Structure and energy transfer in photosystems of oxygenic photosynthesis.

Oxygenic photosynthesis is the principal converter of sunlight into chemical energy on Earth. Cyanobacteria and plants provide the oxygen, food, fuel, fibers, and platform chemicals for life on Earth. The conversion of solar energy into chemical energy is catalyzed by two multisubunit membrane protein complexes, photosystem I (PSI) and photosystem II (PSII). Light is absorbed by the pigment cofactors, and excitation energy is transferred among the antennae pigments and converted into chemical energy at very high efficiency. Oxygenic photosynthesis has existed for more than three billion years, during which its molecular machinery was perfected to minimize wasteful reactions. Light excitation transfer and singlet trapping won over fluorescence, radiation-less decay, and triplet formation. Photosynthetic reaction centers operate in organisms ranging from bacteria to higher plants. They are all evolutionarily linked. The crystal structure determination of photosynthetic protein complexes sheds light on the various partial reactions and explains how they are protected against wasteful pathways and why their function is robust. This review discusses the efficiency of photosynthetic solar energy conversion.

Structure and binding efficiency relations of QB site inhibitors of photosynthetic reaction centres.

Many herbicides employed in agriculture and also some antibiotics bind to a specific site of the reaction centre protein (RC) blocking the photosynthetic electron transport. Crystal structures showed that all these compounds bind at the secondary ubiquinone (QB) site albeit to slightly different places. Different herbicide molecules have different binding affinities (evaluated as inhibition constants, KI, and binding enthalpy values, ΔHbind). The action of inhibitors depends on the following parameters: (i) herbicide molecular structure; (ii) interactions between herbicide and quinone binding site; (iii) protein environment. In our investigations KI and ΔHbind were determined for several inhibitors. Bound herbicide structures were optimized and their intramolecular charge distributions were calculated. Experimental and calculated data were compared to those available from databank crystal structures. We can state that the herbicide inhibition efficiency depends on steric and electronic, i.e. geometry of binding with the protein and molecular charge distribution, respectively. Apolar bulky groups on N-7 atom of the inhibitor molecule (like t-buthyl in terbutryn) are preferable for establishing stronger interactions with QB site, while such substituents are not recommended on N-8. The N-4,7,8 nitrogen atoms maintain a larger electron density so that more effective H-bonds are formed between the inhibitor and the surrounding amino acids of the protein.

Supramolecular organization of photosynthetic membrane proteins in the chlorosome-containing bacterium Chloroflexus aurantiacus.

The arrangement of core antenna complexes (B808-866-RC) in the cytoplasmic membrane of filamentous phototrophic bacterium Chloroflexus aurantiacus was studied by electron microscopy in cultures from different light conditions. A typical nearest-neighbor center-to-center distance of ~18 nm was found, implying less protein crowding compared to membranes of purple bacteria. A mean RC:chlorosome ratio of 11 was estimated for the occupancy of the membrane directly underneath each chlorosome, based on analysis of chlorosome dimensions and core complex distribution. Also presented are results of single-particle analysis of core complexes embedded in the native membrane.

Long-lived quantum coherence in photosynthetic complexes at physiological temperature.

Photosynthetic antenna complexes capture and concentrate solar radiation by transferring the excitation to the reaction center that stores energy from the photon in chemical bonds. This process occurs with near-perfect quantum efficiency. Recent experiments at cryogenic temperatures have revealed that coherent energy transfer--a wave-like transfer mechanism--occurs in many photosynthetic pigment-protein complexes. Using the Fenna-Matthews-Olson antenna complex (FMO) as a model system, theoretical studies incorporating both incoherent and coherent transfer as well as thermal dephasing predict that environmentally assisted quantum transfer efficiency peaks near physiological temperature; these studies also show that this mechanism simultaneously improves the robustness of the energy transfer process. This theory requires long-lived quantum coherence at room temperature, which never has been observed in FMO. Here we present evidence that quantum coherence survives in FMO at physiological temperature for at least 300 fs, long enough to impact biological energy transport. These data prove that the wave-like energy transfer process discovered at 77 K is directly relevant to biological function. Microscopically, we attribute this long coherence lifetime to correlated motions within the protein matrix encapsulating the chromophores, and we find that the degree of protection afforded by the protein appears constant between 77 K and 277 K. The protein shapes the energy landscape and mediates an efficient energy transfer despite thermal fluctuations.

Native architecture of the photosynthetic membrane from Rhodobacter veldkampii.

The photosynthetic membrane in purple bacteria contains several pigment-protein complexes that assure light capture and establishment of the chemiosmotic gradient. The bioenergetic tasks of the photosynthetic membrane require the strong interaction between these various complexes. In the present work, we acquired the first images of the native outer membrane architecture and the supramolecular organization of the photosynthetic apparatus in vesicular chromatophores of Rhodobacter (Rb.) veldkampii. Mixed with LH2 (light-harvesting complex 2) rings, the PufX-containing LH1-RC (light-harvesting complex 1--reaction center) core complexes appear as C-shaped monomers, with random orientations in the photosynthetic membrane. Within the LH1 fence surrounding the RC, a remarkable gap that is probably occupied (or partially occupied) by PufX is visualized. Sequence alignment revealed that one specific region in PufX may be essential for PufX-induced core dimerization. In this region of ten amino acids in length all Rhodobacter species had five conserved amino acids, with the exception of Rb. veldkampii. Our findings provide direct evidence that the presence of PufX in Rb. veldkampii does not directly govern the dimerization of LH1-RC core complexes in the native membrane. It is indicated, furthermore, that the high membrane curvature of Rb. veldkampii chromatophores (Rb. veldkampii features equally small vesicular chromatophores alike Rb. sphaeroides) is not due to membrane bending induced by dimeric RC-LH1-PufX cores, as it has been proposed in Rb. sphaeroides.

PRE-driven protein NMR structures: an alternative approach in highly paramagnetic systems.

Metalloproteins play key roles across biology, and knowledge of their structure is essential to understand their physiological role. For those metalloproteins containing paramagnetic states, the enhanced relaxation caused by the unpaired electrons often makes signal detection unfeasible near the metal center, precluding adequate structural characterization right where it is more biochemically relevant. Here, we report a protein structure determination by NMR where two different sets of restraints, one containing Nuclear Overhauser Enhancements (NOEs) and another containing Paramagnetic Relaxation Enhancements (PREs), are used separately and eventually together. The protein PioC from Rhodopseudomonas palustris TIE-1 is a High Potential Iron-Sulfur Protein (HiPIP) where the [4Fe-4S] cluster is paramagnetic in both oxidation states at room temperature providing the source of PREs used as alternative distance restraints. Comparison of the family of structures obtained using NOEs only, PREs only, and the combination of both reveals that the pairwise root-mean-square deviation (RMSD) between them is similar and comparable with the precision within each family. This demonstrates that, under favorable conditions in terms of protein size and paramagnetic effects, PREs can efficiently complement and eventually replace NOEs for the structural characterization of small paramagnetic metalloproteins and de novo-designed metalloproteins by NMR. DATABASES: The 20 conformers with the lowest target function constituting the final family obtained using the full set of NMR restraints were deposited to the Protein Data Bank (PDB ID: 6XYV). The 20 conformers with the lowest target function obtained using NOEs only (PDB ID: 7A58) and PREs only (PDB ID: 7A4L) were also deposited to the Protein Data Bank. The chemical shift assignments were deposited to the BMRB (code 34487).

Quantum oscillatory exciton migration in photosynthetic reaction centers.

The harvesting of solar energy and its conversion to chemical energy is essential for all forms of life. The primary photon absorption, transport, and charge separation events, which trigger a chain of chemical reactions, take place in membrane-bound photosynthetic complexes. Whether quantum effects, stemming from entanglement of chromophores, persist in the energy transport at room temperature, despite the rapid decoherence effects caused by environment fluctuations, is under current active debate. If confirmed, these may explain the high efficiency of light harvesting and open up numerous applications to quantum computing and information processing. We present simulations of the photosynthetic reaction center of photosystem II that clearly establish oscillatory energy transport at room temperature originating from interference of quantum pathways. These signatures of quantum transport may be observed by two dimensional coherent optical spectroscopy.

Combinatory actions of CP29 phosphorylation by STN7 and stability regulate leaf age-dependent disassembly of photosynthetic complexes.

A predominant physiological change that occurs during leaf senescence is a decrease in photosynthetic efficiency. An optimal organization of photosynthesis complexes in plant leaves is critical for efficient photosynthesis. However, molecular mechanisms for regulating photosynthesis complexes during leaf senescence remain largely unknown. Here we tracked photosynthesis complexes alterations during leaf senescence in Arabidopsis thaliana. Grana stack is significantly thickened and photosynthesis complexes were disassembled in senescing leaves. Defects in STN7 and CP29 led to an altered chloroplast ultrastructure and a malformation of photosynthesis complex organization in stroma lamella. Both CP29 phosphorylation by STN7 and CP29 fragmentation are highly associated with the photosynthesis complex disassembly. In turn, CP29 functions as a molecular glue to facilitate protein complex formation leading phosphorylation cascade and to maintain photosynthetic efficiency during leaf senescence. These data suggest a novel molecular mechanism to modulate leaf senescence via CP29 phosphorylation and fragmentation, serving as an efficient strategy to control photosynthesis complexes.

Photosynthetic system in Blastochloris viridis revisited.

The bacterium Blastochloris viridis carries one of the simplest photosynthetic systems, which includes a single light-harvesting complex that surrounds the reaction center, membrane soluble quinones, and a soluble periplasmic protein cytochrome c(2) that shuttle between the reaction center and the bc(1) complex and act as electron carriers, as well as the ATP synthase. The close arrangement of the photosynthetic membranes in Bl. viridis, along with the extremely tight arrangement of the photosystems within these membranes, raises a fundamental question about the diffusion of the electron carriers. To address this issue, we analyzed the structure and response of the Bl. viridis photosynthetic system to various light conditions, by using a combination of electron microscopy, whole-cell cryotomography, and spectroscopic methods. We demonstrate that in response to high light intensities, the ratio of both cytochrome c(2) and bc(1) complexes to the reaction centers is increased. The shorter membrane stacks, along with the notion that the bc(1) complex is located at the highly curved edges of these stacks, result in a smaller average distance between the reaction centers and the bc(1) complexes, leading to shorter pathways of cytochrome c(2) between the two complexes. Under anaerobic conditions, the slow diffusion rate is further mitigated by keeping most of the quinone pool reduced, resulting in a concentration gradient of quinols that allows for a constant supply of theses electron carriers to the bc(1) complex.

Two-dimensional crystals of photosystem II: biochemical characterization, cryoelectron microscopy and localization of the D1 and cytochrome b559 polypeptides.

Photosystem II (PS II) is a photosynthetic reaction center found in higher plants which has the unique ability to evolve oxygen from water. Several groups have formed two-dimensional PS II crystals or have isolated PS II complexes and studied them by electron microscopy and image analysis. The majority of these specimens have not been well characterized biochemically and have yielded relatively low resolution two-dimensional projection maps with a variety of unit cell sizes. We report the characterization of the polypeptide and lipid content of tubular crystals of PS II. The crystals contain the reaction center core polypeptides D1, D2, cytochrome b559, as well as the chlorophyll-binding polypeptides (CP) CP47, CP43, CP29, CP26, CP24, and CP22. The lipid composition was similar to the lipids found in the stacked portion of thylakoids. We also report a 2.0-nm resolution projection map determined by electron microscopy and image analysis of frozen, hydrated PS II crystals. This projection map includes information on the portion of the complex buried in the lipid bilayer. The unit cell is a dimer with unit vectors of 17.0 and 11.4 nm separated by an angle of 106.6 degrees. In addition, Fab fragments against D1 and cytochrome b559 were used to localize those two polypeptides, and thus the reaction center, within the PS II complex. The results indicate that D1 and cytochrome b559 are found within one of the heaviest densities of the monomeric unit.

Structural changes and lateral redistribution of photosystem II during donor side photoinhibition of thylakoids.

The structural and topological stability of thylakoid components under photoinhibitory conditions (4,500 microE.m-2.s-1 white light) was studied on Mn depleted thylakoids isolated from spinach leaves. After various exposures to photoinhibitory light, the chlorophyll-protein complexes of both photosystems I and II were separated by sucrose gradient centrifugation and analysed by Western blotting, using a set of polyclonals raised against various apoproteins of the photosynthetic apparatus. A series of events occurring during donor side photoinhibition are described for photosystem II, including: (a) lowering of the oligomerization state of the photosystem II core; (b) cleavage of 32-kD protein D1 at specific sites; (c) dissociation of chlorophyll-protein CP43 from the photosystem II core; and (d) migration of damaged photosystem II components from the grana to the stroma lamellae. A tentative scheme for the succession of these events is illustrated. Some effects of photoinhibition on photosystem I are also reported involving dissociation of antenna chlorophyll-proteins LHCI from the photosystem I reaction center.

Structural analysis of photosynthetic membranes by cryo-electron tomography of intact Rhodopseudomonas viridis cells.

During the photosynthetic process, highly organized membranal assemblies convert light into biochemical energy with high efficiency. We have used whole-mount cryo-electron tomography to study the intracellular architecture of the photosynthetic membranes of the anaerobic purple photosynthetic bacterium Rhodopseudomonas viridis, as well as the organization of the photosynthetic units within the membranes. Three-dimensional reconstruction demonstrates a continuity of the plasma membrane with the photosynthetic membranes that form tunnel-like structures with an average diameter of 31 nm+/-8 nm at the connection sites. The spacing between the photosynthetic membranes at their cytoplasmic faces was found to be 11 nm, thus enforcing a highly close packaging of the photosynthetic membranes. Analysis of successive tomographic slices allowed for derivation of the spacing between adjacent photosynthetic core complexes from a single-layered photosynthetic membrane, in situ. This analysis suggests that most, if not all, photosynthetic membranes in R. viridis are characterized by a similar two-dimensional hexagonal lattice organization.

Evidences for interaction of PsbS with photosynthetic complexes in maize thylakoids.

The PsbS subunit of Photosystem II (PSII) has received much attention in the past few years, given its crucial role in photoprotection of higher plants. The exact location of this small subunit in thylakoids is also debated. In this work possible interaction partners of PsbS have been identified by immunoaffinity and immunoprecipitation, performed with mildly solubilized whole thylakoid membrane. The interacting proteins, as identified by mass spectrometry analysis of the immunoaffinity eluate, include CP29, some LHCII components, but also components of Photosystem I, of the cytochrome b(6)f complex as well as of ATP synthase. These proteins can be co-immunoprecipitated by using highly specific anti-PsbS antibodies and, vice-versa, PsbS is co-immunoprecipitated by antisera against components of the interacting complexes. We also find that PsbS co-migrates with bands containing PSII, ATP synthase and cytochrome b(6)f as well as with LHCII-containing bands on non-denaturing Deriphat PAGE. These results suggest multiple location of PsbS in the thylakoid membrane and point to an unexpected lateral mobility of this PSII subunit. As revealed by immunogold labelling with antibody against PsbS, the protein is associated either with granal membranes or prevalently with stroma lamellae in low or high-intensity light-treated intact leaves, respectively. This finding is consistent with the capability of PsbS to interact with complexes located in stroma lamellae, even though the exact physiological condition(s) under which these interactions may take place remain to be clarified.

EMatch: discovery of high resolution structural homologues of protein domains in intermediate resolution cryo-EM maps.

Cryo-EM has become an increasingly powerful technique for elucidating the structure, dynamics, and function of large flexible macromolecule assemblies that cannot be determined at atomic resolution. However, due to the relatively low resolution of cryo-EM data, a major challenge is to identify components of complexes appearing in cryo-EM maps. Here, we describe EMatch, a novel integrated approach for recognizing structural homologues of protein domains present in a 6-10 A resolution cryo-EM map and constructing a quasi-atomic structural model of their assembly. The method is highly efficient and has been successfully validated on various simulated data. The strength of the method is demonstrated by a domain assembly of an experimental cryo-EM map of native GroEL at 6 A resolution.

Photosystem II: a multisubunit membrane protein that oxidises water.

A structure of photosystem II recently determined by X-ray crystallography at 3.8 A resolution complements structural studies using high-resolution electron microscopy and represents a major step towards understanding how photosynthetic organisms use light energy to oxidise water.

Structure of a symmetric photosynthetic reaction center-photosystem.

Reaction centers are pigment-protein complexes that drive photosynthesis by converting light into chemical energy. It is believed that they arose once from a homodimeric protein. The symmetry of a homodimer is broken in heterodimeric reaction-center structures, such as those reported previously. The 2.2-angstrom resolution x-ray structure of the homodimeric reaction center-photosystem from the phototroph Heliobacterium modesticaldum exhibits perfect C2 symmetry. The core polypeptide dimer and two small subunits coordinate 54 bacteriochlorophylls and 2 carotenoids that capture and transfer energy to the electron transfer chain at the center, which performs charge separation and consists of 6 (bacterio)chlorophylls and an iron-sulfur cluster; unlike other reaction centers, it lacks a bound quinone. This structure preserves characteristics of the ancestral reaction center, providing insight into the evolution of photosynthesis.

Ultra-high-resolution structure and charge-density analysis of high-potential iron-sulfur protein.

The high-potential iron-sulfur protein (HiPIP) is a small (~ 80 residues) soluble metalloprotein functioning as an electron carrier in photosynthetic bacteria. HiPIP has one Fe4 S4 cluster at its molecular center. Its electronic structure is important for understanding electron transport. We recently succeeded in determining an ultra-high-resolution structure and analyzing the charge-density of HiPIP by using X-ray diffraction data at 0.48 Å resolution. The distribution of valence electrons in the iron-sulfur cluster and in the protein environment were clearly visualized, which is the first successful case for metalloproteins. In addition, a topological analysis of the charge density provided information about the electronic structure of the cluster.

The structure of Photosystem I from the thermophilic cyanobacterium Synechococcus sp. determined by electron microscopy of two-dimensional crystals.

The structure of the Photosystem I (PS I) complex from the thermophilic cyanobacterium Synechococcus sp. has been investigated by electron microscopy and image analysis of two-dimensional crystals. Crystals were obtained from isolated PS I by removal of detergents with Bio-Beads. After negative staining, either single layers or two superimposed layers with a rotational different orientation were observed. The layers have a rectangular unit cell of 16.0 x 15.0 nm, which contains two PS I monomers. The monomers are arranged alternating up and down in each layer. For double-layer crystals, the images of the two layers could be separately processed by a combination of Fourier-peak-filtering and correlation averaging. Features in the two-dimensional plane can be seen with a resolution up to 1.5-1.8 nm. A model for the PS I structure was obtained by combining three-dimensional reconstructions from three tilt-series. The model shows an asymmetric PS I complex. On one side (presumably the stromal side) there is a 3 nm high ridge. This is most likely comprised of the psaC, psaD and psaE subunits. The other side (presumably the lumenal side) is rather flat, but in the center there is a 3 nm deep indentation, which possibly separates partly the two large subunits psaA and psaB.

Role of subunits in eukaryotic Photosystem I.

Photosystem I (PSI) of eukaryotes has a number of features that distinguishes it from PSI of cyanobacteria. In plants, the PSI core has three subunits that are not found in cyanobacterial PSI. The remaining 11 subunits of the core are conserved but several of the subunits have a different role in eukaryotic PSI. A distinguishing feature of eukaryotic PSI is the membrane-imbedded peripheral antenna. Light-harvesting complex I is composed of four different subunits and is specific for PSI. Light-harvesting complex II can be associated with both PSI and PSII. Several of the core subunits interact with the peripheral antenna proteins and are important for proper function of the peripheral antenna. The review describes the role of the different subunits in eukaryotic PSI. The emphasis is on features that are different from cyanobacterial PSI.

The reaction center of green sulfur bacteria(1).

The composition of the P840-reaction center complex (RC), energy and electron transfer within the RC, as well as its topographical organization and interaction with other components in the membrane of green sulfur bacteria are presented, and compared to the FeS-type reaction centers of Photosystem I and of Heliobacteria. The core of the RC is homodimeric, since pscA is the only gene found in the genome of Chlorobium tepidum which resembles the genes psaA and -B for the heterodimeric core of Photosystem I. Functionally intact RC can be isolated from several species of green sulfur bacteria. It is generally composed of five subunits, PscA-D plus the BChl a-protein FMO. Functional cores, with PscA and PscB only, can be isolated from Prostecochloris aestuarii. The PscA-dimer binds P840, a special pair of BChl a-molecules, the primary electron acceptor A(0), which is a Chl a-derivative and FeS-center F(X). An equivalent to the electron acceptor A(1) in Photosystem I, which is tightly bound phylloquinone acting between A(0) and F(X), is not required for forward electron transfer in the RC of green sulfur bacteria. This difference is reflected by different rates of electron transfer between A(0) and F(X) in the two systems. The subunit PscB contains the two FeS-centers F(A) and F(B). STEM particle analysis suggests that the core of the RC with PscA and PscB resembles the PsaAB/PsaC-core of the P700-reaction center in Photosystem I. PscB may form a protrusion into the cytoplasmic space where reduction of ferredoxin occurs, with FMO trimers bound on both sides of this protrusion. Thus the subunit composition of the RC in vivo should be 2(FMO)(3)(PscA)(2)PscB(PscC)(2)PscD. Only 16 BChl a-, four Chl a-molecules and two carotenoids are bound to the RC-core, which is substantially less than its counterpart of Photosystem I, with 85 Chl a-molecules and 22 carotenoids. A total of 58 BChl a/RC are present in the membranes of green sulfur bacteria outside the chlorosomes, corresponding to two trimers of FMO (42 Bchl a) per RC (16 BChl a). The question whether the homodimeric RC is totally symmetric is still open. Furthermore, it is still unclear which cytochrome c is the physiological electron donor to P840(+). Also the way of NAD(+)-reduction is unknown, since a gene equivalent to ferredoxin-NADP(+) reductase is not present in the genome.