Cryo-EM structure of monomeric photosystem II at 2.78 Å resolution reveals factors important for the formation of dimer.

Photosystem II (PSII) functions mainly as a dimer to catalyze the light energy conversion and water oxidation reactions. However, monomeric PSII also exists and functions in vivo in some cases. The crystal structure of monomeric PSII has been solved at 3.6 Å resolution, but it is still not clear which factors contribute to the formation of the dimer. Here, we solved the structure of PSII monomer at a resolution of 2.78 Å using cryo-electron microscopy (cryo-EM). From our cryo-EM density map, we observed apparent differences in pigments and lipids in the monomer-monomer interface between the PSII monomer and dimer. One ß-carotene and two sulfoquinovosyl diacylglycerol (SQDG) molecules are found in the monomer-monomer interface of the dimer structure but not in the present monomer structure, although some SQDG and other lipid molecules are found in the analogous region of the low-resolution crystal structure of the monomer, or cryo-EM structure of an apo-PSII monomer lacking the extrinsic proteins from Synechocystis sp. PCC 6803. In the current monomer structure, a large part of the PsbO subunit was also found to be disordered. These results indicate the importance of the ß-carotene, SQDG and PsbO in formation of the PSII dimer.

Structural implications for a phycobilisome complex from the thermophilic cyanobacterium Thermosynechococcus vulcanus.

Phycobilisomes (PBSs) are huge, water-soluble light-harvesting complexes used by oxygenic photosynthetic organisms. The structures of some subunits of the PBSs, including allophycocyanin (APC) and phycocyanin (PC), have been solved by X-ray crystallography previously. However, there are few reports on the overall structures of PBS complexes in photosynthetic organisms. Here, we report the overall structure of the PBS complex isolated from the cyanobacterium Thermosynechococcus vulcanus, determined by negative-staining electron microscopy (EM). Intact PBS complexes were purified by trehalose density gradient centrifugation with a high-concentration phosphate buffer and then subjected to a gradient-fixation preparation using glutaraldehyde. The final map constructed by the single-particle analysis of EM images showed a hemidiscoidal structure of the PBS, consisting of APC cores and peripheral PC rods. The APC cores are composed of five cylinders: A1, A2, B, C1, and C2. Each of the cylinders is composed of three (A1 and A2), four (B), or two (C1 and C2) APC trimers. In addition, there are eight PC rods in the PBS: one bottom pair (Rb and Rb'), one top pair (Rt and Rt'), and two side pairs (Rs1/Rs1' and Rs2/Rs2'). Comparison with the overall structures of PBSs from other organisms revealed structural characteristics of T. vulcanus PBS.

Light-adapted charge-separated state of photosystem II: structural and functional dynamics of the closed reaction center.

Photosystem II (PSII) uses solar energy to oxidize water and delivers electrons for life on Earth. The photochemical reaction center of PSII is known to possess two stationary states. In the open state (PSIIO), the absorption of a single photon triggers electron-transfer steps, which convert PSII into the charge-separated closed state (PSIIC). Here, by using steady-state and time-resolved spectroscopic techniques on Spinacia oleracea and Thermosynechococcus vulcanus preparations, we show that additional illumination gradually transforms PSIIC into a light-adapted charge-separated state (PSIIL). The PSIIC-to-PSIIL transition, observed at all temperatures between 80 and 308 K, is responsible for a large part of the variable chlorophyll-a fluorescence (Fv) and is associated with subtle, dark-reversible reorganizations in the core complexes, protein conformational changes at noncryogenic temperatures, and marked variations in the rates of photochemical and photophysical reactions. The build-up of PSIIL requires a series of light-induced events generating rapidly recombining primary radical pairs, spaced by sufficient waiting times between these events-pointing to the roles of local electric-field transients and dielectric relaxation processes. We show that the maximum fluorescence level, Fm, is associated with PSIIL rather than with PSIIC, and thus the Fv/Fm parameter cannot be equated with the quantum efficiency of PSII photochemistry. Our findings resolve the controversies and explain the peculiar features of chlorophyll-a fluorescence kinetics, a tool to monitor the functional activity and the structural-functional plasticity of PSII in different wild-types and mutant organisms and under stress conditions.

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.

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 Šresolution, 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.

Current Understanding of the Mechanism of Water Oxidation in Photosystem II and Its Relation to XFEL Data.

The investigation of water oxidation in photosynthesis has remained a central topic in biochemical research for the last few decades due to the importance of this catalytic process for technological applications. Significant progress has been made following the 2011 report of a high-resolution X-ray crystallographic structure resolving the site of catalysis, a protein-bound Mn4CaOx complex, which passes through ≥5 intermediate states in the water-splitting cycle. Spectroscopic techniques complemented by quantum chemical calculations aided in understanding the electronic structure of the cofactor in all (detectable) states of the enzymatic process. Together with isotope labeling, these techniques also revealed the binding of the two substrate water molecules to the cluster. These results are described in the context of recent progress using X-ray crystallography with free-electron lasers on these intermediates. The data are instrumental for developing a model for the biological water oxidation cycle.