystem I-light-havesting I supercomplex (PSI-LHCI). It forms a functional dimer with Lhca4 but, differently from this complex, it does not contain 'red-forms,' i.e., pigments absorbing above 700 nm. Interestingly, the recent PSI-LHCI structures suggest that Lhca1 is the main point of delivering the energy harvested by the antenna to the core. To identify the excitation energy pathways in Lhca1, we developed a structure-based exciton model based on the simultaneous fit of the low-temperature absorption, linear dichroism, and fluorescence spectra of wild-type Lhca1 and two mutants, lacking chlorophylls contributing to the long-wavelength region of the absorption. The model enables us to define the locations of the lowest energy pigments in Lhca1 and estimate pathways and timescales of energy transfer within the complex and to the PSI core. We found that Lhca1 has a particular energy landscape with an unusual (compared to Lhca4, LHCII, and CP29) configuration of the low-energy states. Remarkably, these states are located near the core, facilitating direct energy transfer to it. Moreover, the low-energy states of Lhca1 are also coupled to the red-most state (red forms) of the neighboring Lhca4 antenna, providing a pathway for effective excitation energy transfer from Lhca4 to the core.
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Complejos de Proteína Captadores de Luz , Complejo de Proteína del Fotosistema I , Complejo de Proteína del Fotosistema I/metabolismo , Complejos de Proteína Captadores de Luz/metabolismo , Proteínas de Unión a Clorofila/metabolismo , Clorofila/metabolismo , Transferencia de EnergíaRESUMEN
Rhodopsins had long been considered non-fluorescent until a peculiar voltage-sensitive fluorescence was reported for archaerhodopsin-3 (Arch3) derivatives. These proteins named QuasArs have been used for imaging membrane voltage changes in cell cultures and small animals, but they could not be applied in living rodents. To develop the next generation of sensors, it is indispensable to first understand the molecular basis of the fluorescence and its modulation by the membrane voltage. Based on spectroscopic studies of fluorescent Arch3 derivatives, we propose a unique photo-reaction scheme with extended excited-state lifetimes and inefficient photoisomerization. Molecular dynamics simulations of Arch3, of the Arch3 fluorescent derivative Archon1, and of several its mutants have revealed different voltage-dependent changes of the hydrogen-bonding networks including the protonated retinal Schiff-base and adjacent residues. Experimental observations suggest that under negative voltage, these changes modulate retinal Schiff base deprotonation and promote a decrease in the populations of fluorescent species. Finally, we identified molecular constraints that further improve fluorescence quantum yield and voltage sensitivity.
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Rodopsinas Microbianas , Bases de Schiff , Animales , Hidrógeno , Enlace de Hidrógeno , Rodopsinas Microbianas/química , Rodopsinas Microbianas/genética , Bases de Schiff/química , Análisis EspectralRESUMEN
Photosynthesis is a fundamental process that converts photons into chemical energy, driven by large protein complexes at the thylakoid membranes of plants, cyanobacteria, and algae. In plants, water-soluble plastocyanin (Pc) is responsible for shuttling electrons between cytochrome b6f complex and the photosystem I (PSI) complex in the photosynthetic electron transport chain (PETC). For an efficient turnover, a transient complex must form between PSI and Pc in the PETC, which implies a balance between specificity and binding strength. Here, we studied the binding frequency and the unbinding force between suitably oriented plant PSI and Pc under redox control using single molecule force spectroscopy (SMFS). The binding frequency (observation of binding-unbinding events) between PSI and Pc depends on their respective redox states. The interaction between PSI and Pc is independent of the redox state of PSI when Pc is reduced, and it is disfavored in the dark (reduced P700) when Pc is oxidized. The frequency of interaction between PSI and Pc is higher when at least one of the partners is in a redox state ready for electron transfer (ET), and the post-ET situation (PSIRed-PcOx) leads to lower binding. In addition, we show that the binding of ET-ready PcRed to PSI can be regulated externally by Mg2+ ions in solution.