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11 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Phycobilisome core architecture influences photoprotective quenching by the Orange Carotenoid Protein

Photosynthetic organisms rely on sophisticated photoprotective mechanisms to prevent oxidative damage under high or fluctuating solar illumination. Cyanobacteria, which have evolved a unique, water-soluble light-harvesting complex—the phycobilisome—achieve photoprotection through a photoactivatable quencher called the Orange Carotenoid Protein (OCP). Phycobilisomes are highly symmetric and modular, formed by hierarchical assembly of conserved subunits into diverse geometries ranging from simple bundles to elaborate fan- or bouquet-like macromolecular architectures. Although OCP is known to provide photoprotection across species of cyanobacteria with different phycobilisome structures, it is not known whether or how these structural variations relate to changes in the photoprotective function of OCP. For example, OCP was recently discovered to bind as a dimer at two specific instances of an abundant structural motif on the tricylindrical phycobilisome of Synechocystis sp. PCC 6803, yet these sites are sterically inaccessible on a more common pentacylindrical phycobilisome ( Anabaena sp. PCC 7120). To understand how structural modularity and binding specificity contribute to conservation of OCP binding sites and function across different phycobilisome architectures, here we compare experimentally measured photophysical states accessible to these prototypical tricylindrical and pentacylindrical phycobilisomes, with and without OCP, at the single-molecule level. Together with Monte Carlo simulations of exciton transfer in OCP-quenched phycobilisomes, our results suggest that OCP binds at distinct and specific sites in each type of phycobilisome, yet provides nearly identical quenching strength to both phycobilisomes. Our findings highlight the utility of modular phycobilisome structures in balancing robust conservation of photoprotective function with adaptability of site-specific binding across species.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Alteration of phycobilisome excitation energy transfer properties in response to attenuations in peripheral electron flow

In Synechocystis sp. PCC 6803 ( S . 6803), two types of phycobilisome (PBS) complexes, CpcG-PBS and CpcL-PBS, function to harvest light energy for photosynthetic reaction centers (RCs), photosystem I (PSI) and photosystem II (PSII). The compositional differences between these two forms of PBS and their specificity for RCs have led to suggestions that they may differ in function. To address this question, we examined how PBS-RC interactions, and the transfer of excitation energy from PBS to RCs, might be adjusted under conditions where electron demand and photon availability are modulated. The CpcG-PBS, CpcL-PBS, and RC complexes were isolated from a S . 6803 strain defective in expression of flavodiiron 1 (oxygen reduction reaction 1, ORR1) grown under varied light regimes. The energy transfer preference from CpcL-PBS to either PSI or PSII was investigated by in vitro crosslinking and 77 K fluorescence emission spectroscopy to assess energy transfer efficiency under photoexcitation. While the results demonstrate that the transfer of excitation energy from CpcL-PBS favors PSI over PSII in WT strains as previously shown, the preference of CpcL-PBS switches from PSI to PSII in ORR1 strains. Surprisingly, this change in preference was reproduced when ORR1 CpcL-PBS was crosslinked with WT RCs, or when WT CpcL-PBS was cross-crosslinked with ORR1 RCs, indicating there are physical modifications to both PBS and RCs that mediate the preference switch. In contrast, the analysis with ORR1 CpcG-PBS shows similar preferences to WT. Additionally, PBS populations in ORR1 shifted to a greater proportion of CpcL-PBS relative to CpcG-PBS. These results demonstrate that under conditions where electron utilization changes, there is a tuning of the excitation energy allocation from CpcL-PBS to RCs to manage the energy distribution for photosynthesis under dynamic flux conditions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Cyanobacteria dynamically regulate phycobilisome-to-photosystem excitation energy transfer

In cyanobacteria and red algae, the phycobilisome (PBS) absorbs light and transfers its energy to the chlorophylls in photosystems II (PSII) and I (PSI). With the help of target analysis of time-resolved emission spectra measured at room temperature (RT) and at 77 K, we establish a general kinetic scheme for excitation energy transfer (EET) and trapping based upon a PBS-PSII-PSI megacomplex. At RT it is found that in the dark-adapted cells (State II), the terminal emitter of PBS, allophycocyanin APC680, transfers energy to PSII and PSI with equal rates of ≈50 ns −1 , and that spillover from PSII to PSI is present with rate ≈6 ns −1 . At 77 K, upon transition from State I to State II the EET rate from APC680 to PSII is constant, whereas the rate to PSI increases by 67%. This indicates that a structural change in EET distance in the PBS-PSII-PSI megacomplex underlies the state transition.

Science & Technology - Other Topics↗
DOE OSTI2025

Underlying data for characterization of phycocyanobilin (PCB) biosynthesis in Galdieria sulphuraria

This dataset contains data for studies of phycocyanobilin (PCB) synthesis in the red alga Galdieria sulphuraria. Red algae such as G. sulphuraria utilize phycobilisomes for light harvesting. The phycobilisomes of early-branching organisms such as G. sulphuraria or Cyanidioschyzon merolae contain PCB chromophores but not phycoerythrobilin (PEB), in contrast to the phycobilisomes of other red algae. The studies reported in this dataset examine biosynthesis of PCB in G. sulphuraria and C. merolae, starting from biliverdin IX-alpha (BV), the last known common precursor for PCB and PEB. In cyanobacteria, phages, green algae, and land plants, conversion of BV into PCB or PEB is carried out by a family of enzymes called ferredoxin-dependent bilin reductases (FDBRs). The current studies demonstrate that G. sulphuraria, but not C. merolae, require the action of an additional isomerase to synthesize PCB.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Biosynthesis of Minimal C-Phycocyanin Chromophore Assemblies in E. coli Provides a Platform to Dissect Protein-Mediated Tuning of Exciton Transfer

Cyanobacteria are arguably among the most evolutionarily successful organisms on Earth, inhabiting a wide range of ocean, freshwater, soil, and even desert environments on every continent. The cyanobacterial phycobilisome consists of stacks of disk-like light-collecting moieties, allophycocyanin (APC) and phycocyanin (CPC), with covalently bound phycocyanobilin (PCB) pigments. The ways in which the energies of the specific chromophores in these complexes are tuned by the protein to achieve its highly efficient and directional energy transfer are not fully understood, as complex combinations of decay pathways are occurring simultaneously and competitively through this elaborate light-harvesting system. This makes it difficult to extract information about isolated protein-pigment interactions. We provide herein a description of a useful new experimental platform in which we have recombinantly expressed a fully functioning CPC complex and selectively created minimal chromophore sets to study their individual contributions to the overall CPC spectra. Structural and computational analysis of this protein system have provided a greater understanding of how the protein environment serves to alter the photophysics of each of these chromophores. Introduction of a quencher into various positions within CPC confirmed the ability of the protein environment to tune the directionality of energy transport in this assembly. Further mutational analysis suggested the roles of key amino acids surrounding the chromophores, showcasing the utility of heterologous expression techniques for understanding the effects of structure on EET mechanisms in the phycobilisome.

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

From cytoplasm to lumen—mapping the free pools of protein subunits of three photosynthetic complexes using quantitative mass spectrometry

The phycobilisome (PBS) captures light energy and transfers it to photosystem I (PSI) and photosystem II (PSII). Which and how many copies of protein subunits in PBSs, PSI, and PSII remain unbound in thylakoids are unknown. Here, quantitative mass spectrometry (QMS) was used to quantify substantial pools of free extrinsic subunits of PSII and PSI. Interestingly, the membrane intrinsic PsaL is 3-fold higher than PsaA/B. This scenario complements the static structures of these complexes as revealed by X-ray crystallography and cryo-EM. Furthermore, the ratios of ApcG and photoprotective OCP over PBS indicate a pool of extra ApcG. The 2.5 ratio of CpcG-PBS over CpcL-PBS improves our understanding of these light-harvesting complexes involved in energy capture and photoprotection in cyanobacteria.

cyanobacteria↗
DOE OSTI2025

The linker protein ApcI regulates light harvesting under red light in Synechocystis sp. PCC 6803

Phycobilisomes (PBSs) are versatile cyanobacterial antenna complexes that harvest light energy to drive photosynthesis. They can adapt to various light conditions; for example, dismantling under high light to prevent photo-oxidation and arranging in rows under low light to increase light harvesting efficiency. Light quality also influences PBS structure and function, as observed under far-red light exposure. Here, we describe a PBS linker protein, ApcI (previously hypothetical protein Sll1911), expressed specifically under red light (620 nm) or upon chemically induced reduction of the plastoquinone pool. We characterized ApcI in Synechocystis sp. PCC 6803 using mutant analyses, PBS binding experiments, and protein interaction studies. Deletion of apcI conferred high light tolerance on Synechocystis sp. PCC 6803 compared to the wild-type strain, leading to reduced energy transfer from PBSs to the photosystems under high light. Binding experiments revealed that ApcI replaces the linker protein ApcG at the membrane-facing side of the PBS core via a paralogous C-terminal motif. Additionally, the N-terminal region of ApcI interacts with photosystem II. Our findings highlight the importance of PBS remodeling for adaptation to different light conditions. The characterization of ApcI provides insight into the mechanisms by which cyanobacteria optimize light harvesting in response to varying light conditions.

Photosystem↗
DOE OSTI2025

Photoactivation Transition State and Dynamical Response of the Orange Carotenoid Protein

The orange carotenoid protein (OCP) regulates light harvesting in cyanobacteria by acting as a photoreceptor in its resting form, OCP O , and by effecting the quenching of bilin excitons upon binding to the core of the phycobilisome in its photoactivated red form, OCP R . We show herein using fluorescence anisotropy measurements and the action spectra for the rate constants of the two light-driven steps in the mechanism that the photoactivation of the OCP from Synechocystis sp. PCC 6803 is triggered by excited-state motions of the canthaxanthin chromophore that yield a twisted and bent conformation. Well-tempered metadynamics simulations reveal that a bicycle-pedal configuration, due to twisting of the two adjacent C=C bonds at the C13−C14 and C15−C15′ positions in the center of canthaxanthin’s π-conjugated isoprenoid backbone, can be accommodated by the binding site in the OCP, with the energy of a captured photon required to cross the local activation energy barriers from the dark equilibrium structure. The bicycle-pedal configuration breaks the conserved hydrogen-bonding interactions between the carbonyl substituent of the β-ionone end ring of canthaxanthin and the adjacent W288 and Y201 residues in the C-terminal domain. The action spectra are modulated by the vibronic excitation prepared by absorption transitions to the S 2 state, indicating that the photoactivation reactions are triggered by the canthaxanthin chromophore well prior to vibrational equilibration. These findings show that an ultrafast structural response of the OCP protein to the excited-state motions of the canthaxanthin chromophore controls the photoactivation yield and the sensing of blue light.

Fluorescence↗
DOE OSTI2025

Plastoquinone redox status influences carboxysome integrity via a RpaA ‐ and reactive oxygen species‐dependent regulatory network

SUMMARY Carboxysomes are bacterial microcompartments that encapsulate Rubisco and are a core component of the cyanobacterial carbon concentration mechanism (CCM). While carboxysome number, size, and spatial organization vary in different environmental conditions (CO 2 , light availability, redox state, temperature, and light quality), the molecular mechanisms underlying this potentially adaptive process remain elusive. Herein, we observe that mutants of the circadian rhythm/metabolism factor, Regulator of Phycobilisome Association A (RpaA), exhibit a striking breakdown of carboxysomes under certain environmental conditions. We find that conditions leading to overreduction of the plastoquinone (PQ) pool (mixotrophic growth, high irradiance, or chemical inhibition of electron transfer from PQ to the cytochromeb 6 fcomplex) are accompanied by an elevated generation of reactive oxygen species (ROS) and correlate with the loss of carboxysome integrity. Carboxysome breakdown is reversed by environmental conditions or chemical inhibitors that prevent PQ overreduction and accompanying ROS generation. Taken together, our data support a novel link between the redox status of the PQ pool and carboxysome integrity. Our results have implications for the fundamental understanding of cyanobacterial energy‐balancing pathways and may indicate new research directions for understanding how the carboxysome is remodeled in response to changing environments.

Plant Sciences↗
DOE OSTI2026

Phycocyanobilin biosynthesis in Galdieria sulphuraria requires isomerization of phycoerythrobilin synthesized by bilin reductases

Phycobiliproteins are essential components of the light-harvesting antennae in cyanobacteria and red algae, requiring covalently bound open-chain tetrapyrrole chromophores (bilins) for proper function. In the red alga Galdieria sulphuraria , the primary chromophore is phycocyanobilin (PCB), despite the apparent presence of only biosynthetic genes for phycoerythrobilin (PEB) biosynthesis (PEBA and PEBB). This observation suggests the presence of an alternative, atypical biosynthetic pathway for PCB. In this study, we confirmed the presence of PEB:PCB isomerase activity in an enriched protein fraction from G. sulphuraria . To further investigate this unusual pathway, we combined in silico analyses with biochemical assays. Phylogenetic analyses confirmed the placement of the G. sulphuraria ferredoxin-dependent bilin reductases within the PEBA and PEBB lineages, typically associated with PEB synthesis, whereas the related red alga Cyanidioschyzon merolae was found to contain only PCYA. This gene distribution presents a functional paradox. G. sulphuraria harbors PEB biosynthesis genes but no detectable PEB chromophores and lacks known PCB-synthesizing enzymes despite containing PCB. Functional characterization of recombinant GsPEBA (G. sulphuraria PEBA) and GsPEBB ( G. sulphuraria PEBB) confirmed their roles in PEB synthesis, demonstrating that these enzymes have not evolved to synthesize PCB or act as isomerases despite their phylogenetic placement. In contrast, Cm PCYA ( C. merolae PCYA) catalyzed direct PCB formation from biliverdin. Together, these findings reveal an atypical isomerase-based pathway for PCB biosynthesis in G. sulphuraria , expanding our understanding of bilin metabolism and providing new insight into the evolutionary flexibility of photosynthetic pigment biosynthesis in Rhodophyta.

14 SOLAR ENERGY↗
DOE OSTI2026

Influence of cellular redox reactions on the structure and function of light harvesting and photosystems

Photosynthesis enables the conversion of one of the most abundant and free forms of energy, sunlight, into chemical bonds through the utilization of highly tailored protein complexes. These enzymes work in unison to absorb, convert, and transform light into high-energy electrons which are used for various functions important to metabolism and cellular protection. Over the last ∼50 years, photosynthetic organisms, such as cyanobacteria, have been adapted and engineered to produce valuable compounds like hydrogen and ethylene, among others. Often this is performed by removing native and/or adding in exogenous energy utilization pathways so that light energy is re-directed towards the synthesis of desired compounds. However, the interplay between primary light capture, conversion reactions, and the downstream electron utilization sinks is not fully understood. Further complicating these strategies are the plethora of compensatory mechanisms that facilitate steady electron flow and the maintenance of photosynthesis under dynamic conditions. This manifests as structural and functional plasticity of the photosynthetic machinery, often seen in modulations of oligomeric compositions or changes in protein-protein interactions and coupling with redox enzymes. Understanding these mechanisms is crucial to biotechnology applications because re-engineering electron utilization sinks has profoundly different effects on the light capture and conversion reactions of photosynthesis. Optimization requires a molecular-level understanding of the functional interrelationships between electron sinks and photosynthetic components that influence photosynthetic efficiencies to realize potential improvements in product yields. Here, we aim to highlight how perturbation of reductive reactions is revealing the functional plasticity in key components of the photosynthetic energy transduction pathway.

59 BASIC BIOLOGICAL SCIENCES↗