Photosynthesis Research (v.111, #1-2)
Synthesis of oligomethylene-strapped chlorophyll derivatives and optical properties of their stereoisomers in a solution by Hitoshi Tamiaki; Hiroshi Takebe; Shin-ichi Sasaki; Yumiko Kataoka (1-8).
Methyl pheophorbide-a/a′ derivatives covalently linked with oligomethylene chains at the 3-CH2OCO– and 132-COO– moieties in a molecule were prepared by modifying chlorophyll-a through intramolecular ring-closing metathesis of vinyl groups. At least, a C10-length between the 33- and 134-positions was necessary for the cyclization and connection of a C12-strap was the most suitable to achieve the highest closure yield. The oligomethylene chain in 132 R-epimers derived from methyl pheophorbide-a covered the α-face of the chlorin π-plane and the strap in the corresponding 132 S-epimers protected the β-face. Synthetic 132 R-epimer with a dodecamethylene chain gave a flat chlorin π-plane, while the decamethylene chain in the 132 R-epimer distorted the π-system due to its shorter linkage. The distortion by strapping in the 132 R-epimer induced a slight blue-shift of Qy peak in dichloromethane. CD spectra of the 132 R-epimers were similarly dependent on the chain length, i.e., the distortion of π-plane. Visible absorption and CD spectra of all the strapped 132 S-epimers were almost identical and only slightly different from those of the unstrapped. The strapping in the 132 S-epimers shifted the Qy peak bathochromically.
Keywords: Circular dichroism spectra; π-plane distortion; Pheophorbide; Ring-closing metathesis; Site energy; Visible absorption spectra
Gene sequencing and characterization of the light-harvesting complex 2 from thermophilic purple sulfur bacterium Thermochromatium tepidum by Fumie Sekine; Kentaro Horiguchi; Yasuhiro Kashino; Yuuki Shimizu; Long-Jiang Yu; Masayuki Kobayashi; Zheng-Yu Wang (9-18).
In this study, gene sequences coding for the light-harvesting (LH) 2 polypeptides from a thermophilic purple sulfur bacterium Thermochromatium tepidum are reported and characterization of the LH2 complex is described. Three sets of pucBA genes have been identified, and the gene products have been analyzed by electrophoresis and reversed-phase chromatography. The result shows that all of the genes are expressed but the distribution of the expression is not uniform. The gene products undergo post-translational modification, where two of the β-polypeptides appear to be N-terminally methylated. Absorption spectrum of the purified LH2 complex exhibits Q y transitions at 800 and 854 nm in dodecyl β-maltopyranoside solution, and the circular dichroism spectrum shows a “molischianum”-like characteristic. No spectral change was observed for the LH2 when the bacterium was cultured under different conditions of light intensity. In lauryl dimethylamine N-oxide (LDAO) solution, significant changes in the absorption spectrum were observed. The B850 peak decreased and blue-shifted with increasing the LDAO concentration, whereas the B800 intensity increased without change in the peak position. The spectral changes can be partially or almost completely reversed by addition of metal ions, and the divalent cations seem to be more effective. The results indicate that ionic interactions may exist between LH2, detergent molecules and metal ions. Possible mechanisms involved in the detergent- and cation-induced spectral changes are discussed.
Keywords: Photosynthesis; Sulfur bacteria; Energy transfer; Membrane protein
Phylogenetic analysis of the light-harvesting system in Chromera velia by Hao Pan; Jan Šlapeta; Dee Carter; Min Chen (19-28).
Chromera velia is a newly discovered photosynthetic eukaryotic alga that has functional chloroplasts closely related to the apicoplast of apicomplexan parasites. Recently, the chloroplast in C. velia was shown to be derived from the red algal lineage. Light-harvesting protein complexes (LHC), which are a group of proteins involved in photon capture and energy transfer in photosynthesis, are important for photosynthesis efficiency, photo-adaptation/accumulation and photo-protection. Although these proteins are encoded by genes located in the nucleus, LHC peptides migrate and function in the chloroplast, hence the LHC may have a different evolutionary history compared to chloroplast evolution. Here, we compare the phylogenetic relationship of the C. velia LHCs to LHCs from other photosynthetic organisms. Twenty-three LHC homologues retrieved from C. velia EST sequences were aligned according to their conserved regions. The C. velia LHCs are positioned in four separate groups on trees constructed by neighbour-joining, maximum likelihood and Bayesian methods. A major group of seventeen LHCs from C. velia formed a separate cluster that was closest to dinoflagellate LHC, and to LHC and fucoxanthin chlorophyll-binding proteins from diatoms. One C. velia LHC sequence grouped with LI1818/LI818-like proteins, which were recently identified as environmental stress-induced protein complexes. Only three LHC homologues from C. velia grouped with the LHCs from red algae.
Keywords: Light-harvesting protein complexes (LHC); Chromera velia (C. velia); Membrane-spanning regions; Chlorophyll-binding protein complexes
Anisotropic circular dichroism signatures of oriented thylakoid membranes and lamellar aggregates of LHCII by Yuliya Miloslavina; Petar H. Lambrev; Tamás Jávorfi; Zsuzsanna Várkonyi; Václav Karlický; Joseph S. Wall; Geoffrey Hind; Győző Garab (29-39).
In photosynthesis research, circular dichroism (CD) spectroscopy is an indispensable tool to probe molecular architecture at virtually all levels of structural complexity. At the molecular level, the chirality of the molecule results in intrinsic CD; pigment–pigment interactions in protein complexes and small aggregates can give rise to excitonic CD bands, while “psi-type” CD signals originate from large, densely packed chiral aggregates. It has been well established that anisotropic CD (ACD), measured on samples with defined non-random orientation relative to the propagation of the measuring beam, carries specific information on the architecture of molecules or molecular macroassemblies. However, ACD is usually combined with linear dichroism and can be distorted by instrumental imperfections, which given the strong anisotropic nature of photosynthetic membranes and complexes, might be the reason why ACD is rarely studied in photosynthesis research. In this study, we present ACD spectra, corrected for linear dichroism, of isolated intact thylakoid membranes of granal chloroplasts, washed unstacked thylakoid membranes, photosystem II (PSII) membranes (BBY particles), grana patches, and tightly stacked lamellar macroaggregates of the main light-harvesting complex of PSII (LHCII). We show that the ACD spectra of face- and edge-aligned stacked thylakoid membranes and LHCII lamellae exhibit profound differences in their psi-type CD bands. Marked differences are also seen in the excitonic CD of BBY and washed thylakoid membranes. Magnetic CD (MCD) spectra on random and aligned samples, and the largely invariable nature of the MCD spectra, despite dramatic variations in the measured isotropic and anisotropic CD, testify that ACD can be measured without substantial distortions and thus employed to extract detailed information on the (supra)molecular organization of photosynthetic complexes. An example is provided showing the ability of CD data to indicate such an organization, leading to the discovery of a novel crystalline structure in macroaggregates of LHCII.
Keywords: Anisotropic circular dichroism; Magnetic circular dichroism; Psi-type circular dichroism; Thylakoid membranes; Grana patches; Light-harvesting complexes
Polarization single complex imaging of circular photosynthetic antenna by Sumera Tubasum; Daniel Thomsson; Richard Cogdell; Ivan Scheblykin; Tõnu Pullerits (41-45).
Single complex fluorescence polarization spectroscopy is applied to study the peripheral light harvesting antenna (LH2) from photosynthetic purple bacterium Rhodopseudomonas (Rps.) acidophila. The measured two-dimensional excitation-emission polarization plots are used to construct geometric representation for the absorbing B800 and emitting B850 as ellipses. The shape and orientation of the ellipses is discussed in terms of tilted LH2 complexes where emission occurs from energetically disordered B850 excitons.
Keywords: LH2 complexes; Single molecule spectroscopy; Polarization spectroscopy; Excitation energy transfer
Theory of excitonic couplings in dielectric media by Thomas Renger; Frank Müh (47-52).
The Poisson-TrEsp method (where TrEsp stands for transition charges from electrostatic potentials) has been successfully applied to calculate excitonic couplings in a variety of pigment–protein complexes. It relies on an isomorphism that allows for relating the excitonic coupling between transition densities in dielectric media to their Coulomb coupling. This isomorphism was derived by Hsu et al. (J. Chem. Phys. 114, 3065, (2001)) using time-dependent density functional response theory. In this article, we provide an alternative and simple derivation by first-order perturbation theory. An application of Poisson-TrEsp to photosystem I trimers reveals that the local field correction/screening factor depends on the mutual orientation of the pigments and on the dielectric boundaries rather than on distance. A mean correction factor of f = 0.69 is determined for this system.
Keywords: Chlorophyll; Dielectric continuum; Excitonic coupling; Perturbation theory; Photosystem I trimers; Pigment-protein complex; Poisson equation; Transition density
Reconstituted CP29: multicomponent fluorescence decay from an optically homogeneous sample by Erica Belgio; Giorgio Tumino; Stefano Santabarbara; Giuseppe Zucchelli; Robert Jennings (53-62).
The multiexponential fluorescence decay of the CP29 complex in which the apoprotein and pigments were reconstituted in vitro was examined. Of the three decay components observed only the two dominant ones, with about 3 and 5 ns lifetimes, were studied. The main question addressed was whether the multicomponent decay was associated with sample optical heterogeneity. To this end, we examined the optical absorption and fluorescence of the CP29 sample by means of two different and independent experimental strategies. This approach was used as the wavelength positions of the absorption/fluorescence spectral forms has recently been shown to be a sensitive indicator of the binding site-induced porphyrin ring deformation (Zucchelli et al. Biophys J 93:2240–2254, 2007) and hence of apoprotein conformational changes. The data indicate that this CP29 sample is optically homogeneous. It is hypothesised that the different lifetimes are explained in terms of multiple detergent/CP29 interactions leading to different quenching states, a suggestion that allows for optical homogeneity.
Keywords: Antenna complexes; CP29; Multicomponent fluorescence decay; Optical homogeneity; Quenched/unquenched conformers
Overexpression of Rhodobacter sphaeroides PufX-bearing maltose-binding protein and its effect on the stability of reconstituted light-harvesting core antenna complex by Shunnsuke Sakai; Akito Hiro; Masaharu Kondo; Toshihisa Mizuno; Toshiki Tanaka; Takehisa Dewa; Mamoru Nango (63-69).
The PufX protein, encoded by the pufX gene of Rhodobacter sphaeroides, plays a key role in the organization and function of the core antenna (LH1)-reaction centre (RC) complex, which collects photons and triggers primary photochemical reactions. We synthesized a PufX/maltose-binding protein (MBP) fusion protein to study the effect of the PufX protein on the reconstitution of B820 subunit-type and LH1-type complexes. The fusion protein was synthesized using an Escherichia coli expression system and purified by affinity chromatography. Reconstitution experiments demonstrated that the MBP-PufX protein destabilizes the subunit-type complex (20°C), consistent with previous reports. Interestingly, however, the preformed LH1-type complex was stable in the presence of MBP-PufX. The MBP-PufX protein did not influence the preformed LH1-type complexes (4°C). The LH1-type complex containing MBP-PufX showed a unique temperature-dependent structural transformation that was irreversible. The predominant form of the complex at 4°C was the LH1-type. When shifted to 20°C, subunit-type complexes became predominant. Upon subsequent cooling back to 4°C, instead of re-forming the LH1-type complexes, the predominant form remained the subunit-type complexes. In contrast, reversible transformation of LH1 (4°C) and subunit-type complexes (20°C) occurs in the absence of PufX. These results are consistent with the suggestion that MBP-PufX interacts with the LH1α- polypeptide in the subunit (α/β)-type complex (at 20°C), preventing oligomerization of the subunit to form LH1-type complexes.
Keywords: PufX; LH1 complex; Maltose-binding protein; Fusion protein; Reconstitution
Modulation of the multilamellar membrane organization and of the chiral macrodomains in the diatom Phaeodactylum tricornutum revealed by small-angle neutron scattering and circular dichroism spectroscopy by Gergely Nagy; Milán Szabó; Renáta Ünnep; György Káli; Yuliya Miloslavina; Petar H. Lambrev; Ottó Zsiros; Lionel Porcar; Peter Timmins; László Rosta; Győző Garab (71-79).
Diatoms possess effective photoprotection mechanisms, which may involve reorganizations in the photosynthetic machinery. We have shown earlier, by using circular dichroism (CD) spectroscopy, that in Phaeodactylum tricornutum the pigment–protein complexes are arranged into chiral macrodomains, which have been proposed to be associated with the multilamellar organization of the thylakoid membranes and shown to be capable of undergoing light-induced reversible reorganizations (Szabó et al. Photosynth Res 95:237, 2008). Recently, by using small-angle neutron scattering (SANS) on the same algal cells we have determined the repeat distances and revealed reversible light-induced reorganizations in the lamellar order of thylakoids (Nagy et al. Biochem J 436:225, 2011). In this study, we show that in moderately heat-treated samples, the weakening of the lamellar order is accompanied by the diminishment of the psi-type CD signal associated with the long-range chiral order of the chromophores (psi, polymer or salt-induced). Further, we show that the light-induced reversible increase in the psi-type CD is associated with swelling in the membrane system, with magnitudes larger in high light than in low light. In contrast, shrinkage of the membrane system, induced by sorbitol, brings about a decrease in the psi-type CD signal; this shrinkage also diminishes the non-photochemical quenching capability of the cells. These data shed light on the origin of the psi-type CD signal, and confirm that both CD spectroscopy and SANS provide valuable information on the macro-organization of the thylakoid membranes and their dynamic properties; these parameters are evidently of interest with regard to the photoprotection in whole algal cells.
Keywords: Circular dichroism; Diatom; Non-photochemical quenching; Small-angle neutron scattering; Thylakoid membranes; Ultrastructure
Ultrafast energy transfer pathways in R-phycoerythrin from Polysiphonia urceolata by Hailong Chen; Wei Dang; Jie Xie; Jingquan Zhao; Yuxiang Weng (81-86).
Energy transfer (ET) processes between chromophores in R-phycoerythrin (R-PE) from Polysiphonia urceolata were studied by use of ultrafast spectroscopic methods. Several primary ET pathways were elaborated. A fluorescence decay component with a time constant of several hundred picoseconds observed by streak camera is tentatively assigned to the reversible formation of exciton traps between α84 and β84 pigment pairs. In order to investigate much faster ET processes in R-PE, a noncollinear optical parametric amplifier based femtosecond time-resolved transient fluorescence spectrometer was employed. The results reveal that the ET between α84 and β84 pigment pair has a time constant of 1–2 ps; the energy migration between α84 and β84 pairs within the R-PE trimer has a time constant of 30–40 ps. We also demonstrated an ET process from phycourobilin to phycoerythrobilin with a time constant as fast as 2.5–3.0 ps, which was directly observed in fluorescence kinetics by selective excitation of the phycourobilin molecules acting as the energy donor.
Keywords: R-phycoerythrin; Ultrafast energy transfer; Time-resolved spectroscopy; Polysiphonia urceolata
Structure-based simulation of linear optical spectra of the CP43 core antenna of photosystem II by Frank Müh; Mohamed El-Amine Madjet; Thomas Renger (87-101).
The linear optical spectra (absorbance, linear dichroism, circular dichroism, fluorescence) of the CP43 (PsbC) antenna of the photosystem II core complex (PSIIcc) pertaining to the S0 → S1 (QY) transitions of the chlorophyll (Chl) a pigments are simulated by applying a combined quantum chemical/electrostatic method to obtain excitonic couplings and local transition energies (site energies) on the basis of the 2.9 Å resolution crystal structure (Guskov et al., Nat Struct Mol Biol 16:334–342, 2009). The electrostatic calculations identify three Chls with low site energies (Chls 35, 37, and 45 in the nomenclature of Loll et al. (Nature 438:1040–1044, 2005). A refined simulation of experimental spectra of isolated CP43 suggests a modified set of site energies within 143 cm−1 of the directly calculated values (root mean square deviation: 80 cm−1). In the refined set, energy sinks are at Chls 37, 43, and 45 in agreement with earlier fitting results (Raszewski and Renger, J Am Chem Soc 130:4431–4446, 2008). The present structure-based simulations reveal that a large part of the redshift of Chl 37 is due to a digalactosyldiacylglycerol lipid. This finding suggests a new role for lipids in PSIIcc, namely the tuning of optical spectra and the creation of an excitation energy funnel towards the reaction center. The analysis of electrostatic pigment–protein interactions is used to identify amino acid residues that are of potential interest for an experimental approach to an assignment of site energies and energy sinks by site-directed mutagenesis.
Keywords: Chlorophyll; Electrostatic interaction; Excitation energy transfer; Lipids; Mutagenesis; Optical spectra
Analysis of heat-induced disassembly process of three different monomeric forms of the major light-harvesting chlorophyll a/b complex of photosystem II by Yajie Zhang; Cheng Liu; Chunhong Yang (103-111).
The temperature-dependent disassembly process of three monomeric isoforms, namely Lhcb1, Lhcb2, and Lhcb3, of the major light-harvesting chlorophyll (Chl) a/b complexes of photosystem II (LHCIIb) were characterized by observing the changes of absorption spectra, circular dichroism (CD), and dissociation processes of the bound pigments to the in vitro reconstituted complexes subjected to high temperatures. Our results suggest that the three isoforms of LHCIIb undergo conformational rearrangements, structural changes, and dissociations of the bound pigments when the ambient temperature increases from 20 to 90°C. The conformation of the complexes changed sensitively to the changing temperatures because the absorption peaks in the Soret region (436 and 471 nm) and the Qy region (650–660 and 680 nm) decreased immediately upon elevating the ambient temperatures. Analyzing temperature-dependent denaturing and pigment dissociation process, we can divide the disassembly process into three stages: The first stage, appeared from 20°C to around 50–60°C, was characterized by the diminishment of the absorption around 650–660 and 680 nm, accompanied by the blue-shift of the peak at 471 nm and disappearance of the absorbance at 436 nm, which is related to changes in the transition energy of the Chl b cluster, and the red-most Chl a cluster in the LHCIIb. The second stage, beginning at about 50–60°C, was signified by the diminishment of the CD signal between (+)483 nm and (−)490 nm, which implied the disturbance of dipole–dipole interaction of pigments, and the onset of the pigment dissociation. The last stage, beginning at about 70–80°C, indicates the complete dissociation of the pigments from the complex. The physiological aspects of the three stages in the denaturing process are also discussed.
Keywords: Major light-harvesting a/b complex of photosystem II; Spectroscopy; Heat-induced disassembly
Dynamic properties of photosystem II membranes at physiological temperatures characterized by elastic incoherent neutron scattering. Increased flexibility associated with the inactivation of the oxygen evolving complex by Gergely Nagy; Jörg Pieper; Sashka B. Krumova; László Kovács; Marcus Trapp; Győző Garab; Judith Peters (113-124).
Elastic incoherent neutron scattering (EINS), a non-invasive technique which is capable of measuring the mean square displacement of atoms in the sample, has been widely used in biology for exploring the dynamics of proteins and lipid membranes but studies on photosynthetic systems are scarce. In this study we investigated the dynamic characteristics of Photosystem II (PSII) membrane fragments between 280 and 340 K, i.e., in the physiological temperature range and in the range of thermal denaturation of some of the protein complexes. The mean square displacement values revealed the presence of a hydration-sensitive transition in the sample between 310 and 320 K, suggesting that the oxygen evolving complex (OEC) plays an important role in the transition. Indeed, in samples in which the OEC had been removed by TRIS- or heat-treatments (323 and 333 K) no such transition was found. Further support on the main role of OEC in these reorganizations is provided by data obtained from differential scanning calorimetry experiments, showing marked differences between the untreated and TRIS-treated samples. In contrast, circular dichroism spectra exhibited only minor changes in the excitonic interactions below 323 K, showing that the molecular organization of the pigment-protein complexes remains essentially unaffected. Our data, along with earlier incoherent neutron scattering data on PSII membranes at cryogenic temperatures (Pieper et al., Biochemistry 46:11398–11409, 2007), demonstrate that this technique can be applied to characterize the dynamic features of PSII membranes, and can be used to investigate photosynthetic membranes under physiologically relevant experimental conditions.
Keywords: Elastic incoherent neutron scattering; Oxygen evolving complex Photosystem II; Protein dynamics; Thermal stability
Differential assembly of polypeptides of the light-harvesting 2 complex encoded by distinct operons during acclimation of Rhodobacter sphaeroides to low light intensity by Kamil Woronowicz; Oluwatobi B. Olubanjo; Hee Chang Sung; Joana L. Lamptey; Robert A. Niederman (125-138).
In order to obtain an improved understanding of the assembly of the bacterial photosynthetic apparatus, we have conducted a proteomic analysis of pigment-protein complexes isolated from the purple bacterium Rhodobacter sphaeroides undergoing acclimation to reduced incident light intensity. Photoheterotrophically growing cells were shifted from 1,100 to 100 W/m2 and intracytoplasmic membrane (ICM) vesicles isolated over 24-h were subjected to clear native polyacrylamide gel electrophoresis. Bands containing the LH2 and reaction center (RC)-LH1 complexes were excised and subjected to in-gel trypsin digestion followed by liquid chromatography (LC)-mass spectroscopy (MS)/MS. The results revealed that the LH2 band contained distinct levels of the LH2-α and -β polypeptides encoded by the two puc operons. Polypeptide subunits encoded by the puc2AB operon predominated under high light and in the early stages of acclimation to low light, while after 24 h, the puc1BAC components were most abundant. Surprisingly, the Puc2A polypeptide containing a 251 residue C-terminal extension not present in Puc1A, was a protein of major abundance. A predominance of Puc2A components in the LH2 complex formed at high light intensity is followed by a >2.5-fold enrichment in Puc1B levels between 3 and 24 h of acclimation, accompanied by a nearly twofold decrease in Puc2A levels. This indicates that the puc1BAC operon is under more stringent light control, thought to reflect differences in the puc1 upstream regulatory region. In contrast, elevated levels of Puc2 polypeptides were seen 48 h after the gratuitous induction of ICM formation at low aeration in the dark, while after 24 h of acclimation to low light, an absence of alterations in Puc polypeptide distributions was observed in the upper LH2-enriched gel band, despite an approximate twofold increase in overall LH2 levels. This is consistent with the origin of this band from a pool of LH2 laid down early in development that is distinct from subsequently assembled LH2-only domains, forming the LH2 gel band.
Keywords: Light-harvesting complexes; Light regulation; puc operon; Proteomics; Rhodobacter sphaeroides
Ca2+-binding reduces conformational flexibility of RC–LH1 core complex from thermophile Thermochromatium tepidum by Selma Jakob-Grun; Jara Radeck; Paula Braun (139-147).
The light-harvesting complex, LH1, of thermophile purple bacteria Thermochromatium tepidum consists of an array of α- and β-polypeptides which assemble the photoactive bacteriochlorophyll and closely interact with the membrane-lipids. In this study, we investigated the effect of calcium and manganese ions on the protein structure and thermostability of the reaction centre (RC)–LH1/lipid complex. The binding of Ca2+, but not Mn2+ is shown to shift the LH1 Q y absorption maximum from ~889 to 915 nm and to significantly raise the thermostability of the RC–LH1 complex. The ATR–FTIR spectra indicate that interaction of Ca2+ as monitored by the carboxylates’ vibration of aspartate residues, but not Mn2+ induces changes in the α-helix packing arrangement. The reduced rate of 1H/2H exchange of proteins’ amide protons shows that the accessibility to 2H2O is significantly lowered in Ca2+-substituted RC–LH1/lipid complexes. In particular, exchange with the associated lipid molecules, is significantly retarded. These results suggest that the thermostability of the RC–LH1 complex is raised by the distinct interaction with calcium cations which reduces the RC–LH1/lipid dynamics, particularly, at the membrane–water interface.
Keywords: Thermochromatium tepidum ; Light-harvesting complex; Thermal stability; Calcium binding; Structural flexibility
Excitation energy transfer and trapping dynamics in the core complex of the filamentous photosynthetic bacterium Roseiflexus castenholzii by Yueyong Xin; Jie Pan; Aaron M. Collins; Su Lin; Robert E. Blankenship (149-156).
The light-harvesting core complex of the thermophilic filamentous anoxygenic phototrophic bacterium Roseiflexus castenholzii is intrinsic to the cytoplasmic membrane and intimately bound to the reaction center (RC). Using ultrafast transient absorption and time-resolved fluorescence spectroscopy with selective excitation, energy transfer, and trapping dynamics in the core complex have been investigated at room temperature in both open and closed RCs. Results presented in this report revealed that the excited energy transfer from the BChl 800 to the BChl 880 band of the antenna takes about 2 ps independent of the trapping by the RC. The time constants for excitation quenching in the core antenna BChl 880 by open and closed RCs were found to be 60 and 210 ps, respectively. Assuming that the light harvesting complex is generally similar to LH1 of purple bacteria, the possible structural and functional aspects of this unique antenna complex are discussed. The results show that the core complex of Roseiflexus castenholzii contains characteristics of both purple bacteria and Chloroflexus aurantiacus.
Keywords: Excitation energy transfer; Trapping; Core complex; Ultrafast spectroscopy; Filamentous anoxygenic photosynthetic bacterium; Roseiflexus castenholzii
Isolation and purification of the major photosynthetic antenna, fucoxanthin-Chl a/c protein, from cultured discoid germilings of the brown Alga, Cladosiphon okamuranus TOKIDA (Okinawa Mozuku) by Ritsuko Fujii; Mamiko Kita; Yoshiro Iinuma; Naohiro Oka; Yuki Takaesu; Tomonori Taira; Masahiko Iha; Richard J. Cogdell; Hideki Hashimoto (157-163).
A chlorophyll c binding membrane intrinsic light-harvesting complex, the fucoxanthin-chlorophyll a/c protein (FCP), was isolated from cultured discoid germilings of an edible Japanese brown alga, Cladosiphon (C.) okamuranus TOKIDA (Okinawa Mozuku in Japanese). The discoid germiling is an ideal source of brown algal photosynthetic pigment-protein complexes in terms of its size and easiness of cultivation on a large scale. Ion-exchange chromatography was crucial for the purification of FCP from solubilized thylakoid proteins. The molecular weight of the purified FCP assembly was estimated to be ~56 kDa using blue native-PAGE. Further subunit analyses using 2D-PAGE revealed that the FCP assembled as a trimer consisting of two distinguishable subunits having molecular weights of 18.2 (H) and 17.5 (L) kDa. Fluorescence and fluorescence-excitation spectra confirmed that the purified FCP assembly was functionally intact.
Keywords: Photosynthetic light-harvesting complex; Membrane protein; Cladosiphon okamuranus TOKIDA; Brown algae; Discoid germiling
The pigment stoichiometry in a chlorophyll a/c type photosynthetic antenna by Ritsuko Fujii; Mamiko Kita; Matsumi Doe; Yoshiro Iinuma; Naohiro Oka; Yuki Takaesu; Tomonori Taira; Masahiko Iha; Tadashi Mizoguchi; Richard J. Cogdell; Hideki Hashimoto (165-172).
The trimeric fucoxanthin–chlorophyll a/c protein (FCP) was purified from a Japanese brown alga, Cladosiphon okamuranus TOKIDA. Its pigment stoichiometry was determined to be chlorophyll (Chl) a:Chl c 1:Chl c 2:fucoxanthin = 4.6:1.1:1.0:5.5 by a combination of binary HPLC and 1H NMR spectroscopy. No violaxanthin found bound to the FCP. The ratio of Chl c/Chl a in this FCP is amongst the highest so far reported.
Keywords: Brown algae; Chlorophyll c ; Fucoxanthin–chlorophyll a/c protein; Photosynthetic pigments; HPLC; 1H NMR
Different phycobilin antenna organisations affect the balance between light use and growth rate in the cyanobacterium Microcystis aeruginosa and in the cryptophyte Cryptomonas ovata by Christfried Kunath; Torsten Jakob; Christian Wilhelm (173-183).
During the recent years, wide varieties of methodologies have been developed up to the level of commercial use to measure photosynthetic electron transport by modulated chlorophyll a-in vivo fluorescence. It is now widely accepted that the ratio between electron transport rates and new biomass (P Fl/B C) is not fixed and depends on many factors that are also taxonomically variable. In this study, the balance between photon absorption and biomass production has been measured in two phycobilin-containing phototrophs, namely, a cyanobacterium and a cryptophyte, which differ in their antenna organization. It is demonstrated that the different antenna organization exerts influence on the regulation of the primary photosynthetic reaction and the dissipation of excessively absorbed radiation. Although, growth rates and the quantum efficiency of biomass production of both phototrophs were comparable, the ratio P Fl/B C was twice as high in the cryptophyte in comparison to the cyanobacterium. It is assumed that this discrepancy is because of differences in the metabolic regulation of cell growth. In the cryptophyte, absorbed photosynthetic energy is used to convert assimilated carbon directly into proteins and lipids, whereas in the cyanobacterium, the photosynthetic energy is preferentially stored as carbohydrates.
Keywords: Biomass formation; Cyanobacteria; Cryptophytes; Electron transport rate; NPQ
PMS: Photosystem I electron donor or fluorescence quencher by Emilie Wientjes; Roberta Croce (185-191).
Light energy harvested by the pigments in Photosystem I (PSI) is used for charge separation in the reaction center (RC), after which the positive charge resides on a special chlorophyll dimer called P700. In studies on the PSI trapping kinetics, P700+ is usually chemically reduced to re-open the RCs. So far, the information available about the reduction rate and possible chlorophyll fluorescence quenching effects of these reducing agents is limited. This information is indispensible to estimate the fraction of open RCs under known experimental conditions. Moreover, it would be important to understand if these reagents have a chlorophyll fluorescence quenching effects to avoid the introduction of exogenous singlet excitation quenching in the measurements. In this study, we investigated the effect of the commonly used reducing agent phenazine methosulfate (PMS) on the RC and fluorescence emission of higher plant PSI–LHCI. We measured the P700+ reduction rate for different PMS concentrations, and show that we can give a reliable estimation on the fraction of closed RCs based on these rates. The data show that PMS is quenching chlorophyll fluorescence emission. Finally, we determined that the fluorescence quantum yield of PSI with closed RCs is 4% higher than if the RCs are open.
Keywords: P700; Phenazine methosulfate; Fluorescence; Quenching
Self-assembly and energy transfer in artificial light-harvesting complexes of bacteriochlorophyll c with astaxanthin by J. Alster; T. Polívka; J. B. Arellano; P. Hříbek; F. Vácha; J. Hála; J. Pšenčík (193-204).
Chlorosomes, the light-harvesting antennae of green photosynthetic bacteria, are based on large aggregates of bacteriochlorophyll molecules. Aggregates with similar properties to those in chlorosomes can also be prepared in vitro. Several agents were shown to induce aggregation of bacteriochlorophyll c in aqueous environments, including certain lipids, carotenes, and quinones. A key distinguishing feature of bacteriochlorophyll c aggregates, both in vitro and in chlorosomes, is a large (>60 nm) red shift of their Qy absorption band compared with that of the monomers. In this study, we investigate the self-assembly of bacteriochlorophyll c with the xanthophyll astaxanthin, which leads to the formation of a new type of complexes. Our results indicate that, due to its specific structure, astaxanthin molecules competes with bacteriochlorophylls for the bonds involved in the aggregation, thus preventing the formation of any significant red shift compared with pure bacteriochlorophyll c in aqueous buffer. A strong interaction between both the types of pigments in the developed assemblies, is manifested by a rather efficient (~40%) excitation energy transfer from astaxanthin to bacteriochlorophyll c, as revealed by fluorescence excitation spectroscopy. Results of transient absorption spectroscopy show that the energy transfer is very fast (<500 fs) and proceeds through the S2 state of astaxanthin.
Keywords: Light-harvesting; Chlorosomes; Self-assembly; Bacteriochlorophyll aggregates; Astaxanthin
Neutron and light scattering studies of light-harvesting photosynthetic antenna complexes by Kuo-Hsiang Tang; Robert E. Blankenship (205-217).
Small-angle neutron scattering (SANS) and dynamic light scattering (DLS) have been employed in studying the structural information of various biological systems, particularly in systems without high-resolution structural information available. In this report, we briefly present some principles and biological applications of neutron scattering and DLS, compare the differences in information that can be obtained with small-angle X-ray scattering (SAXS), and then report recent studies of SANS and DLS, together with other biophysical approaches, for light-harvesting antenna complexes and reaction centers of purple and green phototrophic bacteria.
Keywords: Chlorosomes; Chloroflexus aurantiacus ; Dynamic light scattering; Light-harvesting antennas; Small-angle neutron scattering; Purple bacteria; Reaction centers
Solid-state NMR applied to photosynthetic light-harvesting complexes by Anjali Pandit; Huub J. M. de Groot (219-226).
This short review describes how solid-state NMR has provided a mechanistic and electronic picture of pigment–protein and pigment–pigment interactions in photosynthetic antenna complexes. NMR results on purple bacterial antenna complexes show how the packing of the protein and the pigments inside the light-harvesting oligomers induces mutual conformational stress. The protein scaffold produces deformation and electrostatic polarization of the BChl macrocycles and leads to a partial electronic charge transfer between the BChls and their coordinating histidines, which can tune the light-harvesting function. In chlorosome antennae assemblies, the NMR template structure reveals how the chromophores can direct their self-assembly into higher macrostructures which, in turn, tune the light-harvesting properties of the individual molecules by controlling their disorder, structural deformation, and electronic polarization without the need for a protein scaffold. These results pave the way for addressing the next challenge, which is to resolve the functional conformational dynamics of the lhc antennae of oxygenic species that allows them to switch between light-emitting and light-energy dissipating states.
Keywords: Conformational strain; Electronic structures; Chlorosome; Nonphotochemical quenching
Elucidation of structure–function relationships in plant major light-harvesting complex (LHC II) by nonlinear spectroscopy by Heiko Lokstein; Alexander Betke; Maria Krikunova; Klaus Teuchner; Bernd Voigt (227-235).
Conventional linear and time-resolved spectroscopic techniques are often not appropriate to elucidate specific pigment–pigment interactions in light-harvesting pigment-protein complexes (LHCs). Nonlinear (laser-) spectroscopic techniques, including nonlinear polarization spectroscopy in the frequency domain (NLPF) as well as step-wise (resonant) and simultaneous (non-resonant) two-photon excitation spectroscopies may be advantageous in this regard. Nonlinear spectroscopies have been used to elucidate substructure(s) of very complex spectra, including analyses of strong excitonic couplings between chlorophylls and of interactions between (bacterio)chlorophylls and “optically dark” states of carotenoids in LHCs, including the major antenna complex of higher plants, LHC II. This article shortly reviews our previous study and outlines perspectives regarding the application of selected nonlinear laser-spectroscopic techniques to disentangle structure–function relationships in LHCs and other pigment-protein complexes.
Keywords: Excitonic interactions; Laser spectroscopy; Light-harvesting complex (LHC II); Nonlinear spectroscopy; Pigment–pigment interactions; Two-photon excitation
On the role of excitonic interactions in carotenoid–phthalocyanine dyads and implications for photosynthetic regulation by Pen-Nan Liao; Smitha Pillai; Miroslav Kloz; Devens Gust; Ana L. Moore; Thomas A. Moore; John T. M. Kennis; Rienk van Grondelle; Peter J. Walla (237-243).
In two recent studies, energy transfer was reported in certain phthalocyanine–carotenoid dyads between the optically forbidden first excited state of carotenoids (Car S1) and phthalocyanines (Pcs) in the direction Pc → Car S1 (Kloz et al., J Am Chem Soc 133:7007–7015, 2011) as well as in the direction Car S1 → Pc (Liao et al., J Phys Chem A 115:4082–4091, 2011). In this article, we show that the extent of this energy transfer in both directions is closely correlated in these dyads. This correlation and the additional observation that Car S1 is instantaneously populated after Pc excitation provides evidence that in these compounds excitonic interactions can occur. Besides pure energy transfer and electron transfer, this is the third type of tetrapyrrole–carotenoid interaction that has been shown to occur in these model compounds and that has previously been proposed as a photosynthetic regulation mechanism. We discuss the implications of these models for photosynthetic regulation. The findings are also discussed in the context of a model in which both electronic states are disordered and in which the strength of the electronic coupling determines whether energy transfer, excitonic coupling, or electron transfer occurs.
Keywords: Two-photon excitation; Carotenoids; Chlorophylls; Carotenoid–phthalocyanine dyads; Excitonic interactions
Molecular dynamics of the diatom thylakoid membrane under different light conditions by Bernard Lepetit; Reimund Goss; Torsten Jakob; Christian Wilhelm (245-257).
During the last years significant progress was achieved in unraveling molecular characteristics of the thylakoid membrane of different diatoms. With the present review it is intended to summarize the current knowledge about the structural and functional changes within the thylakoid membrane of diatoms acclimated to different light conditions. This aspect is addressed on the level of the organization and regulation of light-harvesting proteins, the dissipation of excessively absorbed light energy by the process of non-photochemical quenching, and the lipid composition of diatom thylakoid membranes. Finally, a working hypothesis of the domain formation of the diatom thylakoid membrane is presented to highlight the most prominent differences of heterokontic thylakoids in comparison to vascular plants and green algae during the acclimation to low and high light conditions.
Keywords: Diatom; Light acclimation; Lipid composition; NPQ; Pigment–protein complex; Thylakoid membrane; Xanthophyll cycle