Cyanobacterium Thermosynechococcus Elongatus Research Articles

Energetics and proton release in photosystem II from Thermosynechococcus elongatus with a D1 protein encoded by either the psbA2 or psbA3 gene

In the cyanobacterium Thermosynechococcus elongatus, there are three psbA genes coding for the Photosystem II (PSII) D1 subunit that interacts with most of the main cofactors involved in the electron transfers. Recently, the 3D crystal structures of both PsbA2-PSII and PsbA3-PSII have been solved [Nakajima et al., J. Biol. Chem. 298 (2022) 102668.]. It was proposed that the loss of one hydrogen bond of PheD1 due to the D1-Y147F exchange in PsbA2-PSII resulted in a more negative Em of PheD1 in PsbA2-PSII when compared to PsbA3-PSII. In addition, the loss of two water molecules in the Cl-1 channel was attributed to the D1-P173M substitution in PsbA2-PSII. This exchange, by narrowing the Cl-1 proton channel, could be at the origin of a slowing down of the proton release. Here, we have continued the characterization of PsbA2-PSII by measuring the thermoluminescence from the S2QA−/DCMU charge recombination and by measuring proton release kinetics using time-resolved absorption changes of the dye bromocresol purple. It was found that i) the Em of PheD1–/PheD1 was decreased by ∼30 mV in PsbA2-PSII when compared to PsbA3-PSII and ii) the kinetics of the proton release into the bulk was significantly slowed down in PsbA2-PSII in the S2TyrZ• to S3TyrZ and S3TyrZ• → (S3TyrZ•)’ transitions. This slowing down was partially reversed by the PsbA2/M173P mutation and induced by the PsbA3/P173M mutation thus confirming a role of the D1-173 residue in the egress of protons trough the Cl-1 channel.

Crystal structures of photosystem II from a cyanobacterium expressing psbA2 in comparison to psbA3 reveal differences in the D1 subunit

Three psbA genes (psbA1, psbA2, and psbA3) encoding the D1 subunit of photosystem II (PSII) are present in the thermophilic cyanobacterium Thermosynechococcus elongatus and are expressed differently in response to changes in the growth environment. To clarify the functional differences of the D1 protein expressed from these psbA genes, PSII dimers from two strains, each expressing only one psbA gene (psbA2 or psbA3), were crystallized, and we analyzed their structures at resolutions comparable to previously studied PsbA1-PSII. Our results showed that the hydrogen bond between pheophytin/D1 (PheoD1) and D1-130 became stronger in PsbA2- and PsbA3-PSII due to change of Gln to Glu, which partially explains the increase in the redox potential of PheoD1 observed in PsbA3. In PsbA2, one hydrogen bond was lost in PheoD1 due to the change of D1-Y147F, which may explain the decrease in stability of PheoD1 in PsbA2. Two water molecules in the Cl-1 channel were lost in PsbA2 due to the change of D1-P173M, leading to the narrowing of the channel, which may explain the lower efficiency of the S-state transition beyond S2 in PsbA2-PSII. In PsbA3-PSII, a hydrogen bond between D1-Ser270 and a sulfoquinovosyl-diacylglycerol molecule near QB disappeared due to the change of D1-Ser270 in PsbA1 and PsbA2 to D1-Ala270. This may result in an easier exchange of bound QB with free plastoquinone, hence an enhancement of oxygen evolution in PsbA3-PSII due to its high QB exchange efficiency. These results provide a structural basis for further functional examination of the three PsbA variants.

Effects of Esterified Styrene-Maleic Acid Copolymer Degradation on Integral Membrane Protein Extraction.

The detergent-free extraction of integral membrane proteins using styrene-maleic acid copolymers (SMAs) has shown promise as a potentially effective technique to isolate proteins in a more native-like conformation. As the field continues to develop, the protein selectivity and extraction efficiency of many analogues of traditional SMAs are being investigated. Recently, we discovered that the monoesterification of SMAs with alkoxy ethoxylate sidechains drastically affects the bioactivity of these copolymers in the extraction of photosystem I from the cyanobacterium Thermosynechococcus elongatus. However, subsequent investigations also revealed that the conditions under which these esterified SMA polymer analogues are prepared, purified, and stored can alter the structure of the alkoxy ethoxylate-functionalized SMA and perturb the protein extraction process. Herein, we demonstrate that the basic conditions required to solubilize SMA analogues may lead to deleterious saponification side reactions, cleaving the sidechains of an esterified SMA and dramatically decreasing its efficacy for protein extraction. We found that this process is highly dependent on temperature, with polymer samples being prepared and stored at lower temperatures exhibiting significantly fewer saponification side reactions. Furthermore, the effects of small-molecule impurities and exposure to light were also investigated, both of which are shown to have significant effects on the polymer structure and/or protein extraction process.

Mass spectrometry analysis of the photosystem II assembly factor Psb27 revealed variations in its lipid modification

The assembly of large, multi-cofactor membrane protein complexes like photosystem II (PSII) requires a high level of coordination. The process is facilitated by a large network of auxiliary proteins that bind transiently to unassembled subunits, preassembled modules or intermediate states of PSII, which are comprised of a subset of subunits. However, analysis of these immature, partially assembled PSII complexes is hampered by their low abundance and intrinsic instability. In this study, PSII was purified from the thermophilic cyanobacterium Thermosynechococcus elongatus via Twin-Strep-tagged CP43 and further separated by ion exchange chromatography into mature and immature complexes. Mass spectrometry analysis of the immature Psb27-PSII intermediate revealed six different Psb27 proteoforms with distinct lipid modifications. The maturation and functional role of thylakoid localized lipoproteins are discussed.

Production of thermostable phycocyanin in a mesophilic cyanobacterium

Phycocyanin (PC) is a soluble phycobiliprotein found within the light-harvesting phycobilisome complex of cyanobacteria and red algae, and is considered a high-value product due to its brilliant blue colour and fluorescent properties. However, commercially available PC has a relatively low temperature stability. Thermophilic species produce more thermostable variants of PC, but are challenging and energetically expensive to cultivate. Here, we show that the PC operon from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (cpcBACD) is functional in the mesophile Synechocystis sp. PCC 6803. Expression of cpcBACD in an ‘Olive’ mutant strain of Synechocystis lacking endogenous PC resulted in high yields of thermostable PC (112 ± 1 mg g−1 DW) comparable to that of endogenous PC in wild-type cells. Heterologous PC also improved the growth of the Olive mutant, which was further supported by evidence of a functional interaction with the endogenous allophycocyanin core of the phycobilisome complex. The thermostability properties of the heterologous PC were comparable to those of PC from T. elongatus, and could be purified from the Olive mutant using a low-cost heat treatment method. Finally, we developed a scalable model to calculate the energetic benefits of producing PC from T. elongatus in Synechocystis cultures. Our model showed that the higher yields and lower cultivation temperatures of Synechocystis resulted in a 3.5-fold increase in energy efficiency compared to T. elongatus, indicating that producing thermostable PC in non-native hosts is a cost-effective strategy for scaling to commercial production.

Protein Extraction Efficiency and Selectivity of Esterified Styrene-Maleic Acid Copolymers in Thylakoid Membranes.

Amphiphilic styrene-maleic acid copolymers (SMAs) have been shown to effectively extract membrane proteins surrounded by an annulus of native membrane lipids via the formation of nanodiscs. Recent reports have shown that 2-butoxyethanol-functionalized SMA derivatives promote the extraction of membrane proteins from thylakoid membranes, whereas unfunctionalized SMA is essentially ineffective. However, it is unknown how the extent of functionalization and identity of sidechains impact protein solubilization and specificity. Herein, we show that the monoesterification of an SMA polymer with hydrophobic alkoxy ethoxylate sidechains leads to an increased solubilization efficiency (SE) of trimeric photosystem I (PSI) from the membranes of cyanobacterium Thermosynechococcus elongatus. The specific SMA polymer used in this study, PRO 10235, cannot encapsulate single PSI trimers from this cyanobacterium; however, as it is functionalized with alkoxy ethoxylates of increasing alkoxy chain length, a clear increase in the trimeric PSI SE is observed. Furthermore, an exponential increase in the SE is observed when >50% of the maleic acid repeat units are monoesterified with long alkoxy ethoxylates, suggesting that the PSI extraction mechanism is highly dependent on both the number and length of the attached side chains.

Generation and characterization of self-assembled protein nanocages based on β-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 β-carboxysome from the cyanobacterium Thermosynechococcus elongatus BP-1 can lead to the production of β-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.

C-phycocyanin as a highly attractive model system in protein crystallography: unique crystallization properties and packing-diversity screening.

The unique crystallization properties of the antenna protein C-phycocyanin (C-PC) from the thermophilic cyanobacterium Thermosynechococcus elongatus are reported and discussed. C-PC crystallizes in hundreds of significantly different conditions within a broad pH range and in the presence of a wide variety of precipitants and additives. Remarkably, the crystal dimensions vary from a few micrometres, as used in serial crystallography, to several hundred micrometres, with a very diverse crystal morphology. More than 100 unique single-crystal X-ray diffraction data sets were collected from randomly selected crystals and analysed. The addition of small-molecule additives revealed three new crystal packings of C-PC, which are discussed in detail. The high propensity of this protein to crystallize, combined with its natural blue colour and its fluorescence characteristics, make it an excellent candidate as a superior and highly adaptable model system in crystallography. C-PC can be used in technical and methods development approaches for X-ray and neutron diffraction techniques, and as a system for comprehending the fundamental principles of protein crystallography.

Electronic Structure of the Primary Electron Donor P700+• in Photosystem I Studied by Multifrequency HYSCORE Spectroscopy at X- and Q-Band.

The primary electron donor P700 of the photosystem I (PSI) is a heterodimer consisting of two chlorophyll molecules. A series of electron-transfer events immediately following the initial light excitation leads to a stabilization of the positive charge by its cation radical form, P700+•. The electronic structure of P700+• and, in particular, its asymmetry with respect to the two chlorophyll monomers is of fundamental interest and is not fully understood up to this date. Here, we apply multifrequency X- (9 GHz) and Q-band (35 GHz) hyperfine sublevel correlation (HYSCORE) spectroscopy to investigate the electron spin density distribution in the cation radical P700+• of PSI from a thermophilic cyanobacterium Thermosynechococcus elongatus. Six 14N and two 1H distinct nuclei have been resolved in the HYSCORE spectra and parameters of the corresponding nuclear hyperfine and quadrupolar hyperfine interactions were obtained by combining the analysis of HYSCORE spectral features with direct numerical simulations. Based on a close similarity of the nuclear quadrupole tensor parameters, all of the resolved 14N nuclei were assigned to six out of total eight available pyrrole ring nitrogen atoms (i.e., four in each of the chlorophylls), providing direct evidence of spin density delocalization over the both monomers in the heterodimer. Using the obtained experimental values of the 14N electron-nuclear hyperfine interaction parameters, the upper limit of the electron spin density asymmetry parameter is estimated as RA/Bupper = 7.7 ± 0.5, while a tentative assignment of 14N observed in the HYSCORE spectra yields RB/A = 3.1 ± 0.5.

Crystal structures of native cytochrome c6 from Thermosynechococcus elongatus in two different space groups and implications for its oligomerization.

Native cytochrome c6 was purified from an extract of strain BP-1 of the thermophilic cyanobacterium Thermosynechococcus elongatus. The protein was crystallized, and with only slight modifications of the buffer and vapour-diffusion conditions two different space groups were observed, namely H3 and C2. Both crystal structures were solved; they contained three and six molecules per asymmetric unit and were refined to 1.7 and 2.25 Å resolution, respectively. To date, the structure of native cytochrome c6 from T. elongatus has only been reported as a monomer using NMR spectroscopy, i.e. without addressing putative oligomerization, and related structures have only previously been solved using X-ray crystallography after recombinant gene overexpression in Escherichia coli. The reported space groups of related cyanobacterial cytochrome c6 structures differ from those reported here. Interestingly, the protein-protein interfaces that were observed utilizing X-ray crystallography could also explain homo-oligomerization in solution; specifically, trimerization is indicated by infra-red dynamic light scattering and blue native gel electrophoresis in solution. Trimers were also detected by mass spectrometry. Furthermore, there is an indication of post-translational methylation in the crystal structure. Additionally, the possibility of modifying the crystal size and the redox activity in the context of photosynthesis is shaping the investigated cytochrome as a highly suitable model protein for advanced serial crystallography at highly brilliant X-ray free-electron laser sources.

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 β-carotene in the spectral range 500-510nm. 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 502nm assigned to the appearance of CT states of chlorophyll exciplexes. The formation of ion-radical pair P700+A1- at 40ps was limited by energy transfer from LWC to the primary donor P700 and accompanied by carotenoid bleach at 498nm. In PS I from Synechocystis 6803, the excitation at 720nm produced an immediate bidentate bleach at 690/704nm and synchronous carotenoid response at 508nm. 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 ~ 5ps 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 498nm, pointing to fast redistribution of unpaired electron between two branches in favor of the state PB+A1A-.

Structural insights into NDH-1 mediated cyclic electron transfer

NDH-1 is a key component of the cyclic-electron-transfer around photosystem I (PSI CET) pathway, an important antioxidant mechanism for efficient photosynthesis. Here, we report a 3.2-Å-resolution cryo-EM structure of the ferredoxin (Fd)-NDH-1L complex from the cyanobacterium Thermosynechococcus elongatus. The structure reveals three β-carotene and fifteen lipid molecules in the membrane arm of NDH-1L. Regulatory oxygenic photosynthesis-specific (OPS) subunits NdhV, NdhS and NdhO are close to the Fd-binding site whilst NdhL is adjacent to the plastoquinone (PQ) cavity, and they play different roles in PSI CET under high-light stress. NdhV assists in the binding of Fd to NDH-1L and accelerates PSI CET in response to short-term high-light exposure. In contrast, prolonged high-light irradiation switches on the expression and assembly of the NDH-1MS complex, which likely contains no NdhO to further accelerate PSI CET and reduce ROS production. We propose that this hierarchical mechanism is necessary for the survival of cyanobacteria in an aerobic environment.

Current limits of structural biology: The transient interaction between cytochrome c 6 and photosystem I.

Trimeric photosystem I from the cyanobacterium Thermosynechococcus elongatus (TePSI) is an intrinsic membrane protein, which converts solar energy into electrical energy by oxidizing the soluble redox mediator cytochrome c6 (Cyt c6) and reducing ferredoxin. Here, we use cryo-electron microscopy and small angle neutron scattering (SANS) to characterize the transient binding of Cyt c6 to TePSI. The structure of TePSI cross-linked to Cyt c6 was solved at a resolution of 2.9 Å and shows additional cofactors as well as side chain density for 84% of the peptide chain of subunit PsaK, revealing a hydrophobic, membrane intrinsic loop that enables binding of associated proteins. Due to the poor binding specificity, Cyt c6 could not be localized with certainty in our cryo-EM analysis. SANS measurements confirm that Cyt c6 does not bind to TePSI at protein concentrations comparable to those for cross-linking. However, SANS data indicate a complex formation between TePSI and the non-native mitochondrial cytochrome from horse heart (Cyt cHH). Our study pinpoints the difficulty of identifying very small binding partners (less than 5% of the overall size) in EM structures when binding affinities are poor. We relate our results to well resolved co-structures with known binding affinities and recommend confirmatory methods for complexes with KM values higher than 20 μM.

A highly purified C-phycocyanin from thermophilic cyanobacterium Thermosynechococcus elongatus and its cytotoxic activity assessment using an in vitro cell-based approach

ABSTRACT C-phycocyanin (C-PC) is a protein–pigment widely used in nutrition and pharmaceutical applications. High purity of C-PC is a limiting factor required for its medical use; hence purity improvement with successful plausible pharmaceutical application is the state of the art of the present work. Highly purified C-PC (A620/A280 = 6.6) was obtained from Thermosynechococcus elongatus. Combined enzymatic cell wall degradation, cellular exposure to high pressure, sonication, and one-time homogenization was found to be an effective purification protocol. Purified complex was confirmed by SDS-PAGE and UV-absorption spectral analysis. Cytotoxic effect of the obtained C-PC was assessed in vitro using a panel of cancer cells. Results showed that MCF-7 cells were most sensitive to C-PC (IC50 = 158.9 µM) compared to Caco-2 (IC50 = 258.3 µM) and HepG2 (IC50 = 277.5 µM) cells. Our findings shed the light on the potential medical use of C-PC isolated from T. elongatus in controlling cancer cells.

An alternative plant-like cyanobacterial ferredoxin with unprecedented structural and functional properties

Photosynthetic [2Fe-2S] plant-type ferredoxins have a central role in electron transfer between the photosynthetic chain and various metabolic pathways. Several genes are coding for [2Fe2S] ferredoxins in cyanobacteria, with four in the thermophilic cyanobacterium Thermosynechococcus elongatus. The structure and functional properties of the major ferredoxin Fd1 are well known but data on the other ferredoxins are scarce. We report the structural and functional properties of a novel minor type ferredoxin, Fd2 of T. elongatus, homologous to Fed4 from Synechocystis sp. PCC 6803. Remarkably, the midpoint potential of Fd2, Em = −440 mV, is lower than that of Fd1, Em = −372 mV. However, while Fd2 can efficiently react with photosystem I or nitrite reductase, time-resolved spectroscopy shows that Fd2 has a very low capacity to reduce ferredoxin-NADP+ oxidoreductase (FNR). These unique Fd2 properties are discussed in relation with its structure, solved at 1.38 Å resolution. The Fd2 structure significantly differs from other known ferredoxins structures in loop 2, N-terminal region, hydrogen bonding networks and surface charge distributions. UV–Vis, EPR, and Mid- and Far-IR data also show that the electronic properties of the [2Fe2S] cluster of Fd2 and its interaction with the protein differ from those of Fd1 both in the oxidized and reduced states. The structural analysis allows to propose that valine in the motif Cys53ValAsnCys56 of Fd2 and the specific orientation of Phe72, explain the electron transfer properties of Fd2. Strikingly, the nature of these residues correlates with different phylogenetic groups of cyanobacterial Fds. With its low redox potential and its discrimination against FNR, Fd2 exhibits a unique capacity to direct efficiently photosynthetic electrons to metabolic pathways not dependent on FNR.

The N-terminal region of the ε subunit from cyanobacterial ATP synthase alone can inhibit ATPase activity

ATP hydrolysis activity catalyzed by chloroplast and proteobacterial ATP synthase is inhibited by their ϵ subunits. To clarify the function of the ϵ subunit from phototrophs, here we analyzed the ϵ subunit-mediated inhibition (ϵ-inhibition) of cyanobacterial F1-ATPase, a subcomplex of ATP synthase obtained from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. We generated three C-terminal α-helix null ϵ-mutants; one lacked the C-terminal α-helices, and in the other two, the C-terminal conformation could be locked by a disulfide bond formed between two α-helices or an α-helix and a β-sandwich structure. All of these ϵ-mutants maintained ATPase-inhibiting competency. We then used single-molecule observation techniques to analyze the rotary motion of F1-ATPase in the presence of these ϵ-mutants. The stop angular position of the γ subunit in the presence of the ϵ-mutant was identical to that in the presence of the WT ϵ. Using magnetic tweezers, we examined recovery from the inhibited rotation and observed restoration of rotation by 80° forcing of the γ subunit in the case of the ADP-inhibited form, but not when the rotation was inhibited by the ϵ-mutants or by the WT ϵ subunit. These results imply that the C-terminal α-helix domain of the ϵ subunit of cyanobacterial enzyme does not directly inhibit ATP hydrolysis and that its N-terminal domain alone can inhibit the hydrolysis activity. Notably, this property differed from that of the proteobacterial ϵ, which could not tightly inhibit rotation. We conclude that phototrophs and heterotrophs differ in the ϵ subunit-mediated regulation of ATP synthase.

Structure of the complex I-like molecule NDH ofoxygenic photosynthesis.

Cyclic electron flow around photosystem I (PSI) is a mechanism by which photosynthetic organisms balance the levels of ATP and NADPH necessary for efficient photosynthesis1,2. NAD(P)H dehydrogenase-like complex (NDH) is a key component of this pathway in most oxygenic photosynthetic organisms3,4 and is the last large photosynthetic membrane-protein complex for which the structure remains unknown. Related to the respiratory NADH dehydrogenase complex (complex I), NDH transfers electrons originating from PSI to the plastoquinone pool while pumping protons across the thylakoid membrane, thereby increasing the amount of ATP produced per NADP+ molecule reduced4,5. NDH possesses 11 of the 14 core complex I subunits, as well as several oxygenic-photosynthesis-specific (OPS) subunits that are conserved from cyanobacteria to plants3,6. However, the three core complex I subunits that are involved in accepting electrons from NAD(P)H are notably absent in NDH3,5,6, and it is therefore not clear how NDH acquires and transfers electrons to plastoquinone. It is proposed that the OPS subunits-specifically NdhS-enable NDH to accept electrons from its electron donor, ferredoxin3-5,7. Here we report a 3.1 Å structure of the 0.42-MDa NDH complex from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1, obtained by single-particle cryo-electron microscopy. Our maps reveal the structure and arrangement of the principal OPS subunits in the NDH complex, as well as an unexpected cofactor close to the plastoquinone-binding site in the peripheral arm. The location of the OPS subunits supports a role in electron transfer and defines two potential ferredoxin-binding sites at the apex of the peripheral arm. These results suggest that NDH could possess several electron transfer routes, which would serve to maximize plastoquinone reduction and avoid deleterious off-target chemistry of the semi-plastoquinone radical.

Thermosynechococcus as a thermophilic photosynthetic microbial cell factory for CO2 utilisation

Thermophilic unicellular cyanobacterium Thermosynechococcus elongatus PKUAC-SCTE542, has been developed as a thermophilic photosynthetic microbial cell factory for CO2 utilisation. The strain exhibits its highest growth rate around 55 °C, can withstand up to 15% CO2, and up to 0.5 M concentration of sodium bicarbonate. The strain is also capable of resisting a 200 ppm concentration of NO and SO2 in simulated flue gasses, and these compounds have a positive effect on its growth. Whole genome sequencing of the strain revealed the presence of numerous forms of active transport of nutrients and additional chaperones acting as the predominant mechanism of strain adaptation to high temperatures. Based on the sequenced genome, two neutral gene insertion sites have been identified and engineered using modular vectors. Site-specific knock-ins and knock-outs have been performed using the spectinomycin resistance gene and proved functional, enabling future application of the strain to produce biofuels and biochemicals from waste CO2.

Non-detergent isolation of a cyanobacterial photosystem I using styrene maleic acid alternating copolymers

Photosystem I (PSI) from the thermophilic cyanobacterium Thermosynechococcus elongatus (Te) is the largest membrane protein complex to have had its structure solved by X-ray diffraction. This trimeric complex has 36 protein subunits, over 380 non-covalently bound cofactors and a molecular weight of ∼1.2 MDa. Previously, it has been isolated and characterized in a detergent micelle using the non-ionic detergent n-dodecyl-β-d-maltoside (DDM). We have now succeeded in isolating this complex without the use of detergents, using styrene–maleic acid (SMA) alternating copolymer. Intriguingly, a partially esterified copolymer formulation (SMA 1440, Cray Valley) was found to be most efficient in cyanobacterial thylakoid membranes. A host of biochemical, biophysical and functional assays have been applied to characterize this non-detergent form of PSI, referred to as a SMA Lipid Particle (SMALP). The PSI-SMALP has a lower sedimentation coefficient compared to PSI-DDM, suggesting decreased density or a more extended particle shape. We show the 77 K fluorescence maximum for PSI is red shifted in PSI-SMALP compared to PSI-DDM, suggesting a more native orientation of PsaA/B associated chlorophyll. We report that PSI-SMALPs are functional despite the selective loss of one transmembrane subunit, PsaF. This loss may reflect a more labile interaction of the PSI core and PsaF, or a selective displacement during copolymer insertion and/or assembly. PSI-SMALP exhibited decreased reduction kinetics with native recombinant cytochromes c6, while non-native horse heart cytochrome c shows faster reduction of PSI-SMALP compared to PSI-DDM. This is the largest membrane protein isolated using SMA copolymers, and this study expands the potential use of this approach for the isolation and characterization of large supramolecular complexes.

Probing the role of Valine 185 of the D1 protein in the Photosystem II oxygen evolution

In Photosystem II (PSII), the Mn4CaO5-cluster of the active site advances through five sequential oxidation states (S0 to S4) before water is oxidized and O2 is generated. The V185 of the D1 protein has been shown to be an important amino acid in PSII function (Dilbeck et al. Biochemistry 52 (2013) 6824–6833). Here, we have studied its role by making a V185T site-directed mutant in the thermophilic cyanobacterium Thermosynechococcus elongatus. The properties of the V185T-PSII have been compared to those of the WT*3-PSII by using EPR spectroscopy, polarography, thermoluminescence and time-resolved UV–visible absorption spectroscopy. It is shown that the V185 and the chloride binding site very likely interact via the H-bond network linking TyrZ and the halide. The V185 contributes to the stabilization of S2 into the low spin (LS), S = 1/2, configuration. Indeed, in the V185T mutant a high proportion of S2 exhibits a high spin (HS), S = 5/2, configuration. By using bromocresol purple as a dye, a proton release was detected in the S1TyrZ → S2HSTyrZ transition in the V185T mutant in contrast to the WT*3-PSII in which there is no proton release in this transition. Instead, in WT*3-PSII, a proton release kinetically much faster than the S2LSTyrZ → S3TyrZ transition was observed and we propose that it occurs in the S2LSTyrZ → S2HSTyrZ intermediate step before the S2HSTyrZ → S3TyrZ transition occurs. The dramatic slowdown of the S3TyrZ → S0TyrZ transition in the V185T mutant does not originate from a structural modification of the Mn4CaO5 cluster since the spin S = 3 S3 EPR signal is not modified in the mutant. More probably, it is indicative of the strong implication of V185 in the tuning of an efficient relaxation processes of the H-bond network and/or of the protein.