Removal of endocrine disruptor compounds, CO2 fixation, and macromolecules accumulation in Thermosynechococcus sp. CL-1 cultivation.

Recently, concern on several environmental issues including the pollutant discharge and high concentration of CO2 have gained high interest due to its impact on ecosystem and global warming effect, respectively. Implementation of photosynthetic microorganism carries out numerous advantages including high efficiency of CO2 fixation, the great endurance under extreme conditions and generation of valuable bioproducts. Thermosynechococcus sp. CL-1 (TCL-1), a cyanobacterium, has the ability to perform CO2 fixation and accumulation of various byproducts under extreme conditions like high temperature and alkalinity, presence of estrogen, or even using swine wastewater. This study aimed to assess TCL-1 performance under various endocrine disruptor compounds (bisphenol-A, 17-ß-estradiol/E2, and 17-α-ethynilestradiol/EE2), concentrations (0-10 mg/L), light intensities (500-2000 µE/m2/s), and dissolved inorganic carbon/DIC levels (0-113.2 mM). Addition of E2 content even until 10 mg/L carried out insignificant biomass growth interruption along with the improvement in CO2 fixation rate (79.8 ± 0.1 mg/L/h). Besides the influence of E2, application of higher DIC level and light intensity also enhanced the CO2 fixation rate and biomass growth. The highest biodegradation of E2 at 71% was achieved by TCL-1 in the end of 12 h cultivation period. TCL-1 dominantly produced protein (46.7% ± 0.2%), however, production of lipid and carbohydrate (39.5 ± 1.5 and 23.3 ± 0.9%, respectively) also could be considered as the potential source for biofuel production. Thus, this study can provide an efficient strategy in simultaneously dealing with environmental issues with side advantage in production of macromolecules.

CO2 fixation and cultivation of Thermosynechococcus sp. CL-1 for the production of phycocyanin.

Cultivation of Cyanobacteria is preferable for CO2 fixation process due to its efficiency and production of beneficial byproducts like phycocyanin. In this study, Thermosynechococcus sp. CL-1 (TCL-1) was cultivated in a 30 L flat panel photobioreactor using a 3-fold-modified Fitzgerald medium with 113.2 mM dissolved inorganic carbon. The highest CO2 fixation rate of 21.98 ± 1.52 mg/L/h was followed by higher lipid content (49.91 % dry weight content or %dwc) than the generated carbohydrate (24.22 %dwc). TCL-1 also potentially produced phycocyanin that was dominated by C-phycocyanin (98.10 ± 6.67 mg/g) along with a lower amount of allophycocyanin and phycoerythrin under extraction using various types of solvent. Stability of phycocyanin extract was further examined during storage under various temperatures and light illuminations. Extraction with 36 % glucose solvent presented a protective effect to phycocyanin from heat and photo-damage which was proven by the kinetics study of phycocyanin degradation in this study.

Electrospinning for building 3D structured photoactive biohybrid electrodes.

We describe the development of biohybrid electrodes constructed via combination of electrospun (e-spun) 3D indium tin oxide (ITO) with the trimeric supercomplex photosystem I and the small electrochemically active protein cytochrome c (cyt c). The developed 3D surface of ITO has been created by electrospinning of a mixture of polyelthylene oxide (PEO) and ITO nanoparticles onto ITO glass slides followed by a subsequent elimination of PEO by sintering the composite. Whereas the photosystem I alone shows only small photocurrents at these 3D electrodes, the co-immobilization of cyt c to the e-spun 3D ITO results in well-defined photoelectrochemical signals. The scaling of thickness of the 3D ITO layers by controlling the time (10 min and 60 min) of electrospinning results in enhancement of the photocurrent. Several performance parameters of the electrode have been analyzed for different illumination intensities.

Structural insights into cyanobacterial photosystem II intermediates associated with Psb28 and Tsl0063.

Photosystem II (PSII) is a multisubunit pigment-protein complex and catalyses light-induced water oxidation, leading to the conversion of light energy into chemical energy and the release of dioxygen. We analysed the structures of two Psb28-bound PSII intermediates, Psb28-RC47 and Psb28-PSII, purified from a psbV-deletion strain of the thermophilic cyanobacterium Thermosynechococcus vulcanus, using cryo-electron microscopy. Both Psb28-RC47 and Psb28-PSII bind one Psb28, one Tsl0063 and an unknown subunit. Psb28 is located at the cytoplasmic surface of PSII and interacts with D1, D2 and CP47, whereas Tsl0063 is a transmembrane subunit and binds at the side of CP47/PsbH. Substantial structural perturbations are observed at the acceptor side, which result in conformational changes of the quinone (QB) and non-haem iron binding sites and thus may protect PSII from photodamage during assembly. These results provide a solid structural basis for understanding the assembly process of native PSII.

Generation and characterization of self-assembled protein nanocages based on ß-carboxysomes in Escherichia coli.

Self-assembly is a powerful means to create new materials and new catalysts. The advantages of biological self-assembly are based on it being highly programmable and prone to multilevel regulation, which can lead to multiple and complex functions. The self-assembly of carboxysomes in cyanobacteria enables the carboxysomes to enrich carbon dioxide in their interior, resulting in the formation of a highly efficient, multiple-enzyme catalytic system. Here, we show that the construction and coexpression of all genes of the ß-carboxysome from the cyanobacterium Thermosynechococcus elongatus BP-1 can lead to the production of ß-carboxysome-like structures in Escherichia coli. These shell structures were characterized intracellularly and extracellularly by transmission electron microscopy. This work lays a foundation for understanding carboxysome assembly and catalysis and the development of novel carboxysome-based nanomaterials utilizing synthetic biology.

Ultrafast energy transfer dynamics of phycobilisome from Thermosynechococcus vulcanus, as revealed by ps fluorescence and fs pump-probe spectroscopies.

Cyanobacterial photosynthetic systems efficiently capture sunlight using the pigment-protein megacomplexes, phycobilisome (PBS). The energy is subsequently transferred to photosystem I (PSI) and II (PSII), to produce electrochemical potentials. In the present study, we performed picosecond (ps) time-resolved fluorescence and femtosecond (fs) pump-probe spectroscopies on the intact PBS from a thermophilic cyanobacterium, Thermosynechococcus vulcanus, to reveal excitation energy transfer dynamics in PBS. The photophysical properties of the intact PBS were well characterized by spectroscopic measurements covering wide temporal range from femtoseconds to nanoseconds. The ps fluorescence measurements excited at 570 nm, corresponding to the higher energy of the phycocyanin (PC) absorption band, demonstrated the excitation energy transfer from the PC rods to the allophycocyanin (APC) core complex as well as the energy transfer in the APC core complex. Then, the fs pump-probe measurements revealed the detailed energy transfer dynamics in the PC rods taking place in an ultrafast time scale. The results obtained in this study provide the full picture of the funnel-type excitation energy transfer with rate constants of (0.57 ps)-1 → (7.3 ps)-1 → (53 ps)-1 → (180 ps)-1 → (1800 ps)-1.

Cryo-EM structure of a functional monomeric Photosystem I from Thermosynechococcus elongatus reveals red chlorophyll cluster.

A high-resolution structure of trimeric cyanobacterial Photosystem I (PSI) from Thermosynechococcus elongatus was reported as the first atomic model of PSI almost 20 years ago. However, the monomeric PSI structure has not yet been reported despite long-standing interest in its structure and extensive spectroscopic characterization of the loss of red chlorophylls upon monomerization. Here, we describe the structure of monomeric PSI from Thermosynechococcus elongatus BP-1. Comparison with the trimer structure gave detailed insights into monomerization-induced changes in both the central trimerization domain and the peripheral regions of the complex. Monomerization-induced loss of red chlorophylls is assigned to a cluster of chlorophylls adjacent to PsaX. Based on our findings, we propose a role of PsaX in the stabilization of red chlorophylls and that lipids of the surrounding membrane present a major source of thermal energy for uphill excitation energy transfer from red chlorophylls to P700.

High-resolution cryo-EM structure of photosystem II reveals damage from high-dose electron beams.

Photosystem II (PSII) plays a key role in water-splitting and oxygen evolution. X-ray crystallography has revealed its atomic structure and some intermediate structures. However, these structures are in the crystalline state and its final state structure has not been solved. Here we analyzed the structure of PSII in solution at 1.95 Å resolution by single-particle cryo-electron microscopy (cryo-EM). The structure obtained is similar to the crystal structure, but a PsbY subunit was visible in the cryo-EM structure, indicating that it represents its physiological state more closely. Electron beam damage was observed at a high-dose in the regions that were easily affected by redox states, and reducing the beam dosage by reducing frames from 50 to 2 yielded a similar resolution but reduced the damage remarkably. This study will serve as a good indicator for determining damage-free cryo-EM structures of not only PSII but also all biological samples, especially redox-active metalloproteins.

Structural insights into a dimeric Psb27-photosystem II complex from a cyanobacterium Thermosynechococcus vulcanus.

Photosystem II (PSII) is a multisubunit pigment-protein complex and catalyzes light-driven water oxidation, leading to the conversion of light energy into chemical energy and the release of molecular oxygen. Psb27 is a small thylakoid lumen-localized protein known to serve as an assembly factor for the biogenesis and repair of the PSII complex. The exact location and binding fashion of Psb27 in the intermediate PSII remain elusive. Here, we report the structure of a dimeric Psb27-PSII complex purified from a psbV deletion mutant (ΔPsbV) of the cyanobacterium Thermosynechococcus vulcanus, solved by cryo-electron microscopy. Our structure showed that Psb27 is associated with CP43 at the luminal side, with specific interactions formed between Helix 2 and Helix 3 of Psb27 and a loop region between Helix 3 and Helix 4 of CP43 (loop C) as well as the large, lumen-exposed and hydrophilic E-loop of CP43. The binding of Psb27 imposes some conflicts with the N-terminal region of PsbO and also induces some conformational changes in CP43, CP47, and D2. This makes PsbO unable to bind in the Psb27-PSII. Conformational changes also occurred in D1, PsbE, PsbF, and PsbZ; this, together with the conformational changes occurred in CP43, CP47, and D2, may prevent the binding of PsbU and induce dissociation of PsbJ. This structural information provides important insights into the regulation mechanism of Psb27 in the biogenesis and repair of PSII.

Rhythmic adenosine triphosphate release from the cyanobacterial circadian clock protein KaiC revealed by real-time monitoring of bioluminescence using firefly luciferase.

The cyanobacterial circadian clock is composed of three clock proteins, KaiA, KaiB and KaiC. This KaiABC clock system can be reconstituted in vitro in the presence of adenosine triphosphate (ATP) and Mg2+ , and shows circadian rhythms in the phosphorylation level and ATPase activity of KaiC. Previously, we found that ATP regulates a complex formation between KaiB and KaiC, and KaiC releases ATP from KaiC itself (PLoS One, 8, 2013, e80200). In this study, we examined whether the ATP release from KaiC shows any rhythms in vitro. We monitored the release of ATP from wild-type and ATPase motif mutants of KaiC as a bioluminescence in real time using a firefly luciferase assay in vitro and obtained the following results: (a) ATP release from KaiC oscillated even without KaiA and KaiB although period of the oscillation was not 24 hr; (b) ATP was mainly released from the N-terminal domain of KaiC; and (c) the ATP release was enhanced and suppressed by KaiB and KaiA, respectively. These results suggest that KaiC can generate basal oscillation as a core clock without KaiA and KaiB, whereas these two proteins contribute to adjusting and stabilizing the oscillation.

The active site of magnesium chelatase.

The insertion of magnesium into protoporphyrin initiates the biosynthesis of chlorophyll, the pigment that underpins photosynthesis. This reaction, catalysed by the magnesium chelatase complex, couples ATP hydrolysis by a ChlID motor complex to chelation within the ChlH subunit. We probed the structure and catalytic function of ChlH using a combination of X-ray crystallography, computational modelling, mutagenesis and enzymology. Two linked domains of ChlH in an initially open conformation of ChlH bind protoporphyrin IX, and the rearrangement of several loops envelops this substrate, forming an active site cavity. This induced fit brings an essential glutamate (E660), proposed to be the key catalytic residue for magnesium insertion, into proximity with the porphyrin. A buried solvent channel adjacent to E660 connects the exterior bulk solvent to the active site, forming a possible conduit for the delivery of magnesium or abstraction of protons.

Structure of a cyanobacterial photosystem I surrounded by octadecameric IsiA antenna proteins.

Iron-stress induced protein A (IsiA) is a chlorophyll-binding membrane-spanning protein in photosynthetic prokaryote cyanobacteria, and is associated with photosystem I (PSI) trimer cores, but its structural and functional significance in light harvesting remains unclear. Here we report a 2.7-Å resolution cryo-electron microscopic structure of a supercomplex between PSI core trimer and IsiA from a thermophilic cyanobacterium Thermosynechococcus vulcanus. The structure showed that 18 IsiA subunits form a closed ring surrounding a PSI trimer core. Detailed arrangement of pigments within the supercomplex, as well as molecular interactions between PSI and IsiA and among IsiAs, were resolved. Time-resolved fluorescence spectra of the PSI-IsiA supercomplex showed clear excitation-energy transfer from IsiA to PSI, strongly indicating that IsiA functions as an energy donor, but not an energy quencher, in the supercomplex. These structural and spectroscopic findings provide important insights into the excitation-energy-transfer and subunit assembly mechanisms in the PSI-IsiA supercomplex.

Role of PsbV-Tyr137 in photosystem II studied by site-directed mutagenesis in the thermophilic cyanobacterium Thermosynechococcus vulcanus.

PsbV (cytochrome c550) is one of the three extrinsic proteins of photosystem II (PSII) and functions to maintain the stability and activity of the Mn4CaO5 cluster, the catalytic center for water oxidation. PsbV-Y137 is the C-terminal residue of PsbV and is located at the exit of a hydrogen-bond network mediated by the D1-Y161-H190 residue pair. In order to examine the function of PsbV-Y137, four mutants, PsbV-Y137A, PsbV-Y137F, PsbV-Y137G, and PsbV-Y137W, were generated with Thermosynechococcus vulcanus (T. vulcanus). These mutants showed growth rates similar to that of the wild-type strain (WT); however, their oxygen-evolving activities were different. At pH 6.5, the oxygen evolution rates of Y137F and Y137W were almost identical to that of WT, whereas the oxygen evolution rates of the Y137A, Y137G mutants were 64% and 61% of WT, respectively. However, the oxygen evolution in the latter two mutants decreased less at higher pHs, suggesting that higher pHs facilitated oxygen evolution probably by facilitating proton egress in these two mutants. Furthermore, thylakoid membranes isolated from the PsbV-Y137A, PsbV-Y137G mutants exhibited much lower levels of oxygen-evolving activity than that of WT, which was found to be caused by the release of PsbV. In addition, PSII complexes purified from the PsbV-Y137A and PsbV-Y137G mutants lost all of the three extrinsic proteins but instead bind Psb27, an assembly cofactor of PSII. These results demonstrate that the PsbV-Tyr137 residue is required for the stable binding of PsbV to PSII, and the hydrogen-bond network mediated by D1-Y161-H190 is likely to function in proton egress during water oxidation.

Generation of ion-radical chlorophyll states in the light-harvesting antenna and the reaction center of cyanobacterial photosystem I.

The energy and charge-transfer processes in photosystem I (PS I) complexes isolated from cyanobacteria Thermosynechococcus elongatus and Synechocystis sp. PCC 6803 were investigated by pump-to-probe femtosecond spectroscopy. The formation of charge-transfer (CT) states in excitonically coupled chlorophyll a complexes (exciplexes) was monitored by measuring the electrochromic shift of ß-carotene in the spectral range 500-510 nm. The excitation of high-energy chlorophyll in light-harvesting antenna of both species was not accompanied by immediate appearance of an electrochromic shift. In PS I from T. elongatus, the excitation of long-wavelength chlorophyll (LWC) caused a pronounced electrochromic effect at 502 nm assigned to the appearance of CT states of chlorophyll exciplexes. The formation of ion-radical pair P700+A1- at 40 ps was limited by energy transfer from LWC to the primary donor P700 and accompanied by carotenoid bleach at 498 nm. In PS I from Synechocystis 6803, the excitation at 720 nm produced an immediate bidentate bleach at 690/704 nm and synchronous carotenoid response at 508 nm. The bidentate bleach was assigned to the formation of primary ion-radical state PB+Chl2B-, where negative charge is localized predominantly at the accessory chlorophyll molecule in the branch B, Chl2B. The following decrease of carotenoid signal at ~ 5 ps was ascribed to electron transfer to the more distant molecule Chl3B. The reduction of phylloquinone in the sites A1A and A1B was accompanied by a synchronous blue-shift of the carotenoid response to 498 nm, pointing to fast redistribution of unpaired electron between two branches in favor of the state PB+A1A-.

Function of PsbO-Asp158 in photosystem II: effects of mutation of this residue on the binding of PsbO and function of PSII in Thermosynechococcus vulcanus.

PsbO-D158 is a highly conserved residue of the PsbO protein in photosystem II (PSII), and participates in one of the hydrogen-bonding networks connecting the manganese cluster with the lumenal surface. In order to examine the role of PsbO-D158, we mutated it to E, N or K in Thermosynechococcus vulcanus and characterized photosynthetic properties of the mutants obtained. The growth rates of these three mutants were similar to that of the wild type, whereas the oxygen-evolving activity of the three mutant cells decreased to 60-64% of the wild type. Fluorescence kinetics showed that the mutations did not affect the electron transfer from QA to QB, but slightly affected the donor side of PSII. Moreover, all of the three mutant cells were more sensitive to high light and became slower to recover from photoinhibition. In the isolated thylakoid membranes from the three mutants, the PsbU subunit was lost and the oxygen-evolving activity was reduced to a lower level compared to that in the respective cells. PSII complexes isolated from these mutants showed no oxygen-evolving activity, which was found to be due to large or complete loss of PsbO, PsbV and PsbU during the process of purification. Moreover, PSII cores purified from the three mutants contained Psb27, an assembly co-factor of PSII. These results suggest that PsbO-D158 is required for the proper binding of the three extrinsic proteins to PSII and plays an important role in maintaining the optimal oxygen-evolving activity, and its mutation caused incomplete assembly of the PSII complex.

FTIR Microspectroscopic Analysis of the Water Oxidation Reaction in a Single Photosystem II Microcrystal.

Microcrystals of photosystem II (PSII) have recently been used to investigate the intermediate structures of the water oxidizing complex during water oxidation by serial femtosecond crystallography using X-ray free electron lasers. To clarify the water oxidation mechanism, it is crucial to know whether the reaction proceeds properly in the microcrystals. In this work, we monitored the water oxidation reaction in a single PSII microcrystal using Fourier transform infrared (FTIR) microspectroscopy with the transmission method. Flash-induced micro-FTIR difference spectra of S-state transitions in a PSII microcrystal showed features virtually identical to the corresponding spectra previously obtained using the attenuated total reflection method for multiple microcrystals, representing the reactions near the crystal surface, as well as the spectra in solution. This observation indicates that the reaction processes of water oxidation proceed with relatively high efficiencies retaining native intermediate structures in the entire inside of a PSII microcrystal.