RESUMO
The fusion of hydrogenases and photosynthetic reaction centers (RCs) has proven to be a promising strategy for the production of sustainable biofuels. Type I (iron-sulfur-containing) RCs, acting as photosensitizers, are capable of promoting electrons to a redox state that can be exploited by hydrogenases for the reduction of protons to dihydrogen (H2). While both [FeFe] and [NiFe] hydrogenases have been used successfully, they tend to be limited due to either O2 sensitivity, binding specificity, or H2 production rates. In this study, we fuse a peripheral (stromal) subunit of Photosystem I (PS I), PsaE, to an O2-tolerant [FeFe] hydrogenase from Clostridium beijerinckii using a flexible [GGS]4 linker group (CbHydA1-PsaE). We demonstrate that the CbHydA1 chimera can be synthetically activated in vitro to show bidirectional activity and that it can be quantitatively bound to a PS I variant lacking the PsaE subunit. When illuminated in an anaerobic environment, the nanoconstruct generates H2 at a rate of 84.9 ± 3.1 µmol H2 mgchl-1 h-1. Further, when prepared and illuminated in the presence of O2, the nanoconstruct retains the ability to generate H2, though at a diminished rate of 2.2 ± 0.5 µmol H2 mgchl-1 h-1. This demonstrates not only that PsaE is a promising scaffold for PS I-based nanoconstructs, but the use of an O2-tolerant [FeFe] hydrogenase opens the possibility for an in vivo H2 generating system that can function in the presence of O2.
Assuntos
Hidrogênio , Hidrogenase , Luz , Oxigênio , Complexo de Proteína do Fotossistema I , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema I/química , Hidrogenase/metabolismo , Hidrogenase/química , Hidrogênio/metabolismo , Oxigênio/metabolismo , Oxigênio/química , Clostridium beijerinckii/metabolismo , Clostridium beijerinckii/genética , Oxirredução , Proteínas Ferro-Enxofre/metabolismo , Proteínas Ferro-Enxofre/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , FotossínteseRESUMO
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.
Assuntos
Clorofila , Complexos de Proteínas Captadores de Luz , Clorofila A , Complexos de Proteínas Captadores de Luz/química , Clorofila/metabolismo , Fotossíntese , Análise Espectral , Complexo de Proteína do Fotossistema I/química , PlantasRESUMO
Acaryochloris species belong to a special category of cyanobacteria possessing chlorophyll (Chl) d. One of the photosynthetic characteristics of Acaryochloris marina MBIC11017 is that the absorption spectra of photosystem I (PSI) showed almost no bands and shoulders of low-energy Chls d over 740 nm. In contrast, the absorption spectra of other Acaryochloris species showed a shoulder around 740 nm, suggesting that low-energy Chls d within PSI are diversified among Acaryochloris species. In this study, we purified PSI trimer and monomer cores from Acaryochloris sp. NBRC 102871 and examined their protein and pigment compositions and spectral properties. The protein bands and pigment compositions of the PSI trimer and monomer of NBRC102871 were virtually identical to those of MBIC11017. The absorption spectra of the NBRC102871 PSIs exhibited a shoulder around 740 nm, whereas the fluorescence spectra of PSI trimer and monomer displayed maximum peaks at 754 and 767 nm, respectively. These spectral properties were different from those of MBIC11017, indicating the presence of low-energy Chls d within the NBRC102871 PSIs. Moreover, we analyzed the NBRC102871 genome to identify amino acid sequences of PSI proteins and compared them with those of the A. marina MBIC11017 and MBIC10699 strains whose genomes are available. The results showed that some of the sequences in NBRC102871 were distinct from those in MBIC11017 and MBIC10699. These findings provide insights into the variety of low-energy Chls d with respect to the protein environments of PSI cores among the three Acaryochloris strains.
Assuntos
Clorofila , Cianobactérias , Complexo de Proteína do Fotossistema I , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema I/química , Clorofila/metabolismo , Cianobactérias/metabolismo , Cianobactérias/genética , Espectrometria de FluorescênciaRESUMO
Primary excitation energy transfer and charge separation in photosystem I (PSI) from the extremophile desert green alga Chlorella ohadii grown in low light were studied using broadband femtosecond pump-probe spectroscopy in the spectral range from 400 to 850 nm and in the time range from 50 fs to 500 ps. Photochemical reactions were induced by the excitation into the blue and red edges of the chlorophyll Qy absorption band and compared with similar processes in PSI from the cyanobacterium Synechocystis sp. PCC 6803. When PSI from C. ohadii was excited at 660 nm, the processes of energy redistribution in the light-harvesting antenna complex were observed within a time interval of up to 25 ps, while formation of the stable radical ion pair P700+A1- was kinetically heterogeneous with characteristic times of 25 and 120 ps. When PSI was excited into the red edge of the Qy band at 715 nm, primary charge separation reactions occurred within the time range of 7 ps in half of the complexes. In the remaining complexes, formation of the radical ion pair P700+A1- was limited by the energy transfer and occurred with a characteristic time of 70 ps. Similar photochemical reactions in PSI from Synechocystis 6803 were significantly faster: upon excitation at 680 nm, formation of the primary radical ion pairs occurred with a time of 3 ps in ~30% complexes. Excitation at 720 nm resulted in kinetically unresolvable ultrafast primary charge separation in 50% complexes, and subsequent formation of P700+A1- was observed within 25 ps. The photodynamics of PSI from C. ohadii was noticeably similar to the excitation energy transfer and charge separation in PSI from the microalga Chlamydomonas reinhardtii; however, the dynamics of energy transfer in C. ohadii PSI also included slower components.
Assuntos
Chlorella , Transferência de Energia , Complexo de Proteína do Fotossistema I , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema I/química , Chlorella/metabolismo , Synechocystis/metabolismo , Processos Fotoquímicos , Clorofila/metabolismo , Clorofila/química , CinéticaRESUMO
Photosystem I (PS I) is a photosynthetic pigment-protein complex that absorbs light and uses the absorbed energy to initiate electron transfer. Electron transfer has been shown to occur concurrently along two (A- and B-) branches of reaction center (RC) cofactors. The electron transfer chain originates from a special pair of chlorophyll a molecules (P700), followed by two chlorophylls and one phylloquinone in each branch (denoted as A-1, A0, A1, respectively), converging in a single iron-sulfur complex Fx. While there is a consensus that the ultimate electron donor-acceptor pair is P700+A0-, the involvement of A-1 in electron transfer, as well as the mechanism of the very first step in the charge separation sequence, has been under debate. To resolve this question, multiple groups have targeted electron transfer cofactors by site-directed mutations. In this work, the peripheral hydrogen bonds to keto groups of A0 chlorophylls have been disrupted by mutagenesis. Four mutants were generated: PsaA-Y692F; PsaB-Y667F; PsaB-Y667A; and a double mutant PsaA-Y692F/PsaB-Y667F. Contrary to expectations, but in agreement with density functional theory modeling, the removal of the hydrogen bond by Tyr â Phe substitution was found to have a negligible effect on redox potentials and optical absorption spectra of respective chlorophylls. In contrast, Tyr â Ala substitution was shown to have a fatal effect on the PS I function. It is thus inferred that PsaA-Y692 and PsaB-Y667 residues have primarily structural significance, and their ability to coordinate respective chlorophylls in electron transfer via hydrogen bond plays a minor role.
Assuntos
Clorofila , Ligação de Hidrogênio , Complexo de Proteína do Fotossistema I , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema I/química , Complexo de Proteína do Fotossistema I/genética , Clorofila/metabolismo , Clorofila/química , Transporte de Elétrons , Elétrons , Modelos Moleculares , MutaçãoRESUMO
Photosystem I (PSI) serves as a model system for studying fundamental processes such as electron transfer (ET) and energy conversion, which are not only central to photosynthesis but also have broader implications for bioenergy production and biomimetic device design. In this study, we employed electron paramagnetic resonance (EPR) spectroscopy to investigate key light-induced charge separation steps in PSI isolated from several green algal and cyanobacterial species. Following photoexcitation, rapid sequential ET occurs through either of two quasi-symmetric branches of donor/acceptor cofactors embedded within the protein core, termed the A and B branches. Using high-frequency (130 GHz) time-resolved EPR (TR-EPR) and deuteration techniques to enhance spectral resolution, we observed that at low temperatures prokaryotic PSI exhibits reversible ET in the A branch and irreversible ET in the B branch, while PSI from eukaryotic counterparts displays either reversible ET in both branches or exclusively in the B branch. Furthermore, we observed a notable correlation between low-temperature charge separation to the terminal [4Fe-4S] clusters of PSI, termed FA and FB, as reflected in the measured FA/FB ratio. These findings enhance our understanding of the mechanistic diversity of PSI's ET across different species and underscore the importance of experimental design in resolving these differences. Though further research is necessary to elucidate the underlying mechanisms and the evolutionary significance of these variations in PSI charge separation, this study sets the stage for future investigations into the complex interplay between protein structure, ET pathways, and the environmental adaptations of photosynthetic organisms.
Assuntos
Luz , Complexo de Proteína do Fotossistema I , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema I/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Transporte de Elétrons , Cianobactérias/metabolismo , Fotossíntese , Clorófitas/metabolismoRESUMO
In the development of single-molecule spectroscopy, the simultaneous detection of the excitation and emission spectra has been limited. The fluorescence excitation spectrum based on background-free signals is compatible with the fluorescence-emission-based detection of single molecules and can provide insight into the variations in the input energy of the different terminal emitters. Here, we implement single-molecule excitation-emission spectroscopy (SMEES) for photosystem I (PSI) via a cryogenic optical microscope. To this end, we extended our line-focus-based excitation-spectral microscope system to the cryogenic temperature-compatible version. PSI is one of the two photosystems embedded in the thylakoid membrane in oxygen-free photosynthetic organisms. PSI plays an essential role in electron transfer in the photosynthesis reaction. PSIs of many organisms contain a few red-shifted chlorophylls (Chls) with much lower excitation energies than ordinary antenna Chls. The fluorescence emission spectrum originates primarily from the red-shifted Chls, whereas the excitation spectrum is sensitive to the antenna Chls that are upstream of red-shifted Chls. Using SMEES, we obtained the inclining two-dimensional excitation-emission matrix (2D-EEM) of PSI particles isolated from a cyanobacterium, Thermosynechococcus vestitus (equivalent to elongatus), at about 80 K. Interestingly, by decomposing the inclining 2D-EEMs within time course observation, we found prominent variations in the excitation spectra of the red-shifted Chl pools with different emission wavelengths, strongly indicating the variable excitation energy transfer (EET) pathway from the antenna to the terminal emitting pools. SMEES helps us to directly gain information about the antenna system, which is fundamental to depicting the EET within pigment-protein complexes.
Assuntos
Cianobactérias , Complexo de Proteína do Fotossistema I , Complexo de Proteína do Fotossistema I/química , Imagem Individual de Molécula , Espectrometria de Fluorescência , Cianobactérias/química , Temperatura , Clorofila/químicaRESUMO
Cryptophytes are ancestral photosynthetic organisms evolved from red algae through secondary endosymbiosis. They have developed alloxanthin-chlorophyll a/c2-binding proteins (ACPs) as light-harvesting complexes (LHCs). The distinctive properties of cryptophytes contribute to efficient oxygenic photosynthesis and underscore the evolutionary relationships of red-lineage plastids. Here we present the cryo-electron microscopy structure of the Photosystem II (PSII)-ACPII supercomplex from the cryptophyte Chroomonas placoidea. The structure includes a PSII dimer and twelve ACPII monomers forming four linear trimers. These trimers structurally resemble red algae LHCs and cryptophyte ACPI trimers that associate with Photosystem I (PSI), suggesting their close evolutionary links. We also determine a Chl a-binding subunit, Psb-γ, essential for stabilizing PSII-ACPII association. Furthermore, computational calculation provides insights into the excitation energy transfer pathways. Our study lays a solid structural foundation for understanding the light-energy capture and transfer in cryptophyte PSII-ACPII, evolutionary variations in PSII-LHCII, and the origin of red-lineage LHCIIs.
Assuntos
Microscopia Crioeletrônica , Criptófitas , Complexos de Proteínas Captadores de Luz , Complexo de Proteína do Fotossistema II , Complexo de Proteína do Fotossistema II/metabolismo , Complexo de Proteína do Fotossistema II/química , Complexos de Proteínas Captadores de Luz/metabolismo , Complexos de Proteínas Captadores de Luz/química , Criptófitas/metabolismo , Fotossíntese , Modelos Moleculares , Transferência de Energia , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema I/química , Clorofila A/metabolismo , Clorofila A/químicaRESUMO
Photosynthesis converting solar energy to chemical energy is one of the most important chemical reactions on earth. In cyanobacteria, light energy is captured by antenna system phycobilisomes (PBSs) and transferred to photosynthetic reaction centers of photosystem II (PSII) and photosystem I (PSI). While most of the protein complexes involved in photosynthesis have been characterized by in vitro structural analyses, how these protein complexes function together in vivo is not well understood. Here we implemented STAgSPA, an in situ structural analysis strategy, to solve the native structure of PBS-PSII supercomplex from the cyanobacteria Arthrospira sp. FACHB439 at resolution of ~3.5 Å. The structure reveals coupling details among adjacent PBSs and PSII dimers, and the collaborative energy transfer mechanism mediated by multiple super-PBS in cyanobacteria. Our results provide insights into the diversity of photosynthesis-related systems between prokaryotic cyanobacteria and eukaryotic red algae but are also a methodological demonstration for high-resolution structural analysis in cellular or tissue samples.
Assuntos
Cianobactérias , Complexo de Proteína do Fotossistema II , Ficobilissomas , Ficobilissomas/metabolismo , Ficobilissomas/química , Complexo de Proteína do Fotossistema II/metabolismo , Complexo de Proteína do Fotossistema II/química , Cianobactérias/metabolismo , Fotossíntese , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema I/química , Transferência de Energia , Modelos Moleculares , Microscopia CrioeletrônicaRESUMO
Photosystems I and II are the photooxidoreductases central to oxygenic photosynthesis and canonically absorb visible light (400-700 nm). Recent investigations have revealed that certain cyanobacteria can acclimate to environments enriched in far-red light (700-800 nm), yet can still perform oxygenic photosynthesis in a process called far-red light photoacclimation, or FaRLiP. During this process, the photosystem subunits and pigment compositions are altered. Here, the current structural understanding of the photosystems expressed during FaRLiP is described. The design principles may be useful for guiding efforts to engineer shade tolerance in organisms that typically cannot utilize far-red light.
Assuntos
Luz , Complexo de Proteína do Fotossistema I , Complexo de Proteína do Fotossistema II , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema I/química , Complexo de Proteína do Fotossistema II/metabolismo , Complexo de Proteína do Fotossistema II/química , Cianobactérias/metabolismo , Cianobactérias/efeitos da radiação , Fotossíntese , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Aclimatação , Luz VermelhaRESUMO
Photosystem I (PSI) from Fittonia albivenis, an Acanthaceae ornamental plant, is notable among green plants for its red-shifted emission spectrum. Here, we solved the structure of a PSI-light harvesting complex I (LHCI) supercomplex from F. albivenis at 2.46-Å resolution using cryo-electron microscopy. The supercomplex contains a core complex of 14 subunits and an LHCI belt with four antenna subunits (Lhca1-4) similar to previously reported angiosperm PSI-LHCI structures; however, Lhca3 differs in three regions surrounding a dimer of low-energy chlorophylls (Chls) termed red Chls, which absorb far-red beyond visible light. The unique amino acid sequences within these regions are exclusively shared by plants with strongly red-shifted fluorescence emission, suggesting candidate structural elements for regulating the energy state of red Chls. These results provide a structural basis for unraveling the mechanisms of light harvest and transfer in PSI-LHCI of under canopy plants and for designing Lhc to harness longer-wavelength light in the far-red spectral range.
Assuntos
Microscopia Crioeletrônica , Complexos de Proteínas Captadores de Luz , Complexo de Proteína do Fotossistema I , Sequência de Aminoácidos , Clorofila/metabolismo , Clorofila/química , Luz , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/metabolismo , Modelos Moleculares , Complexo de Proteína do Fotossistema I/química , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema I/ultraestrutura , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , AcanthaceaeRESUMO
Phycobilisome (PBS) is a large pigment-protein complex in cyanobacteria and red algae responsible for capturing sunlight and transferring its energy to photosystems (PS). Spectroscopic and structural properties of various PBSs have been widely studied, however, the nature of so-called complex-complex interactions between PBS and PSs remains much less explored. In this work, we have investigated the function of a newly identified PBS linker protein, ApcG, some domain of which, together with a loop region (PB-loop in ApcE), is possibly located near the PBS-PS interface. Using Synechocystis sp. PCC 6803, we generated an ApcG deletion mutant and probed its deletion effect on the energetic coupling between PBS and photosystems. Steady-state and time-resolved spectroscopic characterization of the purified ΔApcG-PBS demonstrated that ApcG removal weakly affects the photophysical properties of PBS for which the spectroscopic properties of terminal energy emitters are comparable to those of PBS from wild-type strain. However, analysis of fluorescence decay imaging datasets reveals that ApcG deletion induces disruptions within the allophycocyanin (APC) core, resulting in the emergence (splitting) of two spectrally diverse subgroups with some short-lived APC. Profound spectroscopic changes of the whole ΔApcG mutant cell, however, emerge during state transition, a dynamic process of light scheme adaptation. The mutant cells in State I show a substantial increase in PBS-related fluorescence. On the other hand, global analysis of time-resolved fluorescence demonstrates that in general ApcG deletion does not alter or inhibit state transitions interpreted in terms of the changes of the PSII and PSI fluorescence emission intensity. The results revealed yet-to-be discovered mechanism of ApcG-docking induced excitation energy transfer regulation within PBS or to Photosystems.
Assuntos
Proteínas de Bactérias , Transferência de Energia , Ficobilissomas , Synechocystis , Ficobilissomas/metabolismo , Ficobilissomas/química , Synechocystis/metabolismo , Synechocystis/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema I/química , Complexo de Proteína do Fotossistema I/genética , Complexo de Proteína do Fotossistema II/metabolismo , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/genética , Peptídeos/metabolismo , Peptídeos/químicaRESUMO
Thylakoid membranes of the cryptophyte Chroomonas sp. strain LT were solubilized with dodecyl-beta-maltoside and subjected to sucrose density gradient centrifugation. The four pigment protein complexes obtained were subsequently characterized by absorption and fluorescence spectroscopy, SDS-PAGE, and Western immunoblotting using antisera against the chlorophyll a/c-binding proteins of the marine cryptophyte Cryptomonas maculata and the reaction-center protein D2 of photosystem II of maize. Band 1 consisted mainly of free pigments, phycobiliproteins, and chlorophyll-a/c-binding proteins. Band 2 represented a major chlorophyll a/c-binding protein fraction. A mixture of photosystem II and photosystem I proteins comprised band 3, whereas band 4 was enriched in proteins of photosystem I. Western immunoblotting demonstrated the presence of chlorophyll a/c-binding proteins and their association with photosystem I in band 4. Phosphorylation experiments showed that chlorophyll a/c-binding proteins became phosphorylated. Negative staining electron microscopy of band B4 revealed photosystem I particles with dimensions of 22 nm. Our work showed that PSI-LHCI complexes of cryptophytes are similar to those of Chlamydomonas rheinhardtii, the diatom Phaeodactylum tricornutum, and higher plants (AU)
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