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Biogenesis of photosystem II (PSII), nature’s water-splitting catalyst, is assisted by auxiliary proteins that form transient complexes with PSII components to facilitate stepwise assembly events. Using cryo-electron microscopy, we solved the structure of such a PSII assembly intermediate from Thermosynechococcus elongatus at 2.94 Å resolution. It contains three assembly factors (Psb27, Psb28 and Psb34) and provides detailed insights into their molecular function. Binding of Psb28 induces large conformational changes at the PSII acceptor side, which distort the binding pocket of the mobile quinone (Q B ) and replace the bicarbonate ligand of non-haem iron with glutamate, a structural motif found in reaction centres of non-oxygenic photosynthetic bacteria. These results reveal mechanisms that protect PSII from damage during biogenesis until water splitting is activated. Our structure further demonstrates how the PSII active site is prepared for the incorporation of the Mn 4 CaO 5 cluster, which performs the unique water-splitting reaction. Photosystems need auxiliary proteins to assist their assembly. Cryo-electron microscopy of a cyanobacterial photosystem II assembly intermediate at 2.94 Å reveals mechanisms protecting against photodamage during vulnerable stages of biogenesis.

Significance Cyanobacteria have sophisticated photosensory systems to adapt to ambient-light conditions to improve oxygenic photosynthesis efficiency. Their genomes contain many genes encoding cyanobacteriochromes (CBCRs), which are the photoreceptors of light-signaling pathways. Although the photochemical properties of many CBCRs have been characterized, whether and how multiple photoreceptors work together are unknown. Herein we describe how three CBCRs work together in a light color-sensitive manner to regulate cyanobacterial cell aggregation. The three CBCRs have distinguishable, but congruent, light color-dependent c-di-GMP synthetic and/or degrading activities. Ours is the first report, to our knowledge, concerning synchronization of distinctive CBCR activities, which emphasizes the underlying need for CBCR photoreceptors with diverse activities. Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors that have diverse spectral properties and domain compositions. Although large numbers of CBCR genes exist in cyanobacterial genomes, no studies have assessed whether multiple CBCRs work together. We recently showed that the diguanylate cyclase (DGC) activity of the CBCR SesA from Thermosynechococcus elongatus is activated by blue-light irradiation and that, when irradiated, SesA, via its product cyclic dimeric GMP (c-di-GMP), induces aggregation of Thermosynechococcus vulcanus cells at a temperature that is suboptimum for single-cell viability. For this report, we first characterize the photobiochemical properties of two additional CBCRs, SesB and SesC. Blue/teal light-responsive SesB has only c-di-GMP phosphodiesterase (PDE) activity, which is up-regulated by teal light and GTP. Blue/green light-responsive SesC has DGC and PDE activities. Its DGC activity is enhanced by blue light, whereas its PDE activity is enhanced by green light. A Δ sesB mutant cannot suppress cell aggregation under teal-green light. A Δ sesC mutant shows a less sensitive cell-aggregation response to ambient light. Δ sesA/ Δ sesB/ Δ sesC shows partial cell aggregation, which is accompanied by the loss of color dependency, implying that a nonphotoresponsive DGC(s) producing c-di-GMP can also induce the aggregation. The results suggest that SesB enhances the light color dependency of cell aggregation by degrading c-di-GMP, is particularly effective under teal light, and, therefore, seems to counteract the induction of cell aggregation by SesA. In addition, SesC seems to improve signaling specificity as an auxiliary backup to SesA/SesB activities. The coordinated action of these three CBCRs highlights why so many different CBCRs exist.

The reduction rate of photo-oxidized Photosystem I (PSI) with various natural and artificial electron donors have been well studied by transient absorption spectroscopy. The electron transfer rate from various donors to P sub(700) super(+) has been measured for a wide range of photosynthetic organisms encompassing cyanobacteria, algae, and plants. PSI can be a limiting component due to tedious extraction and purification methods required for this membrane protein. In this report, we have determined the in vivo, intracellular cytochrome c sub(6) (cyt c sub(6))/PSI ratio in Thermosynechococcus elongatus (T.e.) using quantitative Western blot analysis. This information permitted the determination of P sub(700) super(+) reduction kinetics via recombinant cyt c sub(6) in a physiologically relevant ratio (cyt c sub(6): PSI) with a Joliot-type, LED-driven, pump-probe spectrophotometer. Dilute PSI samples were tested under varying cyt c sub(6) concentration, temperature, pH, and ionic strength, each of which shows similar trends to the reported literature utilizing much higher PSI concentrations with laser-based spectrophotometer. Our results do however indicate kinetic differences between actinic light sources (laser vs. LED), and we have attempted to resolve these effects by varying our LED light intensity and duration. The standardized configuration of this spectrophotometer will also allow a more uniform kinetic analysis of samples in different laboratories. We can conclude that our findings from the LED-based system display an added total protein concentration effect due to multiple turnover events of P sub(700) super(+) reduction by cyt c sub(6) during the longer illumination regime.

Microsecond time-resolved step-scan FTIR difference spectroscopy was used to study photosystem I (PSI) from Thermosynechococcus vestitus BP-1 (T. vestitus, formerly known as T. elongatus) at 77 K. In addition, photoaccumulated (P700+–P700) FTIR difference spectra were obtained at both 77 and 293 K. The FTIR difference spectra are presented here for the first time. To extend upon these FTIR studies nanosecond time-resolved infrared difference spectroscopy was also used to study PSI from T. vestitus at 296 K. Nanosecond infrared spectroscopy has never been used to study PSI samples at physiological temperatures, and here it is shown that such an approach has great value as it allows a direct probe of electron transfer down both branches in PSI. In PSI at 296 K, the infrared flash-induced absorption changes indicate electron transfer down the B- and A-branches is characterized by time constants of 33 and 364 ns, respectively, in good agreement with visible spectroscopy studies. These time constants are associated with forward electron transfer from A1– to FX on the B- and A-branches, respectively. At several infrared wavelengths flash-induced absorption changes at 296 K recover in tens to hundreds of milliseconds. The dominant decay phase is characterized by a lifetime of 128 ms. These millisecond changes are assigned to radical pair recombination reactions, with the changes being associated primarily with P700+ rereduction. This conclusion follows from the observation that the millisecond infrared spectrum is very similar to the photoaccumulated (P700+–P700) FTIR difference spectrum.

Photosystem I in most organisms contains long-wavelength or “Red” chlorophylls (Chls) absorbing light beyond 700 nm. At cryogenic temperatures, the Red Chls become quasi-traps for excitations as uphill energy transfer is blocked. One pathway for de-excitation of the Red Chls is via transfer to the oxidized RC (P700+), which has broad absorption in the near-infrared region. This study investigates the excitation dynamics of Red Chls in Photosystem I from the cyanobacterium Thermostichus vulcanus at cryogenic temperatures (77 K) and examines the role of the oxidized RC in modulating their fluorescence kinetics. Using time-resolved fluorescence spectroscopy, the kinetics of Red Chls were recorded for samples with open (neutral P700) and closed (P700+) RCs. We found that emission lifetimes in the range of 710–720 nm remained unaffected by the RC state, while more red-shifted emissions (>730 nm) decayed significantly faster when the RC was closed. A kinetic model describing the quenching by the oxidized RC was constructed based on simultaneous fitting to the recorded fluorescence emission in Photosystem I with open and closed RCs. The analysis resolved multiple Red Chl forms and variable quenching efficiencies correlated with their spectral properties. Only the most red-shifted Chls, with emission beyond 730 nm, are efficiently quenched by P700+, with rate constants of up to 6 ns−1. The modeling results support the notion that structural and energetic disorder in Photosystem I can have a comparable or larger effect on the excitation dynamics than the geometric arrangement of Chls.

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.Çoruh, Frank et al. report the structure of monomeric Photosystem I from cyanobacteria Thermosynechoccocus elongatus BP-1. They assign monomerization-induced loss of red chlorophylls to a cluster of chlorophylls adjacent to PsaX. This study suggests a role of PsaX in the stabilization of red chlorophylls.

In plants, algae and cyanobacteria, Photosystem II (PSII) catalyzes the light-driven splitting of water at a protein-bound Mn4CaO5-cluster, the water-oxidizing complex (WOC). In the photosynthetic organisms, the light-driven formation of the WOC from dissolved metal ions is a key process because it is essential in both initial activation and continuous repair of PSII. Structural information is required for understanding of this chaperone-free metal-cluster assembly. For the first time, we obtained a structure of PSII from Thermosynechococcus elongatus without the Mn4CaO5-cluster. Surprisingly, cluster-removal leaves the positions of all coordinating amino acid residues and most nearby water molecules largely unaffected, resulting in a pre-organized ligand shell for kinetically competent and error-free photo-assembly of the Mn4CaO5-cluster. First experiments initiating (i) partial disassembly and (ii) partial re-assembly after complete depletion of the Mn4CaO5-cluster agree with a specific bi-manganese cluster, likely a di-µ-oxo bridged pair of Mn(III) ions, as an assembly intermediate.

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.

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 (Q B ) 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. Cryo-electron microscopy structures of two PSII intermediates from psbV -deletion and wild-type strains of cyanobacteria show substantial structural changes. These provide a structural basis for understanding the assembly process of native PSII.

Biological water oxidation, performed by a single enzyme, photosystem II, is a central research topic not only in understanding the photosynthetic apparatus but also for the development of water splitting catalysts for technological applications. Great progress has been made in this endeavor following the report of a high-resolution X-ray crystallographic structure in 2011 resolving the cofactor site (Umena et al. in Nature 473:55–60, 2011), a tetra-manganese calcium complex. The electronic properties of the protein-bound water oxidizing Mn4OxCa complex are crucial to understand its catalytic activity. These properties include: its redox state(s) which are tuned by the protein matrix, the distribution of the manganese valence and spin states and the complex interactions that exist between the four manganese ions. In this short review we describe how magnetic resonance techniques, particularly EPR, complemented by quantum chemical calculations, have played an important role in understanding the electronic structure of the cofactor. Together with isotope labeling, these techniques have also been instrumental in deciphering the binding of the two substrate water molecules to the cluster. These results are briefly described in the context of the history of biological water oxidation with special emphasis on recent work using time resolved X-ray diffraction with free electron lasers. It is shown that these data are instrumental for developing a model of the biological water oxidation cycle.