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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., Competing Interests: Declaration of competing interest The authors have no competing interests to declare that are relevant to the content of this article., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The Orange Carotenoid Protein (OCP) is responsible for photoprotection in many cyanobacteria. Absorption of blue light drives the conversion of the orange, inactive form (OCP O ) to the red, active form (OCP R ). Concomitantly, the N-terminal domain (NTD) and the C-terminal domain (CTD) of OCP separate, which ultimately leads to the formation of a quenched OCP R -PBS complex. The details of the photoactivation of OCP have been intensely researched. Binding site(s) of OCP R on the PBS core have also been proposed. However, the post-binding events of the OCP R -PBS complex remain unclear. Here, we demonstrate that PBS-bound OCP R is not sufficient as a PBS excitation energy quencher. Using site-directed mutagenesis, we generated a suite of single point mutations at OCP Leucine 51 (L51) of Synechocystis 6803. Steady-state and time-resolved fluorescence analyses demonstrated that all mutant proteins are unable to quench the PBS fluorescence, owing to either failed OCP binding to PBS, or, if bound, an OCP-PBS quenching state failed to form. The SDS-PAGE and Western blot analysis support that the L51A (Alanine) mutant binds to the PBS and therefore belongs to the second category. We hypothesize that upon binding to PBS, OCP R likely reorganizes and adopts a new conformational state (OCP 3rd ) different than either OCP O or OCP R to allow energy quenching, depending on the cross-talk between OCP R and its PBS core-binding counterpart., Competing Interests: Declaration of competing interest There is no research interest conflict associated with this research., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The Orange Carotenoid Protein (OCP) is a cytosolic photosensor that is responsible for non-photochemical quenching (NPQ) of the light-harvesting process in most cyanobacteria. Upon photoactivation by blue-green light, OCP binds to the phycobilisome antenna complex, providing an excitonic trap to thermally dissipate excess energy. At present, both the binding site and NPQ mechanism of OCP are unknown. Using an Anti-Brownian ELectrokinetic (ABEL) trap, we isolate single phycobilisomes in free solution, both in the presence and absence of activated OCP, to directly determine the photophysics and heterogeneity of OCP-quenched phycobilisomes. Surprisingly, we observe two distinct OCP-quenched states, with lifetimes 0.09 ns (6% of unquenched brightness) and 0.21 ns (11% brightness). Photon-by-photon Monte Carlo simulations of exciton transfer through the phycobilisome suggest that the observed quenched states are kinetically consistent with either two or one bound OCPs, respectively, underscoring an additional mechanism for excitation control in this key photosynthetic unit.

Photosynthesis starts with absorption of light energy by light-harvesting antenna complexes with subsequent production of energy-rich organic compounds. However, all photosynthetic organisms face the challenge of excess photochemical conversion capacity. In cyanobacteria, non-photochemical quenching (NPQ) performed by the orange carotenoid protein (OCP) is one of the most important mechanisms to regulate the light energy captured by light-harvesting antennas. This regulation permits the cell to meet its cellular energy requirements and at the same time protects the photosynthetic apparatus under fluctuating light conditions. Several reports have revealed that thermal dissipation increases under excess copper in plants. To explore the effects and mechanisms of copper on cyanobacteria NPQ, photoactivation and relaxation of OCP in the presence of copper were examined in this communication. When OCP o (OCP at orange state) is converted into OCP r (OCP at red state), copper ion has no effect on the photoactivation kinetics. Relaxation of OCP r to OCP o , however, is largely delayed-almost completely blocked, in the presence of copper. Even the addition of the fluorescence recovery protein (FRP) cannot activate the relaxation process. Native polyacrylamide gel electrophoresis (PAGE) analysis result indicates the heterogeneous population of Cu 2+ -locked OCP r . The Cu 2+ -OCP binding constant was estimated using a hyperbolic binding curve. Functional roles of copper-binding OCP in vivo are discussed.

Photosystem II (PSII), a large pigment protein complex, undergoes rapid turnover under natural conditions. During assembly of PSII, oxidative damage to vulnerable assembly intermediate complexes must be prevented. Psb28, the only cytoplasmic extrinsic protein in PSII, protects the RC47 assembly intermediate of PSII and assists its efficient conversion into functional PSII. Its role is particularly important under stress conditions when PSII damage occurs frequently. Psb28 is not found, however, in any PSII crystal structure, and its structural location has remained unknown. In this study, we used chemical cross-linking combined with mass spectrometry to capture the transient interaction of Psb28 with PSII. We detected three cross-links between Psb28 and the α- and β-subunits of cytochrome b 559 , an essential component of the PSII reaction-center complex. These distance restraints enable us to position Psb28 on the cytosolic surface of PSII directly above cytochrome b 559 , in close proximity to the Q B site. Protein-protein docking results also support Psb28 binding in this region. Determination of the Psb28 binding site and other biochemical evidence allow us to propose a mechanism by which Psb28 exerts its protective effect on the RC47 intermediate. This study also shows that isotope-encoded cross-linking with the "mass tags" selection criteria allows confident identification of more cross-linked peptides in PSII than has been previously reported. This approach thus holds promise to identify other transient protein-protein interactions in membrane protein complexes.

Taking inspiration from photosynthetic mechanisms in natural systems, we introduced a light-sensitive photo protective quenching element to an artificial light-harvesting antenna model to control the flow of energy as a function of light intensity excitation. The orange carotenoid protein (OCP) is a nonphotochemical quencher in cyanobacteria: under high-light conditions, the protein undergoes a spectral shift, and by binding to the phycobilisome, it absorbs excess light and dissipates it as heat. By the use of DNA as a scaffold, an antenna system made of organic dyes (Cy3 and Cy5) was constructed, and OCP was assembled on it as a modulated quenching element. By controlling the illumination intensity, it is possible to switch the direction of excitation energy transfer from the donor Cy3 to either of two acceptors. Under low-light conditions, energy is transferred from Cy3 to Cy5, and under intense illumination, energy is partially transferred to OCP as well. These results demonstrate the feasibility of controlling the pathway of energy transfer using light intensity in an engineered light-harvesting system.

The orange carotenoid protein (OCP) and fluorescence recovery protein (FRP) are present in many cyanobacteria and regulate an essential photoprotection cycle in an antagonistic manner as a function of light intensity. We characterized the oligomerization states of OCP and FRP by using native mass spectrometry, a technique that has the capability of studying native proteins under a wide range of protein concentrations and molecular masses. We found that dimeric FRP is the predominant state at protein concentrations ranging from 3 to 180 μM and that higher-order oligomers gradually form at protein concentrations above this range. The OCP, however, demonstrates significantly different oligomerization behavior. Monomeric OCP (mOCP) dominates at low protein concentrations, with an observable population of dimeric OCP (dOCP). The ratio of dOCP to mOCP, however, increases proportionally with protein concentration. Higher-order OCP oligomers form at protein concentrations beyond 10 μM. Additionally, native mass spectrometry coupled with ion mobility allowed us to measure protein collisional cross sections and interrogate the unfolding of different FRP and OCP oligomers. We found that monomeric FRP exhibits a one-stage unfolding process, which could be correlated with its C-terminal bent crystal structure. The structural domain compositions of FRP and OCP are compared and discussed.

Chl synthase (ChlG) is an important enzyme of the Chl biosynthetic pathway catalyzing attachment of phytol/geranylgeraniol tail to the chlorophyllide molecule. Here we have investigated the Flag-tagged ChlG (f.ChlG) in a complex with two different high-light inducible proteins (Hlips) HliD and HliC. The f.ChlG-Hlips complex binds a Chl a and three different carotenoids, β-carotene, zeaxanthin and myxoxanthophyll. Application of ultrafast time-resolved absorption spectroscopy performed at room and cryogenic temperatures revealed excited-state dynamics of complex-bound pigments. After excitation of Chl a in the complex, excited Chl a is efficiently quenched by a nearby carotenoid molecule via energy transfer from the Chl a Qy state to the carotenoid S1 state. The kinetic analysis of the spectroscopic data revealed that quenching occurs with a time constant of ~2ps and its efficiency is temperature independent. Even though due to its long conjugation myxoxanthophyll appears to be energetically best suited for role of Chl a quencher, based on comparative analysis and spectroscopic data we propose that β-carotene bound to Hlips acts as the quencher rather than myxoxanthophyll and zeaxanthin, which are bound at the f.ChlG and Hlips interface. The S1 state lifetime of the quencher has been determined to be 13ps at room temperature and 21ps at 77K. These results demonstrate that Hlips act as a conserved functional module that prevents photodamage of protein complexes during photosystem assembly or Chl biosynthesis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Orange Carotenoid Protein (OCP) plays a unique role in protecting many cyanobacteria from light-induced damage. The active form of OCP is directly involved in energy dissipation by binding to the phycobilisome (PBS), the major light-harvesting complex in cyanobacteria. There are two structural modules in OCP, an N-terminal domain (NTD), and a C-terminal domain (CTD), which play different functional roles during the OCP-PBS quenching cycle. Because of the quasi-stable nature of active OCP, structural analysis of active OCP has been lacking compared to its inactive form. In this report, partial proteolysis was used to generate two structural domains, NTD and CTD, from active OCP. We used multiple native mass spectrometry (MS) based approaches to interrogate the structural features of the NTD and the CTD. Collisional activation and ion mobility analysis indicated that the NTD releases its bound carotenoid without forming any intermediates and the CTD is resistant to unfolding upon collisional energy ramping. The unfolding intermediates observed in inactive intact OCP suggest that it is the N-terminal extension and the NTD-CTD loop that lead to the observed unfolding intermediates. These combined approaches extend the knowledge of OCP photo-activation and structural features of OCP functional domains. Combining native MS, ion mobility, and collisional activation promises to be a sensitive new approach for studies of photosynthetic protein-pigment complexes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Photosynthetic cyanobacteria make important contributions to global carbon and nitrogen budgets. A protein known as the orange carotenoid protein (OCP) protects the photosynthetic apparatus from damage by dissipating excess energy absorbed by the phycobilisome, the major light-harvesting complex in many cyanobacteria. OCP binds one carotenoid pigment, but the color of this pigment depends on conditions. It is orange in the dark and red when exposed to light. We modified the orange and red forms of OCP by using isotopically coded cross-linking agents and then analyzed the structural features by using liquid chromatography and tandem mass spectrometry. Unequivocal cross-linking pairs uniquely detected in red OCP indicate that, upon photoactivation, the OCP N-terminal domain (NTD) and C-terminal domain (CTD) reorient relative to each other. Our data also indicate that the intrinsically unstructured loop connecting the NTD and CTD not only is involved in the interaction between the two domains in orange OCP but also, together with the N-terminal extension, provides a structural buffer system facilitating an intramolecular breathing motion of the OCP, thus helping conversion back and forth from the orange to red form during the OCP photocycle. These results have important implications for understanding the molecular mechanism of action of cyanobacterial photoprotection., Competing Interests: The authors declare no competing financial interests.

We report a top-down proteomic analysis of the membrane-bound peripheral light-harvesting complex LH2 isolated from the purple photosynthetic bacterium Rhodobacter sphaeroides. The LH2 complex is coded for by the puc operon. The Rb. sphaeroides genome contains two puc operons, designated puc1BAC and puc2BA. Although previous work has shown consistently that the LH2 β polypeptide coded by the puc2B gene was assembled into LH2 complexes, there are contradictory reports as to whether the Puc2A polypeptides are incorporated into LH2 complexes. Furthermore, post-translational modifications of this protein offer the prospect that it could coordinate bacteriochlorophyll a (Bchl a) by a modified N-terminal residue. Here, we describe the components of the LH2 complex on the basis of electron-capture dissociation fragmentation to confirm the identity and sequence of the protein's subunits. We found that both gene products of the β polypeptides are expressed and assembled in the mature LH2 complex, but only the Puc1A-encoded polypeptide α is observed here. The methionine of the Puc2B-encoded polypeptide is missing, and a carboxyl group is attached to the threonine at the N-terminus. Surprisingly, one amino acid encoded as an isoleucine in both the puc2B gene and the mRNA is found as valine in the mature LH2 complex, suggesting an unexpected and unusual post-translational modification or a specific tRNA recoding of this one amino acid.

The effects of the Hofmeister series of ions on the activation of the orange carotenoid protein (OCP) from the inactive orange form to the active red form were tested. Kosmotropes led to lower OCP activation, whereas chaotropes led to greater OCP activation. Concentrations of thiocyanate exceeding 1.5M dark activate the orange carotenoid protein to its red form. This chemically activated OCP was studied by UV-vis and circular dichroism spectroscopies. The chemically-activated OCP quenches the fluorescence of phycobilisomes in vitro, to a level comparable to that of the light-activated OCP., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The orange carotenoid protein (OCP), a member of the family of blue light photoactive proteins, is required for efficient photoprotection in many cyanobacteria. Photoexcitation of the carotenoid in the OCP results in structural changes within the chromophore and the protein to give an active red form of OCP that is required for phycobilisome binding and consequent fluorescence quenching. We characterized the light-dependent structural changes by mass spectrometry-based carboxyl footprinting and found that an α helix in the N-terminal extension of OCP plays a key role in this photoactivation process. Although this helix is located on and associates with the outside of the β-sheet core in the C-terminal domain of OCP in the dark, photoinduced changes in the domain structure disrupt this interaction. We propose that this mechanism couples light-dependent carotenoid conformational changes to global protein conformational dynamics in favor of functional phycobilisome binding, and is an essential part of the OCP photocycle.

The orange carotenoid protein (OCP) is a 35 kDa water-soluble protein involved in a photoprotective mechanism of the photosynthetic apparatus of cyanobacteria. The OCP protein contains a single molecule of the carotenoid 3'-hydroxyechinenone (3'-hECN). We have performed transient absorption studies at 77 K in the visible and near-infrared spectral ranges on 3'-hECN in solvent glass and in both inactive (orange) and active (red) forms of OCP. In the OCP the cryogenic temperature prohibited the protein from spontaneous conversion between activity stages and allowed us to study well-defined spectral forms of the protein. The studies show that each form of the OCP consists of two protein subpopulations having different photophysical properties of the bound 3'-hECN. At 77 K the inactive OCP reveals two lifetimes of the first excited state of 3'-hECN of 5.2 and 11 ps while in the active form of OCP these are 3.2 and 7.1 ps. We have also determined the energy of the first excited singlet state of 3'-hECN in long-lived subpopulations of both OCP forms at 77 K. This is 13,750 cm(-1) in the inactive OCP and 12,300 cm(-1) in the active OCP. Shortening of the lifetime and decrease of the energy of the first excited singlet state of 3'-hECN confirm the lengthening of the effective conjugation of the carotenoid upon the inactive-to-active conversion of OCP.

PsbQ is a luminal extrinsic protein component that regulates the water splitting activity of photosystem II (PSII) in plants, algae, and cyanobacteria. However, PsbQ is not observed in the currently available crystal structures of PSII from thermophilic cyanobacteria. The structural location of PsbQ within the PSII complex has therefore remained unknown. Here, we report chemical cross-linking followed by immunodetection and liquid chromatography/tandem MS analysis of a dimeric PSII complex isolated from the model cyanobacterium, Synechocystis sp. PCC 6803, to determine the binding site of PsbQ within PSII. Our results demonstrate that PsbQ is closely associated with the PsbO and CP47 proteins, as revealed by cross-links detected between (120)K of PsbQ and (180)K and (59)K of PsbO, and between (102)K of PsbQ and (440)D of CP47. We further show that genetic deletion of the psbO gene results in the complete absence of PsbQ in PSII complexes as well as the loss of the dimeric form of PSII. Overall, our data provide a molecular-level description of the enigmatic binding site of PsbQ in PSII in a cyanobacterium. These results also help us understand the sequential incorporation of the PsbQ protein during the PSII assembly process, as well as its stabilizing effect on the oxygen evolution activity of PSII.

The orange carotenoid protein (OCP) plays a photoprotective role in cyanobacterial photosynthesis similar to that of nonphotochemical quenching in higher plants. Under high-light conditions, the OCP binds to the phycobilisome (PBS) and reduces the extent of transfer of energy to the photosystems. The protective cycle starts from a light-induced activation of the OCP. Detailed information about the molecular mechanism of this process as well as the subsequent recruitment of the active OCP to the phycobilisome are not known. We report here our investigation on the OCP photoactivation from the cyanobacterium Synechocystis sp. PCC 6803 by using a combination of native electrospray mass spectrometry (MS) and protein cross-linking. We demonstrate that native MS can capture the OCP with its intact pigment and further reveal that the OCP undergoes a dimer-to-monomer transition upon light illumination. The reversion of the activated form of the OCP to the inactive, dark form was also observed by using native MS. Furthermore, in vitro reconstitution of the OCP and PBS allowed us to perform protein chemical cross-linking experiments. Liquid chromatography-MS/MS analysis identified cross-linking species between the OCP and the PBS core components. Our result indicates that the N-terminal domain of the OCP is closely involved in the association with a site formed by two allophycocyanin trimers in the basal cylinders of the phycobilisome core. This report improves our understanding of the activation mechanism of the OCP and the structural binding site of the OCP during the cyanobacterial nonphotochemical quenching process.

Photosystem II (PSII), a large multisubunit pigment-protein complex localized in the thylakoid membrane of cyanobacteria and chloroplasts, mediates light-driven evolution of oxygen from water. Recently, a high-resolution X-ray structure of the mature PSII complex has become available. Two PSII polypeptides, D1 and CP43, provide many of the ligands to an inorganic Mn(4)Ca center that is essential for water oxidation. Because of its unusual redox chemistry, PSII often undergoes degradation followed by stepwise assembly. Psb27, a small luminal polypeptide, functions as an important accessory factor in this elaborate assembly pathway. However, the structural location of Psb27 within PSII assembly intermediates has remained elusive. Here we report that Psb27 binds to CP43 in such assembly intermediates. We treated purified genetically tagged PSII assembly intermediate complexes from the cyanobacterium Synechocystis 6803 with chemical cross-linkers to examine intermolecular interactions between Psb27 and various PSII proteins. First, the water-soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was used to cross-link proteins with complementary charged groups in close association to one another. In the His27△ctpAPSII preparation, a 58-kDa cross-linked species containing Psb27 and CP43 was identified. This species was not formed in the HT3△ctpA△psb27PSII complex in which Psb27 was absent. Second, the homobifunctional thiol-cleavable cross-linker 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP) was used to reversibly cross-link Psb27 to CP43 in His27△ctpAPSII preparations, which allowed the use of liquid chromatography/tandem MS to map the cross-linking sites as Psb27K(63)↔CP43D(321) (trypsin) and CP43K(215)↔Psb27D(58)AGGLK(63)↔CP43D(321) (chymotrypsin), respectively. Our data suggest that Psb27 acts as an important regulatory protein during PSII assembly through specific interactions with the luminal domain of CP43.

Photosystem II (PSII) is a large membrane bound molecular machine that catalyzes light-driven oxygen evolution from water. PSII constantly undergoes assembly and disassembly because of the unavoidable damage that results from its normal photochemistry. Thus, under physiological conditions, in addition to the active PSII complexes, there are always PSII subpopulations incompetent of oxygen evolution, but are in the process of undergoing elaborate biogenesis and repair. These transient complexes are difficult to characterize because of their low abundance, structural heterogeneity, and thermodynamic instability. In this study, we show that a genetically tagged Psb27 protein allows for the biochemical purification of two monomeric PSII assembly intermediates, one with an unprocessed form of D1 (His27ΔctpAPSII) and a second one with a mature form of D1 (His27PSII). Both forms were capable of light-induced charge separation, but unable to photooxidize water, largely because of the absence of a functional tetramanganese cluster. Unexpectedly, there was a significant amount of the extrinsic lumenal PsbO protein in the His27PSII, but not in the His27ΔctpAPSII complex. In contrast, two other lumenal proteins, PsbU and PsbV, were absent in both of these PSII intermediate complexes. Additionally, the only cytoplasmic extrinsic protein, Psb28 was detected in His27PSII complex. Based on these data, we have presented a refined model of PSII biogenesis, illustrating an important role of Psb27 as a gate-keeper during the complex assembly process of the oxygen-evolving centers in PSII.