Design of Functionalized Lipids and Evidence for Their Binding to Photosystem II Core Complex by Oxygen Evolution Measurements, Atomic Force Microscopy, and Scanning Near-Field Optical Microscopy (original) (raw)
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Lipids in photosystem II: Interactions with protein and cofactors
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2007
Photosystem II (PSII) is a homodimeric protein-cofactor complex embedded in the thylakoid membrane that catalyses light-driven charge separation accompanied by the oxidation of water during oxygenic photosynthesis. Biochemical analysis of the lipid content of PSII indicates a number of integral lipids, their composition being similar to the average lipid composition of the thylakoid membrane. The crystal structure of PSII at 3.0 Å resolution allowed for the first time the assignment of 14 integral lipids within the protein scaffold, all of them being located at the interface of different protein subunits. The reaction centre subunits D1 and D2 are encircled by a belt of 11 lipids providing a flexible environment for the exchange of D1. Three lipids are located in the dimerization interface and mediate interactions between the PSII monomers. Several lipids are located close to the binding pocket of the mobile plastoquinone Q B , forming part of a postulated diffusion pathway for plastoquinone. Furthermore two lipids were found, each ligating one antenna chlorophyll a. A detailed analysis of lipid-protein and lipid-cofactor interactions allows to derive some general principles of lipid binding pockets in PSII and to suggest possible functional properties of the various identified lipid molecules.
e-Journal of Surface Science and Nanotechnology, 2011
In purple photosynthetic bacteria, light-harvesting complex 2 (LH2) and the light harvesting-reaction center complex (LH1-RC) play the key roles of capturing and transferring light energy and subsequent charge separation. These photosynthetic apparatuses form a molecular assembly; however, how the assembly influences the efficiency of energy conversion is not yet clear. To address this issue, direct observation of the assembly at the molecular level is necessary to analyze its function. In this study, we reconstituted photosynthetic membrane proteins into artificial lipid bilayers and directly observed their assembly by AFM. The absorption spectra of the reconstituted proteins showed characteristic Qy bands of bacteriochlorophyll a that were identical to those of intact proteins. AFM observation of the reconstituted membranes revealed that LH2 and LH1-RC were successfully assembled into the lipid bilayer, and their observed structures were in good agreement with corresponding crystallographic structures. Specifically, binary proteins, i.e., LH2/LH1-RC and LH2/LH1, which form a densely packed molecular assembly, could be clearly identified at the molecular level by this method of observation. Energy transfer from LH2 to LH1-RC in a reconstituted lipid bilayer was observed by steady-state fluorescence spectroscopy. Enhanced energy transfer was confirmed in the membrane phase compared to that in a homogeneous micellar solution. Such reconstituted molecular assemblies are useful experimental platforms to investigate the relationship between supramolecular arrays and function.
Lipids in photosystem II: Multifunctional cofactors
Journal of Photochemistry and Photobiology B: Biology, 2011
To maintain its functionality, photosystem II (PSII) employs several types of auxiliary molecules (cofactors). As shown for PSII from Thermosynechococcus elongatus, lipids previously thought to play mostly the role of a hydrophobic matrix for embedding the membrane proteins, must be considered as a new, multifunctional type of cofactors, playing a vital role in the fine tuning of PSII and in its overall operation. The 2.9 Å resolution crystal structure of cyanobacterial homodimeric PSII showed the position of 25 lipid molecules per monomer, and allowed detailed analysis of individual binding sites as well as functional aspects related to lipids. The positions of the bound lipids suggest that they are essential for the assembly and disassembly of PSII, provide the proper environment for plastoquinone exchange, might tune electron transfer through contacts with chlorophylls and carotenoids, and might serve as an oxygen-outlet system from the lumen.
Photosynthesis Research, 2000
Basic structural elements of the two photosystems and their component electron donors, acceptors, and carriers were revealed by newly developed spectroscopic methods in the 1960s and subsequent years. The spatial organization of these constituents within the functional membrane was elucidated by electrochromic band shift analysis, whereby the membrane-spanning chlorophyll-quinone couple of Photosystem (PS) II emerged as reaction center and as a model relevant also to other photosystems. A further step ahead for improved structural information was realized with the use of thermophilic cyanobacteria instead of plants which led to isolation of supramolecular complexes of the photosystems and their identification as PS I trimers and PS II dimers. The preparation of crystals of the PS I trimer, started in the late 1980s. Genes encoding the 11 subunits of PS I from Synechococcus elongatus were isolated and the predicted sequences of amino acid residues formed a basis for the interpretation of X-ray structure analysis of the PS I crystals. The crystallization of PS I was optimized by introduction of the 'reverse of salting in' crystallization with water as precipitating agent. On this basis the PS I structure was successively established from 6 Å resolution in the early 1990s up to a model at 2.5 Å resolution in 2001. The first crystals of the PS II dimer, capable of water oxidation, were prepared in the late 1990s; a PS II model at 3.8-3.6 Å resolution was presented in 2001. Implications of the PS II structure for the mechanism of transmembrane charge separation are discussed. With the availability of PS I and PS II crystals, new directional structural results became possible also by application of different magnetic resonance techniques through measurements on single crystals in different orientations. Abbreviations: BChl-bacteriochlorophyll; Bpheo-bacteriopheophytin; Car-carotenoid; Chl-chlorophyll; CP43,47-inner antenna system of PS II, with Mr of 43 and 47 kDa; Cyt-cytochrome; D1-D1 subunit of the PS II reaction center; D2-D2 subunit of the PS II reaction center; ENDOR-electron nuclear double resonance; EPR-electron paramagnetic resonance; ETC-electron transfer chain; EXAFS-extended X-ray absorption fine structure; P D1/D2-Chl coordinated by His 198/197 of D1/D2; Pheo-pheophytin; P680-primary electron donor of PS II; P700-primary electron donor of PS I; Q-plastoquinone of a pool; Q A-stable primary quinone acceptor of PS II RC; RC-reaction center; Y D-Tyr 160 of D2; Y Z-Tyr 161 of D1
Biophysical Journal, 2003
The main function of the transmembrane light-harvesting complexes in photosynthetic organisms is the absorption of a light quantum and its subsequent rapid transfer to a reaction center where a charge separation occurs. A combination of freeze-thaw and dialysis methods were used to reconstitute the detergent-solubilized Light Harvesting 2 complex (LH2) of the purple bacterium Rhodopseudomonas acidophila strain 10050 into preformed egg phosphatidylcholine liposomes, without the need for extra chemical agents. The LH2-containing liposomes opened up to a flat bilayer, which were imaged with tapping and contact mode atomic force microscopy under ambient and physiological conditions, respectively. The LH2 complexes were packed in quasicrystalline domains. The endoplasmic and periplasmic sides of the LH2 complexes could be distinguished by the difference in height of the protrusions from the lipid bilayer. The results indicate that the complexes entered in intact liposomes. In addition, it was observed that the most hydrophilic side, the periplasmic, enters first in the membrane. In contact mode the molecular structure of the periplasmic side of the transmembrane pigment-protein complex was observed. Using Fö ster's theory for describing the distance dependent energy transfer, we estimate the dipole strength for energy transfer between two neighboring LH2s, based on the architecture of the imaged unit cell.
Recent Progress in the Crystallographic Studies of Photosystem II
ChemPhysChem, 2010
The photosynthetic oxygen-evolving photosystem II (PSII) is the only known biochemical system that is able to oxidize water molecules and thereby generates almost all oxygen in the Earth's atmosphere. The elucidation of the structural and mechanistic aspects of PSII keeps scientists all over the world engaged since several decades. In this Minireview, we outline the progress in understanding PSII based on the most recent crystal structure at 2.9 resolution. A likely position of the chloride ion, which is known to be required for the fast turn-over of water oxidation, could be determined in native PSII and is compared with work on bromide and iodide substituted PSII. Moreover, eleven new integral lipids could be assigned, emphasizing the importance of lipids for the perfect function of PSII. A third plastoquinone molecule (Q C ) and a second quinone transfer channel are revealed, making it possible to consider different mechanisms for the exchange of plastoquinone/ plastoquinol molecules. In addition, possible transport channels for water, dioxygen and protons are identified.
Structure of photosystem II and substrate binding at room temperature
Nature, 2016
Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has generated most of the dioxygen in the atmosphere. PS II, a membrane-bound multi-subunit pigment protein complex, couples the one-electron photochemistry at the reaction centre with the four-electron redox chemistry of water oxidation at the Mn4CaO5 cluster in the oxygen-evolving complex (OEC). Under illumination, the OEC cycles through five intermediate S-states (S0 to S4), in which S1 is the dark-stable state and S3 is the last semi-stable state before O-O bond formation and O2 evolution. A detailed understanding of the O-O bond formation mechanism remains a challenge, and will require elucidation of both the structures of the OEC in the different S-states and the binding of the two substrate waters to the catalytic site. Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage-free, room temperature structures of dark-adapted (S1), two-fla...
Chemical Physics, 2013
In purple photosynthetic bacteria, light-harvesting complex 2 (LH2) and light harvesting/reaction centre core complex (LH1-RC) play the key roles of capturing and transferring light energy and subsequent charge separation. These photosynthetic apparatuses form a supramolecular assembly; however, how the assembly influences the efficiency of energy conversion is not yet clear. We addressed this issue by evaluating the energy transfer in reconstituted photosynthetic protein complexes LH2 and LH1-RC and studying the structures and the membrane environment of the LH2/LH1-RC assemblies, which had been embedded into various lipid bilayers. Thus, LH2 and LH1-RC from Rhodopseudomonas palustris 2.1.6 were reconstituted in phosphatidylglycerol (PG), phosphatidylcholine (PC), and phosphatidylethanolamine (PE)/PG/cardiolipin (CL). Efficient energy transfer from LH2 to LH1-RC was observed in the PC and PE/PG/CL membranes. Atomic force microscopy revealed that LH2 and LH1-RC were heterogeneously distributed to form clusters in the PC and PE/PG/CL membranes. The results indicated that the phospholipid species influenced the cluster formation of LH2 and LH1-RC as well as the energy transfer efficiency.
Photochemistry and Photobiology, 1997
The photosystem II (PSII) reaction center (RC) is a hydrophobic intrinsic protein complex that drives the water-oxidation process of photosynthesis. Unlike the bacterial RC complex, an X-ray crystal structure of the PSII RC is not available. In order to determine the physical dimensions of the isolated PSII RC complex, we applied Langmuir techniques to determine the cross-sectional area of an isolated RC in a condensed monolayer film. Low-angle X-ray diffraction results obtained by examining Langmuir-Blodgett multilayer films of alternating PSII RC/Cd stearate monolayers were used to determine the length (or height; z-direction, perpendicular to the plane of the original membrane) of the complex. The values obtained for a PSII RC monomer were 26 nm2 and 4.8 nm, respectively, and the structural integrity of the RC in the multilayer film was confirmed by several approaches. Assuming a cylindrical-type RC structure, the above dimensions lead to a predicted volume of about 125 nm3. This value is very close to the expected volume of 118 nm3, calculated from the known molecular weight and partial specific volume of the PSII RC proteins. This same type of comparison was also made with the Rhodobacter sphaeroides RC based on published data, and we conclude that the PSII RC is much shorter in length and has a more regular solid geometric structure than the bacterial RC. Furthermore, the above dimensions of the PSII RC and those of PSII core (RC plus proximal antenna) proteins protruding outside the plane of the PSII membrane into the lumenal space as imaged by scanning tunneling microscopy (Seibert, Aust. J. Pl. Physiol. 22, 161-166, 1995) fit easily into the known dimensions of the PSII core complex visualized by others as electron-density projection maps. From this we conclude that the in situ PSII core complex is a dimeric structure containing two copies of the PSII RC.
2011
Mamoru Nango‡ Department of Frontier Materials, Graduate School of Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan Department of Physics, Graduate School of Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan and CREST, Japan Science and Technology Agency, Japan (Received 30 September 2010; Accepted 16 December 2010; Published 29 January 2011)