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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Computing the Relative Affinity of Chlorophylls a and b to Light-Harvesting Complex II

In plants and algae, the primary antenna protein bound to photosystem II is light-harvesting complex II (LHCII), a pigment–protein complex that binds eight chlorophyll (Chl) a molecules and six Chl b molecules. Chl a and Chl b differ only in that Chl a has a methyl group (–CH 3 ) on one of its pyrrole rings, while Chl b has a formyl group (–CHO) at that position. This blue-shifts the Chl b absorbance relative to Chl a . It is not known how the protein selectively binds the right Chl type at each site. Knowing the selection criteria would allow the design of light-harvesting complexes that bind different Chl types, modifying an organism to utilize the light of different wavelengths. The difference in the binding affinity of Chl a and Chl b in pea and spinach LHCII was calculated using multiconformation continuum electrostatics and free energy perturbation. Both methods have identified some Chl sites where the bound Chl type ( a or b ) has a significantly higher affinity, especially when the protein provides a hydrogen bond for the Chl b formyl group. However, the Chl a sites often have little calculated preference for one Chl type, so they are predicted to bind a mixture of Chl a and b . The electron density of the spinach LHCII was reanalyzed, which, however, confirmed that there is negligible Chl b in the Chl a -binding sites. Finally, it is suggested that the protein chooses the correct Chl type during folding, segregating the preferred Chl to the correct binding site.

chemical calculations↗
DOE OSTI2026

Exploring Local Vibrational Structure in Protein-Bound Chlorophyll a: Isotope-Enrichment Experiments and Electrostatic Analysis

Local chlorophyll (Chl) vibrations play an essential role in biological photosynthesis by facilitating rapid energy transfer between pigments. In research studies, they also provide a useful spectroscopic probe of the local protein environment that surrounds each pigment. However, measuring the complete vibrational absorption spectrum of a protein-bound Chl molecule is much more difficult than, for example, Chl in neat solvent due to overlap with protein vibrations that typically drown out Chl vibrational features. Resonance Raman and fluorescence spectroscopies provide a way around this problem for Franck–Condon active vibrations, but these often rely on cryogenic measurement conditions and fail to capture vibrational signatures from, e.g., ester-group vibrations that lack coupling to a convenient electronic transition. In the present contribution, we use 13 C-enrichment of the protein backbone to shift protein background signals to lower frequency, providing a largely clean spectral window in which to study local Chl a C═O stretch modes. A room-temperature absorption spectrum for Chl a in the water-soluble chlorophyll protein (WSCP) of Lepidium virginicum is thus extracted as a difference between protein-plus-pigment and protein-only vibrational spectra. Excellent agreement in the molecular fingerprint region with the vibrational spectrum of Chl a in organic solvents confirms that the resulting spectrum represents the response of the protein-bound Chl a molecule. Furthermore, the ester group resonance is observed to shift in response to the S53P mutation that eliminates a 17 3 ester group hydrogen bond from the protein environment. Finally, we analyze these experimental results using MD-based electrostatic analysis, finding that electric-field mapping at the C atom of the ester group provides a satisfactory explanation for the observed frequency shifts between organic solvent and protein environment. MD analysis further suggests that a red-shifted ester peak observed experimentally for the S53P mutant results from solvation of the 17 3 Chl ester group due increase water penetration into the Chl-binding pocket relative to wild-type WSCP. Here, we anticipate that these results will prove useful both for benchmarking future simulation work and as a reference for interpreting Chl vibrational spectra as a probe of pigment–protein interactions.

14 SOLAR ENERGY↗
DOE OSTI2023

Energy dissipation efficiency in the CP43 assembly intermediate complex of photosystem II

Photosystem II in oxygenic organisms is a large membrane bound rapidly turning over pigment protein complex. During its biogenesis, multiple assembly intermediates are formed, including the CP43-preassembly complex (pCP43). Here, to understand the energy transfer dynamics in pCP43, we first engineered a His-tagged version of the CP43 in a CP47-less strain of the cyanobacterium Synechocystis 6803. Isolated pCP43 from this engineered strain was subjected to advanced spectroscopic analysis to evaluate its excitation energy dissipation characteristics. These included measurements of steady-state absorption and fluorescence emission spectra for which correlation was tested with Stepanov relation. Comparison of fluorescence excitation and absorptance spectra determined that efficiency of energy transfer from β-carotene to chlorophyll a is 39 %. Time-resolved fluorescence images of pCP43-bound Chl a were recorded on streak camera, and fluorescence decay dynamics were evaluated with global fitting. These demonstrated that the decay kinetics strongly depends on temperature and buffer used to disperse the protein sample and fluorescence decay lifetime was estimated in 3.2–5.7 ns time range, depending on conditions. The pCP43 complex was also investigated with femtosecond and nanosecond time-resolved absorption spectroscopy upon excitation of Chl a and β-carotene to reveal pathways of singlet excitation relaxation/decay, Chl a triplet dynamics and Chl a → β-carotene triplet state sensitization process. The latter demonstrated that Chl a triplet in the pCP43 complex is not efficiently quenched by carotenoids. Finally, detailed kinetic analysis of the rise of the population of β-carotene triplets determined that the time constant of the carotenoid triplet sensitization is 40 ns.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Chlorophyllase from Arabidopsis thaliana Reveals an Emerging Model for Controlling Chlorophyll Hydrolysis

Chlorophyll (Chl) is one of Nature’s most complex pigments to biosynthesize and derivatize. This pigment is vital for survival and also paradoxically toxic if overproduced or released from a protective protein scaffold. Therefore, along with the mass production of Chl, organisms also invest in mechanisms to control its degradation and recycling. One important enzyme that is involved in these latter processes is chlorophyllase. This enzyme is employed by numerous photosynthetic organisms to hydrolyze the phytol tail of Chl. Although traditionally thought to catalyze the first step of Chl degradation, recent work suggests that chlorophyllase is instead employed during times of abiotic stress or conditions that produce reactive oxygen species. However, the molecular details regarding how chlorophyllases are regulated to function under such conditions remain enigmatic. Here, we investigate the Arabidopsis thaliana chlorophyllase isoform AtCLH2 using site-directed mutagenesis, mass spectrometry, dynamic light scattering, size-exclusion multiangle light scattering, and both steady-state enzyme kinetic and thermal stability measurements. Through these experiments, we show that AtCLH2 exists as a monomer in solution and contains two disulfide bonds. One disulfide bond putatively maps to the active site, whereas the other links two N-terminal Cys residues together. These disulfide bonds are cleaved by chemical or chemical and protein-based reductants, respectively, and are integral to maintaining the activity, stability, and substrate scope of the enzyme. This work suggests that Cys residue oxidation in chlorophyllases is an emerging regulatory strategy for controlling the hydrolysis of Chl pigments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Elucidating Photoinduced Processes of Photosystem I Via Multidimensional Electronic and Vibrational Spectroscopies

This project was motivated by an overarching goal to elucidate the mechanism of energy and electron transfer that governs the efficient charge separation in photosystem I (PSI) complexes. PSI is a natural light harvesting complex that drives oxygenic photosynthesis in plants, algae, and cyanobacteria. It uses ~300 tightly packed chlorophylls (Chls) to absorb photons, transfer the excitation energy to the reaction center (RC), and generate a charge separated state with near unity quantum efficiency (QE). A better understanding of the mechanism of energy transfer and charge separation in PSI is required for understanding the high QE of natural light harvesting complexes, and it could lead to the further development of artificial photosynthetic systems for solar energy conversion and modification of light harvesting complexes to improve crop yields. We applied two-dimensional optical spectroscopies to different cyanobacterial photosystem I complexes, including PSI complexes that contain Chl f molecules, to map energy transfer pathways and gain insight into the efficient light harvesting of PSI. We used two-dimensional electronic spectroscopies (2DES) to map energy transfer in Chl a and Chl f containing PSI complexes. To investigate the Chl f PSI complexes, we modified our spectrometer to probe the lower energy states associated with Chl f molecules. We interpreted the 2DES spectra through global analysis procedures to generate maps of energy transfer. We also constructed a two-dimensional electronic vibrational (2DEV) spectrometer that will be used to investigate charge transfer transitions and dynamics within PSI complexes. Measurements were performed on model systems to establish general data analysis procedures for interpreting 2D spectra and gain insight into protein cofactor interactions.

14 SOLAR ENERGY↗
DOE OSTI2023

Electronic structure and energetics of a heterodimeric BChl g ′/Chl a ′ special pair generated by exposure of Heliomicrobium modesticaldum to dioxygen

Heliobacteria are anoxygenic phototrophs that have a Type I homodimeric reaction center containing bacteriochlorophyll g (BChl g). Previous experimental studies have shown that in the presence of light and dioxygen, BChl g is converted into 8 1 -OH-chlorophyll a F (hereafter Chl a F ), with an accompanying loss of light-driven charge separation. These studies suggest that the reaction center only loses the ability to transfer electrons once both BChl g′ molecules of the P 800 special pair have been converted to Chl a F ′. Here, the present work confirms that the partially converted BChl g′/Chl a F ′ special pair remains functional in samples exposed to dioxygen by demonstrating its presence using hyperfine couplings obtained from Q-band 1 H ENDOR, 2D 14 N HYSCORE and DFT methods. The DFT calculations of the BChl g′/BChl g′ homodimeric primary donor, which are based on the recently published X-ray crystal structure, predict that the unpaired electron spin is equally delocalized over both BChl g′ molecules and provide an excellent match to the experimental hyperfine couplings of the anaerobic samples. Exposure to dioxygen leads to substantial changes in the hyperfine interactions, indicative of greater localization of the unpaired electron spin. The measured hyperfine couplings are reproduced in the DFT calculations by replacing one of the BChl g′ molecules of the primary donor with a Chl a F ′ molecule. The calculations reveal that the spin density becomes localized on BChl g′ in the heterodimeric primary donor. Time-dependent DFT calculations demonstrate that conversion of either or both of the accessory BChl g molecules and/or one of the BChl g′ molecules of P800 to Chl a F ′ results in minor effects on the energy of the charge-separated states. In contrast, if both of the BChl g′ molecules of P 800 are converted a large increase in the energy of the charge-separated state occurs. This suggests that the reaction center remains functional when only one half of the dimer is converted, however, conversion of both halves of the P 800 dimer leads to loss of function

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Insights into the year-round vertical distribution of chlorophyll concentration in high-latitude Arctic Ocean: implications for primary production

Climate-induced rapid changes in the Arctic Ocean, such as decreasing sea ice extent and increasing water temperature, are altering nutrient and light availability, profoundly impacting primary producer growth. However, access to the high-latitude Arctic Ocean is limited, and satellite data are primarily available only during summer, making continuous in-situ data collection challenging. We collected year-round chlorophyll-a (Chl-a) concentration data in high-latitude regions using a mooring system and performed a comparative analysis with reanalysis data. Unlike previous satellite-based studies, which typically rely on surface measurements, we used the annual vertical distribution of Chl-a. These data were applied to the vertically generalized production model to accurately estimate annual primary production. The moored Chl-a concentration data showed that phytoplankton exhibited a typical subsurface chlorophyll maximum (SCM) layer as sea ice retreated in June. Contrary to the gradually deepening SCM distribution predicted by model-based reanalysis data, the SCM layer persisted for approximately 4 months. This indicates that light and nutrient conditions within the SCM layer remained stable, sustaining continuous phytoplankton growth. Annual primary production, reflecting this vertical distribution of Chl-a concentration, was 6.85 gC m −2 yr −1 . This exceeded satellite-based estimates by at least two-fold, highlighting the significant underestimation of primary production by satellite approaches. Estimating primary production while accounting for the vertical distribution of phytoplankton and light is essential for improving ecological models to better understand carbon cycle and food web changes in the Arctic Ocean, with important implications for climate change predictions.

Arctic Ocean↗
DOE OSTI2024

Geranylgeranylated-chlorophyll-protein complexes in lhl3 mutant of the green alga Chlamydomonas reinhardtii

Chlorophylls a and b (Chl a and b) are involved in light harvesting, photochemical reactions, and electron transfer reactions in plants and green algae. The core complexes of the photosystems (PSI and PSII) associate with Chl a, while the peripheral antenna complexes (LHCI and LHCII) bind Chls a and b. One of the final steps of Chl biosynthesis is the conversion of geranylgeranylated Chls (Chls GG ) to phytylated Chls by geranylgeranyl reductase (GGR). Here, we isolated and characterized a pale green mutant of the green alga Chlamydomonas reinhardtii that was very photosensitive and was unable to grow photoautotrophically. This mutant has a 16-bp deletion in the LHL3 gene, which resulted in the loss of LHL3 and GGR and accumulated only Chls GG . The lhl3 mutant cells grown in the dark accumulated PSII and PSI proteins at 25–50% of WT levels, lacked PSII activity, and retained a decreased PSI activity. The PSII and PSI proteins were depleted to trace amounts in the mutant cells grown in light. In contrast, the accumulation of LHCI and LHCII was unaffected except for LHCA3. Our results suggest that the replacement of Chls with Chls GG strongly affects the structural and functional integrity of PSII and PSI complexes but their associating LHC complexes to a lesser extent. Affinity purification of HA-tagged LHL3 confirmed the formation of a stable LHL3-GGR complex, which is vital for GGR stability. The LHL3-GGR complex contained a small amount of PSI complex assembly factors, suggesting a putative coupling between Chl synthesis and PSI complex assembly.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Allosterically coupled conformational dynamics in solution prepare the sterol transfer protein StarD4 to release its cargo upon interaction with target membranes

Complex mechanisms regulate the cellular distribution of cholesterol, a critical component of eukaryote membranes involved in regulation of membrane protein functions directly and through the physiochemical properties of membranes. StarD4, a member of the steroidogenic acute regulator-related lipid-transfer (StART) domain (StARD)-containing protein family, is a highly efficient sterol-specific transfer protein involved in cholesterol homeostasis. Its mechanism of cargo loading and release remains unknown despite recent insights into the key role of phosphatidylinositol phosphates in modulating its interactions with target membranes. We have used large-scale atomistic Molecular dynamics (MD) simulations to study how the dynamics of cholesterol bound to the StarD4 protein can affect interaction with target membranes, and cargo delivery. We identify the two major cholesterol (CHL) binding modes in the hydrophobic pocket of StarD4, one near S136&S147 (the Ser-mode), and another closer to the putative release gate located near W171, R92&Y117 (the Trp-mode). We show that conformational changes of StarD4 associated directly with the transition between these binding modes facilitate the opening of the gate. To understand the dynamics of this connection we apply a machine-learning algorithm for the detection of rare events in MD trajectories (RED), which reveals the structural motifs involved in the opening of a front gate and a back corridor in the StarD4 structure occurring together with the spontaneous transition of CHL from the Ser-mode of binding to the Trp-mode. Further analysis of MD trajectory data with the information-theory based NbIT method reveals the allosteric network connecting the CHL binding site to the functionally important structural components of the gate and corridor. Mutations of residues in the allosteric network are shown to affect the performance of the allosteric connection. These findings outline an allosteric mechanism which prepares the CHL-bound StarD4 to release and deliver the cargo when it is bound to the target membrane.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Driving Electron Transfer in Photosystem I Using Far-Red Light: Overall Perspectives

Photosystem I (PSI) is a photosynthetic protein–pigment complex that, upon photoexcitation, transfers electrons to ferredoxin, facilitating the production of NADPH. Isolated PSI reaction centers (RCs) have also been used in hybrid systems to reduce protons and produce ‘biohydrogen’. This review article examines how various cyanobacteria with similar photosynthetic machinery utilize different wavelengths of light to execute photosynthetic electron transport through PSI. Key factors, such as, the structure of the electron transfer cofactors, the protein environment surrounding the primary donor pigments and hydrogen-bonding interactions with the surrounding protein matrix are analyzed to understand their roles in maintaining efficient electron transfer when it is driven using photons of different energies. We compare PSI complexes with known atomic structures from four species of cyanobacteria, Thermosynechococcus elongatus, Acaryochloris marina, Halomicronema hongdechloris, and Fischerella thermalis. T. elongatus is typical of most oxygenic photosynthetic organisms in that it requires visible light and uses only chlorophyll a (Chl a ) in PSI. In contrast, H. hongdechloris and F. thermalis are photoacclimating species capable of producing Chl f and Chl d that use red light when little visible light is available. A. marina , on the other hand, is adapted to red light conditions and consistently utilizes Chl d as its primary photosynthetic pigment, maintaining a stable pigment composition. Here, we explore the structural and functional differences between the PSI RCs of these organisms and the impact of these differences on electron transport. The structural differences in the cofactors influence both the absorption wavelengths of the cofactors and the energy levels of the intermediate states of electron transfer. An analysis of the surrounding protein shows how it has been adapted and underscores the interplay between the pigment structure, protein environment, and hydrogen bonding networks in tuning the efficiency and adaptability of photosynthetic mechanisms across different species of cyanobacteria.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Nonpigmented PsbR is involved in the integrity of excitation landscape in higher plant photosystem II, a case study in Arabidopsis thaliana and a mutant

PsbR is a nonpigmented 10 kDa protein in Photosystem II (PSII) in algae and plants. A recent structural study clarified its enigmatic structural location in a Photosystem II megacomplex that has baffled the community for more than four decades. Our current study interrogates whether absence of PsbR affects the overall dynamics of excitation energy migration within light harvesting complexes (LHC) and PSII super assemblies using highly-active PSII membrane particles, so-called BBY particles, isolated from a PsbR deletion mutant (ΔPsbR) of Arabidopsis thaliana. A femto-second (fs)-time-resolved transient absorption experimentation recorded at 77 K with selective excitation of Chl b which is exclusively present in LHCs enabled us to resolve the temporal differences in LHC→LHC and LHC→PSII excitation energy transfer steps. By applying specific target spectro-kinetic models to the transient absorption datasets, we demonstrated that the time constants of Chl a LHC → Chl a LHC excitation transfer significantly elongates in the ΔPsbR LHC-PSII particles, suggestive of the decreased aggregation level of photosynthetic proteins in the mutant. These findings highlight excitation energy transfer integrity in LHC-PSII assembly is not only determined by the pigmented light-harvesting complexes, but also synergistically by the nonpigmented PSII components. Furthermore, the disturbed integrity in dynamics of excitation energy transfer pathway within LHC-PSII supercomplex is discussed in the context of the altered LHC-PSII megacomplexes type I and II architectures which result from the absence of the PsbR protein in higher plant PSII.

Chlorophyll↗
DOE OSTI2025

Functional Connectivity of Red Chlorophylls in Cyanobacterial Photosystem I Revealed by Fluence-Dependent Transient Absorption

External stressors modulate the oligomerization state of photosystem I (PSI) in cyanobacteria. The number of red chlorophylls (Chls), pigments lower in energy than the P700 reaction center, depends on the oligomerization state of PSI. Here, we use ultrafast transient absorption spectroscopy to interrogate the effective connectivity of the red Chls in excitonic energy pathways in trimeric PSI in native thylakoid membranes of the model cyanobacterium Synechocystis sp. PCC 6803, including emergent dynamics, as red Chls increase in number and proximity. Fluence-dependent dynamics indicate singlet–singlet annihilation within energetically connected red Chl sites in the PSI antenna but not within bulk Chl sites on the picosecond time scale. These data support picosecond energy transfer between energetically connected red Chl sites as the physical basis of singlet–singlet annihilation. The time scale of this energy transfer is faster than predicted by Förster resonance energy transfer calculations, raising questions about the physical mechanism of the process. Our results indicate distinct strategies to steer excitations through the PSI antenna; the red Chls present a shallow reservoir that direct excitations away from P 700 , extending the time to trapping by the reaction center.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

An efficient protocol for excited states of large biochromophores

Efficient energy transport in photosynthetic antenna is a long-standing source of inspiration for artificial light harvesting materials. However, characterizing the excited states of the constituent chromophores poses a considerable challenge to mainstream quantum chemical and semiempirical excited state methods due to their size and complexity and the accuracy required to describe small but functionally important changes in their properties. In this paper, we explore an alternative approach to calculating the excited states of large biochromophores, exemplified by a specific method for calculating the Q y transition of bacteriochlorophyll a, which we name Chl-xTB. Using a diagonally dominant approximation to the Casida equation and a bespoke parameterization scheme, Chl-xTB can match time-dependent density functional theory’s accuracy and semiempirical speed for calculating the potential energy surfaces and absorption spectra of chlorophylls. We demonstrate that Chl-xTB (and other prospective realizations of our protocol) can be integrated into multiscale models, including concurrent excitonic and point-charge embedding frameworks, enabling the analysis of biochromophore networks in a native environment. We exploit this capability to probe the low-frequency spectral densities of excitonic energies and interchromophore interactions in the light harvesting antenna protein LH2 (light harvesting complex 2). The impact of low-frequency protein motion on interchromophore coupling and exciton transport has routinely been ignored due to the prohibitive costs of including it in simulations. Our results provide a more rigorous basis for continued use of this approximation by demonstrating that exciton transition energies are unaffected by low-frequency vibrational coupling to exciton interaction energies.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Impacts of abiotic and biotic factors on tundra productivity near Utqiaġvik, Alaska

Abstract Earlier snowmelt, warmer temperatures and herbivory are among the factors that influence high-latitude tundra productivity near the town of Utqiaġvik in northern Alaska. However, our understanding of the potential interactions between these factors is limited. MODIS observations provide cover fractions of vegetation, snow, standing water, and soil, and fractional absorption of photosynthetically active radiation by canopy chlorophyll (fAPAR chl ) per pixel. Here, we evaluated a recent time-period (2001–2014) that the tundra experienced large interannual variability in vegetation productivity metrics (i.e. fAPAR chl and APAR chl ), which was explainable by both abiotic and biotic factors. We found earlier snowmelt to increase soil and vegetation cover, and productivity in June, while warmer temperatures significantly increased monthly productivity. However, abiotic factors failed to explain stark decreases in productivity during August of 2008, which coincided with a severe lemming outbreak. MODIS observations found this tundra ecosystem to completely recover two years later, resulting in elevated productivity. This study highlights the potential roles of both climate and herbivory in modulating the interannual variability of remotely retrieved plant productivity metrics in Arctic coastal tundra ecosystems.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2025

Switchable client specificity in a dual functional chaperone coordinates light-harvesting complex biogenesis

The proper assembly of light-harvesting complexes (LHCs) is critical for photosynthesis and requires the biogenesis of light-harvesting chlorophylla,b-binding proteins (LHCPs) to be coordinated with chlorophyll (Chl) biosynthesis. The mechanism underlying this coordination is not well understood. Here, we show that a conserved molecular chaperone, chloroplast signal recognition particle 43-kDa protein (cpSRP43), provides a molecular thermostat that helps maintain this coordination. cpSRP43 undergoes a conformational rearrangement between a well-folded closed state and a partially disordered open state. Closed cpSRP43 is dedicated to the biogenesis of LHCPs, whereas open cpSRP43 protects multiple Chl biosynthesis enzymes from heat-induced destabilization. Rising temperature shifts cpSRP43 to the open state, enabling it to protect heat-destabilized Chl biosynthesis enzymes. Our results reveal the molecular basis of a posttranslational mechanism for the thermoadaptation of LHC biogenesis. They also demonstrate how an adenosine triphosphate–independent chaperone uses conformational dynamics to switch its activity and client selectivity, thereby adapting to different proteostatic demands under shifting environmental conditions.

Science & Technology - Other Topics↗
DOE OSTI2023

Light Energy Transduction in Green Sulfur Bacteria

Green sulfur bacteria (GSB) are exquisitely adapted for growth at extraordinarily low light intensities. They are important primary producers of biomass in many anoxic environments, and they contribute significantly to the biogeochemical cycling of carbon, nitrogen, and sulfur on Earth. Green bacteria more generally share the property of using chlorosomes for light-harvesting, and these unusual organelles have many unique features. These include the presence of a monolayer lipid-protein envelope, self-assembling bacteriochlorophylls (BChl) in which pigment-pigment interactions predominate, and the presence of redox components ([2Fe-2S] ferredoxins and quinones) that play a role in regulating excitation energy transfer to the type-1 homodimeric reaction centers. The reaction centers of GSB are related to Photosystem I of cyanobacteria and higher plants but also exhibit several unique structural and functional features. The long-term objectives of this research program are to understand the structure, functions, and biogenesis of the chlorosomes, reaction centers, and electron transport chains that carry out the photochemical transduction of light energy into chemical energy in the model green sulfur bacterium, Chlorobaculum (formerly Chlorobium) tepidum. Over a period of 26 years, we characterized chlorosomes in detail. We identified the proteins in the chlorosome envelopes of diverse organisms, identified the nearest neighbors of those proteins, and characterized the chlorosomes of mutant strains lacking one to five of these proteins. After the genome sequence of Cba. tepidum became available, we identified all genes encoding enzymes for BChl a , BChl c/d/e/ƒ , and Chl a biosynthesis Cba. tepidum . We additionally identified all genes encoding enzymes for carotenoid biosynthesis. By constructing a bchQRU mutant strain that produces [Et, Me]-BChl d , together with collaborators who are specialists in solid-state NMR and cryo-electron microscopy, we solved the structure of the BChls in chlorosomes. Using similar methods and chlorosomes containing [Et, Me]-BChl c , we then showed that alternative structures were possible using the same syn-anti BChl dimer. The structures were refined by introducing spectroscopic data from single-chlorosome measurements. Over the course of this project, we sequenced the genomes of approximately twenty GSB strains, which provided important information for comparative analyses. Through analyses of metagenomic data from Mushroom and Octopus Springs in Yellowstone National Park, we identified a novel chlorophototroph belonging to the phylum Acidobacteriota. We successfully isolated an axenic culture of this organism. We characterized the photosynthetic apparatus of this bacterium, named Chloracidobacterium thermophilum, in significant detail, in particular its type-1, homodimeric reaction centers. Surprisingly, these reaction centers contain three types of Chls, BChl a , Chl a , and Zn-BChl a' . We showed that a dimer of Zn-BChl a' is the primary donor and Chl a the primary acceptor of electrons in this reaction center by using advanced spectroscopic techniques. We isolated eight additional strains of Chloracidobacterium spp. from Mushroom Spring and Rupite hot springs in Bulgaria. Comparative genomes showed that these represent three species, Cab. thermophilum , Cab. aggregatum , and Cab. validum . By applying comparative genomics, genetic, biochemical biophysical and physiological approaches to study green bacteria, we produced a wealth of new information about the remarkable light-harvesting and energy transduction capabilities of these poorly characterized microorganisms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Two-Dimensional Electronic Spectroscopy of the Far-Red-Light Photosystem II Reaction Center

Understanding the role of specific pigments in primary energy conversion in the photosystem II (PSII) reaction center has been impeded by the spectral overlap of its constituent pigments. When grown in far-red light, some cyanobacteria incorporate chlorophyll-f and chlorophyll-d into PSII, relieving the spectral congestion. Here we employ two-dimensional electronic spectroscopy to study PSII at 77 K from Synechococcus sp. PCC 7335 cells that were grown in far-red light (FRL-PSII). We observe the formation of a radical pair within ~3 ps that we assign to Chl D1 •– P D1 •+ . While Pheo D1 is thought to act as the primary electron acceptor in PSII from cells grown in visible light, we see no evidence of its involvement, which we attribute to its reduction by dithionite treatment in our samples. Our work demonstrates that primary charge separation occurs between Chl D1 and P D1 in FRL-PSII, suggesting that P D1 /P D2 may play an underappreciated role in PSII’s charge separation mechanism.

14 SOLAR ENERGY↗
DOE OSTI2024

Light-induced H 2 generation in a photosystem I-O 2 -tolerant [FeFe] hydrogenase nanoconstruct

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 (H 2 ). While both [FeFe] and [NiFe] hydrogenases have been used successfully, they tend to be limited due to either O 2 sensitivity, binding specificity, or H 2 production rates. In this study, we fuse a peripheral (stromal) subunit of Photosystem I (PS I), PsaE, to an O 2 -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 H 2 at a rate of 84.9 ± 3.1 µmol H 2 mg chl –1 h –1 . Further, when prepared and illuminated in the presence of O 2 , the nanoconstruct retains the ability to generate H 2 , though at a diminished rate of 2.2 ± 0.5 µmol H 2 mg chl –1 h –1 . This demonstrates not only that PsaE is a promising scaffold for PS I-based nanoconstructs, but the use of an O 2 -tolerant [FeFe] hydrogenase opens the possibility for an in vivo H 2 generating system that can function in the presence of O 2 .

Hydrogenase↗