Photosynthesis Research (v.95, #2-3)

Introduction by Richard Cogdell; Conrad Mullineaux (117-117).

On the basis of the recent progress in the resolution of the structure of the antenna light harvesting complex II (LHC II) of the photosystem II, we propose a microscopically motivated theory to predict excitation intensity-dependent spectra. We show that optical Bloch equations provide the means to include all 2 N excited states of an oligomer complex of N coupled two-level systems and analyze the effects of Pauli Blocking and exciton–exciton annihilation on pump-probe spectra. We use LHC Bloch equations for 14 Coulomb coupled two-level systems, which describe the S 0 and S 1 level of every chlorophyll molecule. All parameter introduced into the Hamiltonian are based on microscopic structure and a quantum chemical model. The derived Bloch equations describe not only linear absorption but also the intensity dependence of optical spectra in a regime where the interplay of Pauli Blocking effects as well as exciton–exciton annihilation effects are important. As an example, pump-probe spectra are discussed. The observed saturation of the spectra for high intensities can be viewed as a relaxation channel blockade on short time scales due to Pauli blocking. The theoretical investigation is useful for the interpretation of the experimental data, if the experimental conditions exceed the low intensity pump limit and effects like strong Pauli Blocking and exciton–exciton annihilation need to be considered. These effects become important when multiple excitations are generated by the pump pulse in the complex.
Keywords: Bloch equations; Non-linear optical response; Exciton–exciton annihilation; Pauli blocking

Significance of molecular crowding in grana thylakoids of higher plants on photosystem II function was studied by ‘titrating’ the naturally high protein density by fusing unilamellar liposomes of the native lipid mixture with isolated grana membranes (BBY). The incorporation of lipids was monitored by equilibrium density gradient centrifugation and two-dimensional thin layer chromatography. The excitonic coupling between light-harvesting (LHC) II and photosystem (PS) II was analysed by chlorophyll a fluorescence spectroscopy. The fluorescence parameters Fv/Fm and Fo clearly depend on the protein density indicating the importance of molecular crowding for establishing an efficient excitonic protein network. In addition the strong dependency of Fo on the protein density reveals weak interactions between LHCII complexes which could be important for dynamic adjustment of the photosynthetic apparatus in higher plants.
Keywords: Grana membranes; Lipids; Liposome; Molecular crowding; PSII

Association of small CAB-like proteins (SCPs) of Synechocystis sp. PCC 6803 with Photosystem II by Galyna Kufryk; Miguel A. Hernandez-Prieto; Thomas Kieselbach; Helder Miranda; Wim Vermaas; Christiane Funk (135-145).
The cyanobacterial small CAB-like proteins (SCPs) are one-helix proteins with compelling similarity to the first and third transmembrane helix of proteins belonging to the CAB family of light-harvesting complex proteins in plants. The SCP proteins are transiently expressed at high light intensity and other stress conditions but their exact function remains largely unknown. Recently we showed association of ScpD with light-stressed, monomeric Photosystem II in Synechocystis sp. PCC 6803 (Yao et al. J Biol Chem 282:267–276, 2007). Here we show that ScpB associates with Photosystem II at normal growth conditions. Moreover, upon introduction of a construct into Synechocystis so that ScpB is expressed continuously under normal growth conditions, ScpE was detected under non-stressed conditions as well, and was copurified with tagged ScpB and Photosystem II. We also report on a one-helix protein, Slr1544, that is somewhat similar to the SCPs and whose gene is cotranscribed with that of ScpD; Slr1544 is another member of the extended light-harvesting-like (Lil) protein family, and we propose to name it LilA.
Keywords: Antenna; Chlorophyll-binding protein; Cyanobacteria; Early-light induced proteins (ELIPs); High-light induced proteins (HLIPs); Light-harvesting complex; Synechocystis sp. PCC 6803

Nomenclature for membrane-bound light-harvesting complexes of cyanobacteria by Min Chen; Yinan Zhang; Robert E. Blankenship (147-154).
Accessory chlorophyll-binding proteins (CBP) in cyanobacteria have six transmembrane helices and about 11 conserved His residues that might participate in chlorophyll binding. In various species of cyanobacteria, the CBP proteins bind different types of chlorophylls, including chlorophylls a, b, d and divinyl-chlorophyll a, b. The CBP proteins do not belong to the light-harvesting complexes (LHC) superfamily of plant and algae. The proposed new name of CBP for this class of proteins, which is a unique accessory light-harvesting superfamily in cyanobacteria, clarifies the confusion of names of prochlorophytes chlorophyll binding protein (Pcb), PSII-like light-harvesting proteins and iron-stress-induced protein A (IsiA). The CBP complexes are a member of a larger family that includes the chlorophyll a-binding proteins CP43 and CP47 that function as core antennas of photosystem II.
Keywords: Light-harvesting protein complexes; Chlorophyll-binding protein complexes; Cyanobacteria; Acaryochloris; Prochlorophytes

Red antenna states of photosystem I from Synechococcus sp. PCC 7002 by Marc Brecht; Jana B. Nieder; Hauke Studier; Eberhard Schlodder; Robert Bittl (155-162).
Absorption, fluorescence and single-molecule spectroscopy at low temperatures were used to elucidate spectral properties, heterogeneities and dynamics of the red-shifted chlorophyll a (Chla) molecules responsible for the fluorescence in photosystem I (PSI) from the cyanobacterium Synechoccocus sp. PCC 7002. The 77 K absorption spectrum indicates the presence of 2–3 red-shifted Chla’s absorbing at about 708 nm. The fluorescence emission spectrum is dominated by a broad band at 714 nm. The emission spectra of single PSI complexes show zero-phonon lines (ZPLs) as well as a broad intensity distribution without ZPLs. The spectral region below 710 nm often shows ZPLs, they form a spectral band with a maximum at 698 nm (F698). The region above 710 nm is dominated by broad intensity distributions and the observation of ZPLs is less frequent. The broad distributions are due to the emission of the C708 Chla’s and the emission from F698 stems from a Chla species absorbing at the blue side of P700. The properties of these two emissions show a close relation to those of the C708 and C719 pools observed in T. elongatus. Therefore an assignment of F698 and C708 to Chla-species with similarities to C708 and C719 in T. elongatus is proposed.
Keywords: Photosystem I; Single-molecule spectroscopy; Synechoccocus sp. PCC 7002; Protein-cofactor interaction; Spectral diffusion; Energy landscape

Intermediate binding of phycocyanobilin to the lyase, CpeS1, and transfer to apoprotein by Jun-Ming Tu; Michaela Kupka; Stephan Böhm; Matthias Plöscher; Lutz Eichacker; Kai-Hong Zhao; Hugo Scheer (163-168).
The phycobilin: Cysteine-84-phycobiliprotein lyase, CpeS1, catalyzes phycocyanobilin (PCB) and phycoerythrobilin attachment to nearly all cysteine-84 (consensus sequence) binding sites of phycoerythrin, phycoerythrocyanin, phycocyanin and allophycocyanin (Zhao et al. (2007) Proc Natl Acad Sci 104:14300–14305). We now show that CpeS1 can bind PCB, as assayed by Ni2+ chelating affinity chromatography. Binding is rapid, and the chromophore is bound in an extended conformation similar to that in phycobiliproteins but only poorly fluorescent. Upon addition of apo-biliproteins, the chromophore is transferred to the latter much slower (∼1 h), indicating that chromophorylated CpeS1 is an intermediate in the enzymatic reaction. In addition, imidazole is bound to PCB, as shown by mass spectroscopy of tryptic digests of the intermediate CpeS1–PCB complex.
Keywords: Cyanobacteria; Phycobiliproteins; Phycocyanin; Phycoerythrocyanin; Lyase; Phycocyanobilin

Structure and organization of phycobilisomes on membranes of the red alga Porphyridium cruentum by Ana A. Arteni; Lu-Ning Liu; Thijs J. Aartsma; Yu-Zhong Zhang; Bai-Cheng Zhou; Egbert J. Boekema (169-174).
In the present work, electron microscopy and single particle averaging was performed to investigate the supramolecular architecture of hemiellipsoidal phycobilisomes from the unicellular red alga Porphyridium cruentum. The dimensions were measured as 60 × 41 × 34 nm (length × width × height) for randomly ordered phycobilisomes, seen under high-light conditions. The hemiellipsoidal phycobilisomes were found to have a relatively flexible conformation. In closely packed semi-crystalline arrays, observed under low-light conditions, the width is reduced to 31 or 35 nm, about twice the width of the phycobilisome of the cyanobacterium Synechocystis sp. PCC 6803. Since the latter size matches the width of dimeric PSII, we suggest that one PBS lines up with one PSII dimer in cyanobacteria. In red algae, a similar 1:1 ratio under low-light conditions may indicate that the red algal phycobilisome is enlarged by a membrane-bound peripheral antenna which is absent in cyanobacteria.
Keywords: Phycobilisome; Red alga; Porphyridium cruentum ; Electron microscopy; Single particle analysis

The phycobilisome is a remarkable light-harvesting antenna that combines high efficiency with functional flexibility and the ability to capture light across a broad spectral range. A combination of biochemical, structural and spectroscopic studies has given an excellent picture of the structure and function of isolated phycobilisomes. However, we still know remarkably little about the interaction of the phycobilisome with the thylakoid membrane and the reaction centres. This article will discuss the various current ideas about this question and explain the things we need to know more about. As a working model, I propose that the phycobilisome is attached to the membrane by multiple weak charge–charge interactions with lipid head-groups and/or proteins, and that the core-membrane linker polypeptide ApcE provides a flexible surface allowing interaction with multiple membrane components.
Keywords: ApcE; Cyanobacterium; Light-harvesting; Photosystem I; Photosystem II; Phycobilisome; State transition; Thylakoid membrane

Effect of quinones on formation and properties of bacteriochlorophyll c aggregates by Jan Alster; Anita Zupcanova; Frantisek Vacha; Jakub Psencik (183-189).
Chlorosomes of green photosynthetic bacterium Chlorobium tepidum contain aggregates of bacteriochlorophyll c (BChl c) with carotenoids and isoprenoid quinones. BChl aggregates with very similar optical properties can be prepared also in vitro either in non-polar solvents or in aqueous buffers with addition of lipids and/or carotenoids. In this work, we show that the aggregation of BChl c in aqueous buffer can be induced also by quinones (vitamin K1 and K2), provided they are non-polar due to a hydrophobic side-chain. Polar vitamin K3, which possess the same functional group as K1 and K2, does not induce the aggregation. The results confirm a principal role of the hydrophobic interactions as a driving force for the aggregation of chlorosomal BChls. The chlorosomal quinones play an important role in a redox-dependent excitation quenching, which may protect the cells against damage under oxygenic conditions. We found that aggregates of BChl c with vitamin K1 and K2 exhibit an excitation quenching as well. The amplitude of the quenching depends on quinone concentration, as determined from fluorescence measurements. No lipid is necessary to induce the quenching, which therefore originates mainly from interactions of BChl c with quinones incorporated in the aggregate structure. In contrast, only a weak quenching was observed for dimers of BChl c in buffer (either with or without vitamin K3) and also for BChl c aggregates prepared with a lipid (lecithin). Thus, the weak quenching seems to be a property of BChl c itself.
Keywords: Bacteriochlorophyll aggregates; Chlorosomes; Green sulphur bacteria; Redox-dependent excitation quenching; Quinones

We have extracted polar lipids and waxes from isolated chlorosomes from the green sulfur bacterium Chlorobium tepidum and determined the fatty acid composition of each lipid class. Polar lipids amounted to 4.8 mol per 100 mol bacteriochlorophyll in the chlorosomes, while non-polar lipids (waxes) were present at a ratio of 5.9 mol per 100 mol bacteriochlorophyll. Glycolipids constitute 60 % of the polar lipids while phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, and an aminoglycosphingolipid make up respectively 15, 3, 8 and 12 %. A novel glycolipid was identified as a rhamnose derivative of monogalactosyldiacylglycerol, while the other major glycolipid was monogalactosyldiacylglycerol. Tetradecanoic acid was the major fatty acid in the aminoglycosphingolipid, while the other polar lipids contained predominantly hexandecanoic acid. The chlorosome waxes are esters of unbranched fatty acids and fatty alcohols with 14 or 16 carbon atoms, joined to form molecules with between 28 and 32 carbon atoms. The stoichiometry between lipids and bacteriochlorophyll suggests that much of the chlorosome surface is covered by protein.
Keywords: Aminoglycosphingolipid; Glycolipid; Green sulfur bacteria; Light-harvesting antenna

Calculation of pigment transition energies in the FMO protein by Julian Adolphs; Frank Müh; Mohamed El-Amine Madjet; Thomas Renger (197-209).
The Fenna–Matthews–Olson (FMO) protein of green sulfur bacteria represents an important model protein for the study of elementary pigment–protein couplings. We have previously used a simple approach [Adolphs and Renger (2006) Biophys J 91:2778–2797] to study the shift in local transition energies (site energies) of the FMO protein of Prosthecochloris aestuarii by charged amino acid residues, assuming a standard protonation pattern of the titratable groups. Recently, we have found strong evidence that besides the charged amino acids also the neutral charge density of the protein is important, by applying a combined quantum chemical/electrostatic approach [Müh et al. (2007) Proc Natl Acad Sci USA, in press]. Here, we extract the essential parts from this sophisticated method to obtain a relatively simple method again. It is shown that the main contribution to the site energy shifts is due to charge density coupling (CDC) between the pigments and their pigment, protein and water surroundings and that polarization effects for qualitative considerations can be approximated by screening the Coulomb coupling by an effective dielectric constant.
Keywords: Pigment–protein complex; Excitonic coupling; Prosthecochloris aestuarii ; FMO protein; Site energies; Genetic algorithm

Calculation of pigment transition energies in the FMO protein by Julian Adolphs; Frank Müh; Mohamed El-Amine Madjet; Thomas Renger (211-211).

Composition and localization of bacteriochlorophyll a intermediates in the purple photosynthetic bacterium Rhodopseudomonas sp. Rits by Jiro Harada; Tadashi Mizoguchi; Sayaka Yoshida; Megumi Isaji; Hirozo Oh-oka; Hitoshi Tamiaki (213-221).
Rhodopseudomonas sp. Rits is a recently isolated new species of photosynthetic bacteria and found to accumulate a significantly high amount of bacteriochlorophyll (BChl) a intermediates possessing non-, di- and tetra-hydrogenated geranylgeranyl groups at the 17-propionate as well as normal phytylated BChl a (Mizoguchi T et al. (2006) FEBS Lett 580:137–143). A phylogenetic analysis showed that this bacterium was closely related to Rhodopseudomonas palustris. The strain Rits synthesizes light-harvesting complexes 2 and 4 (LH2/4), as peripheral antennas, as well as the reaction center and light-harvesting 1 core complex (RC-LH1 core). The amounts of these complexes were dependent upon the incident light intensities, which was also a typical behavior of Rhodopseudomonas palustris. HPLC analyses of extracted pigments indicated that all four BChls a were associated with the purified photosynthetic pigment–protein, as complexes described above. The results suggested that this bacterium could use these pigments as functional molecules within the LH2/4 and RC-LH1 core. Pigment compositional analyses in several purple photosynthetic bacteria showed that such BChl a intermediates were always detected and were more widely distributed than expected. Long chains in the propionate moiety of BChl a would be one of the important factors for assembly of LH systems in purple photosynthetic bacteria.
Keywords: Geranylgeranyl; Light-harvesting assembly; Phytyl; Purple non-sulfur bacteria; Rhodopseudomonas palustris

Zinc complexes of 3-hydroxymethyl-13/15-carbonyl-chlorins having a six-membered lactone as the E-ring were prepared by modifying purpurin-18 as models of bacteriochlorophyll-d, one of the chlorophyllous pigments in the main light-harvesting antenna systems (chlorosomes) of green photosynthetic bacteria. The synthetic 13-carbonylated compound self-aggregated in 1%(v/v) tetrahydrofuran and hexane to give large oligomers possessing red-shifted and broadened electronic absorption bands and intense circular dichroism bands at the shifted Q y region, indicating that the supramolecular structure of the resulting self-aggregate was similar to those of natural and artificial chlorosomal aggregates. The red-shift value observed here was smaller than the reported values in chlorosomal pigments having a five-membered keto-ring, which was ascribable to a weaker intermolecular hydrogen-bonding of 13-C=O with 31-OH in a supramolecule of the former self-aggregate and suppression of the π–π interaction among the composite chlorins. On the other hand, the isomeric 15-carbonylated molecule was monomeric even in the nonpolar organic solvent, confirming the reported proposal that the linear orientation of three interactive moieties, OH, C=O and Zn, in a molecule is requisite for its chlorosomal self-aggregation.
Keywords: Bacteriochlorophyll; Chlorophyll; Chlorosome; Photosynthetic antenna; Self-aggregate

The fluorescence yield of isolated fucoxanthin chlorophyll proteins, serving as light harvesting proteins in diatoms, was compared to the amount of diatoxanthin bound. Diatoxanthin was earlier shown to be involved in the xanthophyll cycle in diatoms as a functional analogue of zeaxanthin in higher plants. By growing cells under different light conditions, the amount of diatoxanthin in both the trimeric FCPa as well as the oligomeric FCPb of the diatom Cyclotella meneghiniana was increased. In the trimeric FCPa, the fluorescence yield decreased with increasing diatoxanthin content, whereas in the oligomeric FCPb fluorescence was generally lower, albeit constant. No pH dependence of fluorescence yield could be demonstrated except for artificially aggregated FCPa. Thus, diatoxanthin is able to quench fluorescence in FCPa, but the yield is also influenced by pH when the protein becomes aggregated.
Keywords: Light-harvesting complex; LI818; Non-photochemical quenching (NPQ); Xanthophyll cycle; Aggregation

Structurally flexible macro-organization of the pigment–protein complexes of the diatom Phaeodactylum tricornutum by Milán Szabó; Bernard Lepetit; Reimund Goss; Christian Wilhelm; László Mustárdy; Győző Garab (237-245).
By means of circular dichroism (CD) spectroscopy, we have characterized the organization of the photosynthetic complexes of the diatom Phaeodactylum tricornutum at different levels of structural complexity: in intact cells, isolated thylakoid membranes and purified fucoxanthin chlorophyll protein (FCP) complexes. We found that the CD spectrum of whole cells was dominated by a large band at (+)698 nm, accompanied by a long tail from differential scattering, features typical for psi-type (polymerization or salt-induced) CD. The CD spectrum additionally contained intense (−)679 nm, (+)445 nm and (−)470 nm bands, which were also present in isolated thylakoid membranes and FCPs. While the latter two bands were evidently produced by excitonic interactions, the nature of the (−)679 nm band remained unclear. Electrochromic absorbance changes also revealed the existence of a CD-silent long-wavelength (∼545 nm) absorbing fucoxanthin molecule with very high sensitivity to the transmembrane electrical field. In intact cells the main CD band at (+)698 nm appeared to be associated with the multilamellar organization of the thylakoid membranes. It was sensitive to the osmotic pressure and was selectively diminished at elevated temperatures and was capable of undergoing light-induced reversible changes. In isolated thylakoid membranes, the psi-type CD band, which was lost during the isolation procedure, could be partially restored by addition of Mg-ions, along with the maximum quantum yield and the non-photochemical quenching of singlet excited chlorophyll a, measured by fluorescence transients.
Keywords: Circular dichroism spectroscopy; Diatoms; Electrochromic absorbance changes; Macrodomains; Light-harvesting complexes; Phaeodactylum tricornutum ; Thylakoid membranes

Relative binding affinities of chlorophylls in peridinin–chlorophyll–protein reconstituted with heterochlorophyllous mixtures by T. H. P. Brotosudarmo; S. Mackowski; E. Hofmann; R. G. Hiller; C. Bräuchle; H. Scheer (247-252).
Peridinin–chlorophyll–protein (PCP), containing differently absorbing chlorophyll derivatives, are good models with which to study energy transfer among monomeric chlorophylls (Chls) by both bulk and single-molecule spectroscopy. They can be obtained by reconstituting the N-terminal domain of the protein (N-PCP) with peridinin and chlorophyll mixtures. Upon dimerization of these “half-mers”, homo- and heterochlorophyllous complexes are generated, that correspond structurally to monomeric protomers of native PCP from Amphidinium carterae. Heterochlorophyllous complexes contain two different Chls in the two halves of the complete structure. Here, we report reconstitution of N-PCP with binary mixtures of Chl a, Chl b, and [3-acetyl]-Chl a. The ratios of the pigments were varied in the reconstitution mixture, and relative binding constants were determined from quantification of these pigments in the reconstituted PCPs. We find higher affinities for both Chl b and [3-acetyl]-Chl a than for the native pigment, Chl a.
Keywords: Dinoflagellate; Light-harvesting; Peridinin–chlorophyll–protein; Chlorophyll; Binding affinity

Fluorescence spectroscopy of reconstituted peridinin–chlorophyll–protein complexes by S. Mackowski; S. Wörmke; T. H. P. Brotosudarmo; H. Scheer; C. Bräuchle (253-260).
Peridinin–chlorophyll–proteins (PCP) were reconstituted with binary 1:1 chlorophyll (Chl) mixtures of Chl a, Chl b, [3–acetyl]-Chl a (acChl a), and studied by bulk and single-molecule fluorescence spectroscopy. The latter provides a way to distinguish in a given sample hetero-chlorophyllous complexes that contain two different Chls from homo-chlorophyllous ones containing the same Chl in both binding sites. The results are compared with those of homo-chlorophyllous PCP reconstituted with pure Chl a, Chl b, or acChl a. Relative intensities of the Chl fluorescence in hetero-chlorophyllous complexes were obtained and modeled using the Förster description of energy transfer combined with known variations of peridinin (Per)–Chl excitation transfer rates for the different Chl pigments. In the case of hetero-chlorophyllous complexes containing acChl a, the energy transfer is unidirectional in the energetically preferable direction, while it is bi-directional in the sample reconstituted with Chl a and Chl b.
Keywords: Light-harvesting complexes; Fluorescence; Single molecule spectroscopy; Energy transfer; Chlorophyll reconstitution

Phototrophic purple sulfur bacteria as heat engines in the South Andros Black Hole by Rodney A. Herbert; Andrew Gall; Takashi Maoka; Richard J. Cogdell; Bruno Robert; Shinichi Takaichi; Stephanie Schwabe (261-268).
Photosynthetic organisms normally endeavor to optimize the efficiency of their light-harvesting apparatus. However, here we describe two bacterial isolates belonging to the genera Allochromatium and Thiocapsa that demonstrate a novel adaptation by optimizing their external growth conditions at the expense of photosynthetic efficiency. In the South Andros Black Hole, Bahamas, a dense l-m thick layer of these anoxygenic purple sulfur bacteria is present at a depth of 17.8 m. In this layer the water temperature increases sharply to 36°C as a consequence of the low-energy transfer efficiency of their carotenoids (ca. 30%). These include spirilloxanthin, and related polyene molecules and a novel chiral carotenoid identified as spirilloxanthin-2-ol, not previously reported in purple bacteria. To our knowledge, this study presents the first evidence of such a bacterial mass significantly increasing the ambient water temperature. The transduction of light to heat energy to excess heat may provide these anoxygenic phototropic bacteria with a competitive advantage over non-thermotolerant species, which would account for their predominance within the microbial layer.
Keywords: Anoxygenic purple sulfur bacteria; Allochromatium ; Thiocapsa ; South Andros Black Hole; Carotenoid; Spirilloxanthin; Energy-transfer

Recent topographs of the intracytoplasmic membrane (ICM) of purple bacteria obtained by atomic force microscopy (AFM) have provided the first surface views of the native architecture of a multicomponent biological membrane at submolecular resolution, representing an important landmark in structural biology. A variety of species-dependent, closely packed arrangements of light-harvesting (LH) complexes was revealed: the most highly organized was found in Rhodobacter sphaeroides in which the peripheral LH2 antenna was seen either in large clusters or in fixed rows interspersed among ordered arrays of dimeric LH1-reaction center (RC) core complexes. A more random organization was observed in other species containing both the LH1 and LH2 complexes, as typified by Rhododspirillum photometricum with randomly packed monomeric LH1-RC core complexes intermingled with large, paracrystalline domains of LH2 antenna. Surprisingly, no structures that could be identified as the ATP synthase or cytochrome bc 1 complexes were observed, which may reflect their localization at ICM vesicle poles or in curved membrane areas, out of view from the flat regions imaged by AFM. This possible arrangement of energy transducing complexes has required a reassessment of energy tranduction mechanisms which place the cytochrome bc 1 complex in close association with the RC. Instead, more plausible proposals must account for the movement of quinone redox species over considerable membrane distances on appropriate time scales. AFM, together with atomic resolution structures are also providing the basis for molecular modeling of the ICM that is leading to an improved picture of the supramolecular organization of photosynthetic complexes, as well as the forces that drive their segregation into distinct domains.
Keywords: Light harvesting complexes; Membrane protein; Rhodobacter sphaeroides ; Rhodospirillum photometricum

Rhodobacter capsulatus contains lhaA and pucC genes that have been implicated in light-harvesting complex 1 and 2 (LH1 and LH2) assembly. The proteins encoded by these genes, and homologues in other photosynthetic organisms, have been classified as the bacteriochlorophyll delivery (BCD) family of the major facilitator superfamily. A new BCD family phylogenetic tree reveals that several PucC, LhaA and Orf428-related sequences each form separate clusters, while plant and cyanobacterial homologues cluster more distantly. The PucC protein is encoded in the pucBACDE superoperon which also codes for LH2 α (PucA) and β (PucB) proteins. PucC was previously shown to be necessary for formation of LH2. This article gives evidence indicating that PucC has a shepherding activity that keeps the homologous α and β proteins of LH1 and LH2 apart, allowing LH1 to assemble properly. This shepherding function was indicated by a 62% reduction in LH1 levels in ΔLHII strains carrying plasmids encoding pucBA along with a C-terminally truncated pucC gene. More severe reductions in LH1 were seen when the truncated pucC gene was co-expressed in the presence of C-terminal PucC::PhoA fusion proteins. It appears that interaction between truncated PucC::PhoA fusion proteins and the truncated PucC protein disrupts LH1 assembly, pointing towards a PucC dimeric or multimeric functional unit.
Keywords: Photosynthesis; Purple bacteria; Light-harvesting; LH1; LH2; Rhodobacter ; PucC; LhaA; Bacteriochlorophyll delivery homologues; Complex-specific assembly factors

Spectral diffusion of the lowest exciton component in the core complex from Rhodopseudomonas palustris studied by single-molecule spectroscopy by Martin F. Richter; Jürgen Baier; June Southall; Richard J. Cogdell; Silke Oellerich; Jürgen Köhler (285-290).
We have recorded fluorescence-excitation spectra from individual RC–LH1 complexes from Rhodopseudomonas palustris. The spectra feature a few broad bands accompanied by a sharp line at the low-energy side of the spectrum which is ascribed to the lowest exciton state of the BChl a assembly. Recording several fluorescence-excitation spectra from the same individual complex in rapid succession reveals that the linewidth of the lowest exciton transition is determined by spectral diffusion which increases for higher excitation energies.
Keywords: Bacterial photosynthesis; Light-harvesting complex; Single-molecule spectroscopy; Spectral diffusion; Purple bacteria

Comparison of the fluorescence kinetics of detergent-solubilized and membrane-reconstituted LH2 complexes from Rps. acidophila and Rb. sphaeroides by Tobias Pflock; Manuela Dezi; Giovanni Venturoli; Richard J. Cogdell; Jürgen Köhler; Silke Oellerich (291-298).
Picosecond time-resolved fluorescence spectroscopy has been used in order to compare the fluorescence kinetics of detergent-solubilized and membrane-reconstituted light-harvesting 2 (LH2) complexes from the purple bacteria Rhodopseudomonas (Rps.) acidophila and Rhodobacter (Rb.) sphaeroides. LH2 complexes were reconstituted in phospholipid model membranes at different lipid:protein-ratios and all samples were studied exciting with a wide range of excitation densities. While the detergent-solubilized LH2 complexes from Rps. acidophila showed monoexponential decay kinetics (τf = 980 ps) for excitation densities of up to 3·1013 photons/(pulse·cm2), the membrane-reconstituted LH2 complexes showed multiexponential kinetics even at low excitation densities and high lipid:protein-ratios. The latter finding indicates an efficient clustering of LH2 complexes in the phospholipid membranes. Similar results were obtained for the LH2 complexes from Rb. sphaeroides.
Keywords: Bacterial photosynthesis; Fluorescence; Light-harvesting 2 complex; Membrane reconstitution; Phospholipids; Purple bacteria; Time-resolved spectroscopy

Four-wave mixing signals from β-carotene and its n = 15 homologue by Mitsuru Sugisaki; Masazumi Fujiwara; Kazuhiro Yanagi; Richard J. Cogdell; Hideki Hashimoto (299-308).
The third-order nonlinear optical responses of β-carotene and its homologue having a conjugation-double bond n = 15 have been investigated using sub-20 fs ultra-short optical pulses in order to clarify the dissipation processes of excess energy. Using the four-wave mixing spectroscopy, we observed a clear coherent oscillation with a period of a few tens of femtoseconds. The spectral density of these molecules was estimated that allowed the theoretical linear and nonlinear optical signals to be directly compared with the experimental data. Calculations based on the Brownian oscillator model were performed under the impulsive excitation limit. We show that the memory of the vibronic coherence generated upon the excitation into the S2 state is lost via the relaxation process including the S1 state. The vibronic decoherence lifetime of the system was estimated to be 1 ps, which is about 5 times larger than the life time of the S2 state (∼150 fs) determined in previous studies. The role of coherence and the efficient energy transfer in the light-harvesting antenna complexes are discussed.
Keywords: Nonlinear spectroscopy; Four-wave mixing; β-carotene; Brownian oscillator model

Linear and nonlinear optical responses in bacteriochlorophyll a by Mitsuru Sugisaki; Ritsuko Fujii; Richard J. Cogdell; Hideki Hashimoto (309-316).
Nonlinear optical responses of bacteriochlorophyll a (BChl a) were investigated by means of the three-pulse four-wave mixing (FWM) technique under the resonant excitation into the Q y band. The experimental results are explained by a theoretical model calculation including the Brownian oscillation mode of the solvent. We have determined the spectral density, which is the most important function with which to calculate optical signals. The linear absorption spectrum can be reproduced fairly well when the vibronic oscillation modes of the solvent together with those of BChl a are properly taken into consideration. The FWM signal was also calculated using the spectral density. It was found that a simple two-level model could not explain the experimental result. The effect of the higher-order interactions is discussed.
Keywords: Nonlinear spectroscopy; Four-wave mixing; BChl a ; Brownian oscillator model; Spectral density

Aggregation of photosynthetic light-harvesting complexes strongly influences their spectroscopic properties. Fluorescence yield and excited state lifetimes of the main light-harvesting complex (LHC II) of higher plants strongly depend on its aggregation state. Detergents are commonly used to solubilize membrane proteins and/or to circumvent their aggregation in aqueous environments. Nonlinear polarization spectroscopy in the frequency domain (NLPF) was performed with LHC II over a wide concentration range of the mild detergent n-dodecyl β-d-maltoside (β-DM). Additionally, conventional absorption-, fluorescence- and circular dichroism-spectra were measured.The results indicate that: (i) conventional spectroscopic techniques are not well suited to investigate aggregation effects. NLPF provides a novel approach to overcome this problem: NLPF spectra display dramatic alterations upon even minor β-DM concentration changes. (ii) Commonly used detergent concentrations (around or slightly above the critical micellar concentration) apparently do not lead to complete trimerization of LHC II. A long-wavelength species in the NLPF spectra (peaking at about 685 nm), indicative of residual aggregation, persists up to DM-concentrations of 0.06%. (iii) High-resolution NLPF spectra indicate the existence of a species with a considerably shortened excited state lifetime. (iv) No indication of denaturation was found even at the highest β-DM concentrations used. (v) A specific change in interaction between certain chlorophyll(s) b and a xanthophyll molecule, probably neoxanthin, was detected upon aggregation as well as at higher β-DM concentrations. The results are discussed with respect to the still elusive mechanism of nonradiative dissipation of excess excitation energy in the antenna system.
Keywords: Aggregation; Laser spectroscopy; LHC II; Light-harvesting complex; Nonlinear polarization spectroscopy in the frequency domain; Non-photochemical quenching; Pigment–pigment interactions

Typical purple bacterial photosynthetic units consist of supra-molecular arrays of peripheral (LH2) and core (LH1-RC) antenna complexes. Recent atomic force microscopy pictures of photosynthetic units in intact membranes have revealed that the architecture of these units is variable (Scheuring et al. (2005) Biochim Bhiophys Acta 1712:109–127). In this study, we describe methods for the construction of heterologous photosynthetic units in lipid-bilayers from mixtures of purified LH2 (from Rhodopseudomonas acidophila) and LH1-RC (from Rhodopseudomonas viridis) core complexes. The architecture of these reconstituted photosynthetic units can be varied by controlling ratio of added LH2 to core complexes. The arrangement of the complexes was visualized by electron-microscopy in combination with Fourier analysis. The regular trigonal array of the core complexes seen in the native photosynthetic membrane could be regenerated in the reconstituted membranes by temperature cycling. In the presence of added LH2 complexes, this trigonal symmetry was replaced with orthorhombic symmetry. The small lattice lengths for the latter suggest that the constituent unit of the orthorhombic lattice is the LH2. Fluorescence and fluorescence-excitation spectroscopy was applied to the set of the reconstituted membranes prepared with various proportions of LH2 to core complexes. Remarkably, even though the LH2 complexes contain bacteriochlorophyll a, and the core complexes contain bacteriochlorophyll b, it was possible to demonstrate energy transfer from LH2 to the core complexes. These experiments provide a first step along the path toward investigating how changing the architecture of purple bacterial photosynthetic units affects the overall efficiency of light-harvesting.
Keywords: Bacteriochlorophyll b ; Energy transfer from LH2 to LH1; Fluorescence; Fluorescence excitation; Lipid bilayer; Photosynthetic bacteria; Reconstituted membrane

Probing binding site of bacteriochlorophyll a and carotenoid in the reconstituted LH1 complex from Rhodospirillum rubrum S1 by Stark spectroscopy by Katsunori Nakagawa; Satoru Suzuki; Ritsuko Fujii; Alastair T. Gardiner; Richard J. Cogdell; Mamoru Nango; Hideki Hashimoto (339-344).
Stark spectroscopy is a powerful technique to investigate the electrostatic interactions between pigments as well as between the pigments and the proteins in photosynthetic pigment–protein complexes. In this study, Stark spectroscopy has been used to determine two nonlinear optical parameters (polarizability change Tr(Δα) and static dipole-moment change |Δμ| upon photoexcitation) of isolated and of reconstituted LH1 complexes from the purple photosynthetic bacterium, Rhodospirillum (Rs.) rubrum. The integral LH1 complex was prepared from Rs. rubrum S1, while the reconstituted complex was assembled by addition of purified carotenoid (all-trans-spirilloxanthin) to the monomeric subunit of LH1 from Rs. rubrum S1. The reconstituted LH1 complex has its Qy absorption maximum at 878 nm. This is shifted to the blue by 3 nm in comparison to the isolated LH1 complex. The energy transfer efficiency from carotenoid to bacteriochlorophyll a (BChl a), which was determined by fluorescence excitation spectroscopy of the reconstituted LH1 complex, is increased to 40%, while the efficiency in the isolated LH1 complex is only 28%. Based on the differences in the values of Tr(Δα) and |Δμ|, between these two preparations, we can calculate the change in the electric field around the BChl a molecules in the two situations to be E Δ ≈ 3.4 × 105 [V/cm]. This change can explain the 3 nm wavelength shift of the Qy absorption band in the reconstituted LH1 complex.
Keywords: Bacteriochlorophyll a ; Binding-site; Carotenoid; LH1 complex; Purple photosynthetic bacterium; Reconstitution; Rhodospirillum rubrum ; Stark spectroscopy

Electrostatic effect of surfactant molecules on bacteriochlorophyll a and carotenoid binding sites in the LH1 complex isolated from Rhodospirillum rubrum S1 probed by Stark spectroscopy by Katsunori Nakagawa; Satoru Suzuki; Ritsuko Fujii; Alastair T. Gardiner; Richard J. Cogdell; Mamoru Nango; Hideki Hashimoto (345-351).
The LH1 complexes were isolated from the purple photosynthetic bacterium Rhodospirillum rubrum strain S1. They were initially solubilized using LDAO and then purified in the presence of Triton X-100. The purified complexes were then either used directly or following an exchange into LDAO. Stark spectroscopy was applied to probe the electrostatic field around the bacteriochlorophyll a (BChl a) and carotenoid binding sites in the LH1 complexes surrounded by these two different surfactant molecules. Polarizabilty change ( $$Updeltavarvec{upalpha}$$ ) and dipole moment change ( $$Updeltavarvec{upmu}$$ ) upon photoexcitation were determined for the BChl a Qy band. Both of these parameters show smaller values in the presence of LDAO than in Triton X-100. This indicates that polar detergent molecules, like LDAO, affect the electrostatic environment around BChl a, and modify the nonlinear optical parameters ( $$Updeltavarvec{upalpha}$$ and $$Updeltavarvec{upmu}$$ values). The electrostatic field around the BChl a binding site, which is generated by the presence of LDAO, was determined to be |E L | = ∼3.9 × 105 [V/cm]. Interestingly, this kind of electrostatic effect was not observed for the carotenoid-binding site. The present study demonstrates a unique electrostatic interaction between the polar detergent molecules surrounding the LH1 complex and the Qy absorption band of BChl a that is bound to the LH1 complex.
Keywords: Bacteriochlorophyll; Carotenoid; LH1 complex; Non-polar detergent; Polar detergent; Purple photosynthetic bacteria; Stark spectroscopy

Molecular assembly of Zn porphyrin complexes using synthetic light-harvesting model polypeptides by Tsuyoshi Ochiai; Takahide Asaoka; Tomoya Kato; Shinichiro Osaka; Takehisa Dewa; Keiji Yamashita; Alastair T. Gardiner; Hideki Hashimoto; Mamoru Nango (353-361).
Synthetic single α-helix hydrophobic polypeptides, which have similar amino acid sequences to the hydrophobic core in the native light-harvesting 1-β polypeptide from Rhodobacter sphaeroides, formed Zn porphyrin complexes on a gold electrode, as well as in n-octyl-β-glucoside micelles: this process is dependent on the structure of the pigments and the polypeptides. Interestingly, an enhanced photoelectric current was observed when Zn mesoporphyrin monomer complexed with the synthetic light-harvesting model polypeptide in an α-helical configuration was assembled with a defined orientation onto the electrode. Analog of these light-harvesting model complexes are also useful in providing insights into the effect of polypeptide structure on the formation of light-harvesting complexes on and off electrodes.
Keywords: Zn porphyrin complex; Light-harvesting model polypeptide; Reconstitution; Self-assembling Monolayer (SAM); Photocurrent

Mimicking the photosynthetic system with strong hydrogen bonds to promote proton electron concerted reactions by Isamu Kinoshita; Hideki Hashimoto; Takanori Nishioka; Riichi Miyamoto; Naoto Kuwamura; Yuki Yoshida (363-371).
A Copper(2+) complex with a CuII–C bond containing sp3 configuration was used to investigate the role of strong hydrogen bonds in proton coupled electron transfer (PCET) reactions. The only example of a CuII–C system realized so far is that using tris-(pyridylthio)methyl (tptm) as a tetradentate tripodal ligand. Using this ligand, [CuF(tptm)] and [Cu(tptm)(OH)] have been prepared. The former complex forms supra-molecular arrays of layers of the complex between which hydroquinone is intercalated in the crystalline phase. This hydroquinone intercalation crystal was prepared via the photochemical conversion of quinone during the crystallization process. This conversion reaction probably involves a proton coupled electron transfer process. The nuclear magnetic resonance spectroscopic analysis of the reaction mixture shows the presence of Cu(III) during the conversion reaction. These results strongly suggest the presence of the molecular aggregate of the [CuF(tptm)] complex, water and quinone in the solution phase during the quinone to hydroquinone conversion. The presence of this type of aggregate requires a strong hydrogen bond between the [CuF(tptm)] complex and water. The presence of this particular hydrogen bond is a unique character of such a complex that has the CuII–C bond. This complex is used as a model for photosynthetic water splitting since the photoconversion of quinone to hydroquinone also involves the production of oxygen from water.
Keywords: Proton coupled electron transfer; Quinone hydroquinone conversion; Copper complex