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We present a molecular-scale model of Bacteriochlorophyll a (BChl a) binding to the chlorosome protein A (CsmA) of Chlorobaculum tepidum, and the aggregated pigment–protein dimer, as determined from protein–ligand docking and quantum chemistry calculations. Our calculations provide strong evidence that the BChl a molecule is coordinated to the His25 residue of CsmA, with the magnesium center of the bacteriochlorin ring situated

Photosystem II (PSII) initiates photosynthesis in plants through the absorption of light and subsequent conversion of excitation energy to chemical energy via charge separation. The pigment binding proteins associated with PSII assemble in the grana membrane into PSII supercomplexes and surrounding light harvesting complex II trimers. To understand the high efficiency of light harvesting in PSII requires quantitative insight into energy transfer and charge separation in PSII supercomplexes. We have constructed the first structure-based model of energy transfer in PSII supercomplexes. This model shows that the kinetics of light harvesting cannot be simplified to a single rate limiting step. Instead, substantial contributions arise from both excitation diffusion through the antenna pigments and transfer from the antenna to the reaction center (RC), where charge separation occurs. Because of the lack of a rate-limiting step, fitting kinetic models to fluorescence lifetime data cannot be used to derive mechanistic insight on light harvesting in PSII. This model will clarify the interpretation of chlorophyll fluorescence data from PSII supercomplexes, grana membranes, and leaves.

Light harvesting for oxygenic photosynthesis is regulated to prevent the formation of harmful photoproducts by activation of photoprotective mechanisms safely dissipating the energy absorbed in excess. Lumen acidification is the trigger for the formation of quenching states in pigment binding complexes. With the aim to uncover the photoprotective functional states responsible for excess energy dissipation in green algae and mosses, we compared the fluorescence dynamic properties of the light-harvesting complex stress-related (LHCSR1) protein, which is essential for fast and reversible regulation of light use efficiency in lower plants, as compared to the major LHCII antenna protein, which mainly fulfills light harvesting function. Both LHCII and LHCSR1 had a chlorophyll fluorescence yield and lifetime strongly dependent on detergent concentration but the transition from long- to short-living states was far more complete and fast in the latter. Low pH and zeaxanthin binding enhanced the relative amplitude of quenched states in LHCSR1, which were characterized by the presence of 80 ps fluorescence decay components with a red-shifted emission spectrum. We suggest that energy dissipation occurs in the chloroplast by the activation of 80 ps quenching sites in LHCSR1 which spill over excitons from the photosystem II antenna system.

Anthocyanins were isolated from blueberry wine lees using Sephadex LH-20 column chromatography and semipreparative high-performance liquid chromatography (semipreparative HPLC) and then identified by HPLC-DAD-ESI-MS/MS. Our results show that malvidin-3-hexose (Mv-3-hex) and malvidin-3-(6'acetyl)-hexose (Mv-3-ace-hex) are the major components in the anthocyanin extracts of blueberry wine lees (90%). The binding characteristics of Mv-3-hex and Mv-3-ace-hex with β-glucosidase were investigated by fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular docking. Spectroscopic analysis revealed that β-glucosidase fluorescence quenched by Mv-3-hex and Mv-3-ace-hex follows a static mode. Binding of Mv-3-hex and Mv-3-ace-hex to β-glucosidase mainly depends on electrostatic force. The result from CD spectra shows that adaptive structure rearrangement and increase of β-sheet structure occur only in the presence of Mv-3-ace-hex. A molecular docking study suggests that Mv-3-ace-hex has stronger binding with β-glucosidase than Mv-3-hex.

NADPH:protochlorophyllide oxidoreductase (POR) is a key enzyme for the light-induced greening of etiolated angiosperm plants. It belongs to the 'RED' family of reductases, epimerases and dehydrogenases. All POR proteins characterized so far contain evolutionarily conserved cysteine residues implicated in protochlorophyllide (Pchlide)-binding and catalysis. cDNAs were constructed by site-directed mutagenesis that encode PORB mutant proteins with defined Cys→Ala exchanges. These cDNAs were expressed in transgenic plants of a PORB-deficient knock-out mutant (porB) of Arabidopsis thaliana. Results show that porB plants expressing PORB mutant proteins with Ala substitutions of Cys276 or Cys303 are hypersensitive to high-light conditions during greening. Hereby, failure to assemble higher molecular weight complexes of PORB with its twin isoenzyme, PORA, as encountered with (Cys303→Ala)-PORB plants, caused more severe effects than replacing Cys276 by an Ala residue in the active site of the enzyme, as encountered in (Cys276→Ala)-PORB plants. Our results are consistent with the presence of two distinct pigment binding sites in PORB, with Cys276 establishing the active site of the enzyme and Cys303 providing a second, low affinity pigment binding site that is essential for the assembly of higher molecular mass light-harvesting PORB::PORA complexes and photoprotection of etiolated seedlings. Failure to assemble such complexes provoked photodynamic damage through the generation of singlet oxygen. Together, our data highlight the importance of PORB for Pchlide homoeostasis and greening in Arabidopsis.

International audience; Resonance Raman spectroscopy was used to evaluate the structure of light-harvesting chlorophyll (Chl) a/b complexes of photosystem II (LHCII), reconstituted from wild-type (WT) and mutant apoproteins over-expressed in Escherichia coli. The point mutations involved residue S123, exchanged for either P (S123P) or G (S123G). In all reconstituted proteins, lutein 2 displayed a distorted conformation, as it does in purified LHCII trimers. Reconstituted WT and S123G also exhibited a conformation of bound neoxanthin (Nx) molecules identical to the native protein, while the S123P mutation was found to induce a change in Nx conformation. This structural change of neoxanthin is accompanied by a blue shift of the absorption of this carotenoid molecule. The interactions assumed by (and thus the structure of the binding sites of) the bound Chls b were found identical in all the reconstituted proteins, and only marginally perturbed as compared to purified LHCII. The interactions assumed by bound Chls a were also identical in purified LHCII and the reconstituted WT. However, the keto carbonyl group of one Chl a, originally free-from-interactions in WT LHCII, becomes involved in a strong H-bond with its environment in LHCII reconstituted from the S123P apoprotein. As the absorption in the Qy region of this protein is identical to that of the LHCII reconstituted from the WT apoprotein, we conclude that the interaction state of the keto carbonyl of Chl a does not play a significant role in tuning the binding site energy of these molecules.

We studied the localization of diadinoxanthin cycle pigments in the diatoms Cyclotella meneghiniana and Phaeodactylum tricornutum. Isolation of pigment protein complexes revealed that the majority of high-light-synthesized diadinoxanthin and diatoxanthin is associated with the fucoxanthin chlorophyll protein (FCP) complexes. The characterization of intact cells, thylakoid membranes, and pigment protein complexes by absorption and low-temperature fluorescence spectroscopy showed that the FCPs contain certain amounts of protein-bound diadinoxanthin cycle pigments, which are not significantly different in high-light and low-light cultures. The largest part of high-light-formed diadinoxanthin cycle pigments, however, is not bound to antenna apoproteins but located in a lipid shield around the FCPs, which is copurified with the complexes. This lipid shield is primarily composed of the thylakoid membrane lipid monogalactosyldiacylglycerol. We also show that the photosystem I (PSI) fraction contains a tightly connected FCP complex that is enriched in protein-bound diadinoxanthin cycle pigments. The peripheral FCP and the FCP associated with PSI are composed of different apoproteins. Tandem mass spectrometry analysis revealed that the peripheral FCP is composed mainly of the light-harvesting complex protein Lhcf and also significant amounts of Lhcr. The PSI fraction, on the other hand, shows an enrichment of Lhcr proteins, which are thus responsible for the diadinoxanthin cycle pigment binding. The existence of lipid-dissolved and protein-bound diadinoxanthin cycle pigments in the peripheral antenna and in PSI is discussed with respect to different specific functions of the xanthophylls.

The one-hour intraocular pressure (IOP) response to topical ocularly applied timolol was studied in darkly and lightly pigmented eyes. One drop of 0.25% 1-timolol was applied to one eye of ten subjects with brown and ten subjects with blue iris color. In the treated and untreated eyes with blue iris color the IOP fell significantly from the baseline value, whereas no pressure reduction was obtained either in the treated or untreated eyes with brown iris color. Beta-blocking agents are known to accumulate in pigment-containing ocular structures. The lack of a one-hour pressure response to timolol in darkly pigmented eyes observed in our study may refer to the reduction of 1-timolol in the biophase due to excessive pigment binding.

Systemic absorption, ocular distribution and ocular metabolism in albino and pigmented rabbit of topically applied 1% [3H]l-adrenaline eye drops was studied by liquid scintillation counting and thin layer chromatography. The effect of adrenaline on the pupillary diameter was also registered. The peak plasma level of adrenaline was reached at 150 min. The corneal adrenaline metabolism was substantial, but did not vary with the rabbit strain. At 30 min after instillation, the total adrenaline concentration of pigmented iris and ciliary body exceeded that of corresponding albino tissues. At 180 min the drug concentration of the iris and ciliary body did not differ between the rabbit strains. The time course of adrenaline induced mydriasis was equal in both rabbit strains. Since neither total adrenaline concentration at 180 min nor the time course of adrenaline induced mydriasis differed between the rabbit strains, the higher initial adrenaline uptake by pigmented tissues is not explained by pigment binding. It is proposed that the increased initial binding of adrenaline by pigmented tissues is caused by a difference between pigmented and albino tissues in the number of adrenergic neurones; this is also supported by the literature.

In this work, Photosystem I supercomplexes have been purified from Lhca-deficient lines of Arabidopsis thaliana using a mild detergent treatment that does not induce loss of outer antennas. The complexes have been studied by integrating biochemical analysis with electron microscopy. This allows the direct correlation of changes in protein content with changes in supramolecular structure of Photosystem I to get information about the position of the individual Lhca subunits, the association of the antenna to the core, and the influence of the individual subunits on the stability of the system. Photosystem I complexes with only two or three antenna complexes were purified, showing that the binding of Lhca1/4 and Lhca2/3 dimers to the core is not interdependent, although weak binding of Lhca2/3 to the core is stabilized by the presence of Lhca4. Moreover, Lhca2 and Lhca4 can be associated with the core in the absence of their “dimeric partners.” The structure of Photosystem I is very rigid, and the absence of one antenna complex leaves a “hole” in the structure that cannot be filled by other Lhcas, clearly indicating that the docking sites for the individual subunits are highly specific. There is, however, an exception to the rule: Lhca5 can substitute for Lhca4, yielding highly stable PSI supercomplexes with a supramolecular organization identical to the WT.

The major chlorophyll (Chl) a/b complexes of photosystem II (LHCIIb), in addition to their primary light-harvesting function, play key roles in the organization of the granal ultrastructure of the thylakoid membranes and in various regulatory processes. These functions depend on the structural stability and flexibility of the complexes. The lumenal side of LHCIIb is exposed to broadly variable pH environments, due to the build-up and decay of the pH gradient during photosynthesis. Therefore, the negatively charged amino acids in the lumenal loop might be of paramount importance for adjusting the structure and functions of LHCIIb. In order to clarify the structural roles of these residues, we investigated the pigment stoichiometries, absorption, linear and circular dichroism spectra of the reconstituted LHCIIb complexes, in which the negatively charged amino acids in the lumenal loop were exchanged to neutral ones (E94G, E107V and D111V). The mutations influenced the pigment binding and the molecular architecture of the complexes. Exchanging E94 to G destabilized the 310 helix in the lumenal loop structure and led to an acquired pH sensitivity of the LHCIIb structure. We conclude that these amino acids are important not only for pigment binding in the complexes, but also in stabilizing the conformation of LHCIIb at different pHs.

Stereoselective recognition of bilirubin and biliverdin by poly(L-lysine) (PLL), poly(D-lysine) (PDL), and poly(L-arginine) (PLA) and their micelles with dodecanoate ions (C(12)) at different pH has been studied using a combination of vibrational and electronic circular dichroism. Biliverdin has been found to be more sensitive to pH in its complexes with the polypeptides. In acidic media in the complexes with PLL-C(12) and PDL-C(12) the conformation becomes more closed than the characteristic one found at physiological pH. Partial flattening and chiral self-association of bilirubin molecules takes place at higher concentrations with PLL and PDL. For both pigments, inversions of the ECD signals are observed in the systems with PLA at pHor = 8.5. This study was carried out in order to clarify the role of Lys and Arg residues in pigment binding to serum albumin. The circular dichroism spectra obtained for bilirubin bound to different mammalian serum albumins have been compared with the homology within the IIA principal ligand-binding structural domains. Analysis suggests that the chiroptical properties of the pigment in the complexes with serum albumins depend on the location of Lys and/or Arg at positions 222 and 199 in the binding site.

NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR) A is the only known example thus far of a nucleus-encoded plastid protein that is imported to its final destination in a substrate-dependent, Pchlide-regulated manner. Previous work has shown that the cytosolic PORA precursor (pPORA) does not utilize the general import site but uses a distinct translocon designated the Pchlide-dependent translocon complex. Here we demonstrate that a pentapeptide motif, threonine-threonine-serine-proline-glycine (TTSPG) in pPORA's transit peptide (transA), is involved in Pchlide-dependent transport. Deletion of this motif from the COOH-terminal end of transA abolished both Pchlide binding and protein import. Incorporation of the TTSPG motif into normally non-Pchlide-responsive transit sequences conferred the pigment binding properties onto the engineered chimeric precursors but was insufficient to render protein import substrate dependent. An additional motif was identified in the NH2-terminal part of transA that was needed for binding of the precursor to the Pchlide-dependent translocon complex. Point mutations of the TTSPG motif identified glycine as the Pchlide binding site. By analogy to the major light-harvesting chlorophyll a/b binding protein of photosystem II, we propose that the peptidyl carbonyl oxygen of glycine may bind directly or via a water molecule to the central Mg atom of the pigment.

The formation of photosynthetic complexes in facultatively photosynthetic bacteria is controlled by the oxygen tension in the environment. In Rhodobacter capsulatus the two-component system RegB/RegA plays a major role in the redox control of photosynthesis genes but also controls other redox-dependent systems. The response regulator RegA is phosphorylated under low oxygen tension and activates the puf and puc operons, which encode pigment binding proteins, by binding to their promoter regions. Data from a yeast two-hybrid analysis as well as an in vitroanalysis indicate that RegA interacts with the NtrX protein, the response regulator of the NtrY/NtrX two-component system which is believed to be involved in regulation of nitrogen fixation genes. Our further analysis revealed that NtrX is indeed involved in the regulation of the puf and puc operons. Furthermore, we showed that an altered NtrX protein, which is predicted to adopt the conformation of phosphorylated NtrX protein, binds within the puf promoter region close to the RegA binding sites. We conclude that a direct interaction of two response regulators connects the regulatory systems for redox control and nitrogen control.

International audience; The structures and environments of the protein-bound peridinins (Pers) and chlorophylls (Chls) a/c2 in the membrane-intrinsic major light-harvesting complex of the dinoflagellate Amphidinium carterae (LHCAmph) are characterised using resonance Raman (RR) spectroscopy with 11 excitation wavelengths, at 77K. The excitation-dependent variation in the CC stretching mode (ν1) suggests the presence of three Pers with conjugation lengths over 8 double bonds (dBs), and one diadinoxanthin, between 413.7 and 528.7nm. Two Perred species are revealed on excitation at 550 and 560nm. These Perred species exhibit anomalously low ν1 values, together with notable resonant enhancement of lactone ring-breathing and -deformation modes. To discern protein-specific effects, the RR spectra are compared to that of Per in polar (acetonitrile), polarisable (toluene) and polar-protic (ethanol) solvents. Resonantly enhanced lactone, ring-breathing (942cm(-1)) and ring-deformation (~650cm(-1)), modes are identified both in solution, and in the protein, and discussed in the context of the mixing of the S1 and S2 states, and formation of the intramolecular charge-transfer (ICT) state. In the Chl-absorbing region, two sets of Chl c2's, and (at least) six Chl a's can be differentiated. With a pigment ratio of 5-6 (Chl a):2 (Chl c2):5-6 (Per):1 Ddx determined from the fit to the RT absorption and 77K RR spectra, sequence comparison of LHCAmp to LHCII, and the diatom LHC, fucoxanthin-chlorophyll-a/c-protein (FCP), a template for the conserved pigment binding sites is proposed, to fill the paucity of structural information in the absence of a crystal structure for LHCAmph.

Eleven proteins belonging to photosystem II (PSII) bind photosynthetic pigments in the form of thylakoid membrane-associated pigment-protein complexes. Five of them (PsbA, PsbB, PsbC, PsbD and PsbS) are assigned to PSII core complex while the remaining six (Lhcb1, Lhcb2, Lhcb3, Lhcb4, Lhcb5 and Lhcb6) constitute, along with their pigments, functional complexes situated more distantly with regard to P680 - the photochemical center of PSII. The main function of the pigment-binding proteins is to harvest solar energy and deliver it, in the form of excitation energy, ultimately to P680 although individual pigment-proteins may be engaged in other photosynthesis-related processes as well. The aim of this review is to present the current state of knowledge regarding the structure, functions and degradation of this family of proteins.

NADPH:protochlorophyllide oxidoreductase (POR) B is a key enzyme for the light-induced greening of etiolated angiosperm plants. It is nucleus-encoded, imported into the plastids posttranslationally, and assembled into larger light-harvesting POR:protochlorophyllide complexes termed LHPP (Reinbothe et al., Nature 397:80-84, 1999). An in vitro-mutagenesis approach was taken to study the role of the evolutionarily conserved Cys residues in pigment binding. Four Cys residues are present in the PORB of which two, Cys276 and Cys303, established distinct pigment binding sites, as shown by biochemical tests, protein import studies, and in vitro-reconstitution experiments. While Cys276 constituted the Pchlide binding site in the active site of the enzyme, Cys303 established a second, low affinity pigment binding site that was involved in the assembly and stabilization of imported PORB enzyme inside etioplasts.

Anthocyanins from a Cabernet Mitos grape pomace were extracted without sulfitation, purified and concentrated on a laboratory scale by adsorption to a styrene–divinylbenzene copolymerisate. The pigment loss during sample loading and washing of the column with water was negligible. Elution was performed with acidified methanol, ethanol and 2-propanol, respectively. The recovery rates ranged from 96 to 100% of anthocyanins when methanol was used, from 86 to 96% for ethanol and from 78 to 88% when pigments were eluted with 2-propanol. The temperature during adsorption and desorption (25 and 50 °C) did not significantly influence the pigment binding capacity. The recovery rates were not affected by the scale-up from laboratory to pilot-plant adsorption experiments. The use of polymeric resins facilitates nonthermal anthocyanin concentration since highly pigmented alcoholic eluates were obtained which can further be concentrated under mild conditions. The present study provides systematic data on adsorber technology increasingly used for the recovery of plant secondary metabolites.

A new potential light-harvesting protein, named Lhca5, was recently detected in higher plants. Because of the low amount of Lhca5 in thylakoid membranes, the isolation of a native Lhca5 pigment-protein complex has not been achieved to date. Therefore, we used in vitro reconstitution to analyze whether Lhca5 binds pigments and is actually an additional light-harvesting protein. By this approach we could demonstrate that Lhca5 binds pigments in a unique stoichiometry. Analyses of pigment requirements for light-harvesting complex formation by Lhca5 revealed that chlorophyll b is the only indispensable pigment. Fluorescence measurements showed that ligated chlorophylls and carotenoids are arranged in a way that allows directed energy transfer within the light-harvesting complex. Reconstitutions of Lhca5 together with other Lhca proteins resulted in the formation of heterodimers with Lhca1. This result demonstrates that Lhca5 is indeed a protein belonging to the light-harvesting antenna of photosystem I. The properties of Lhca5 are compared with those of previously characterized Lhca proteins, and the consequences of an additional Lhca protein for the composition of the light-harvesting antenna of photosystem I are discussed in view of the recently published photosystem I structure of the pea.

Raman and infrared microscopy have been used to characterise the black pigments on prehistoric Southwest American black-on-white pottery. Conclusive spectroscopic evidence for the use of carbon-based paints on these sherds has been provided using the Raman technique. Maghaemite (γ-Fe 2 O 3 ) and magnetite (Fe 3 O 4 ), found alternatively or mixed with a carbonaceous pigment, were also identified on some sherds. Infrared measurements indicated that little, if any, organic material from biogenic precursors of the black pigment or from pigment binding agents remained in the paints. These spectroscopic results were obtained rapidly and non-destructively on unprepared samples, and the resulting data complement, and in some instances correct, paint type conclusions drawn from studies involving techniques such as XRD, SEM-EDX, and XPS.

The light-harvesting chlorophyll a/b complex (LHCIIb) spontaneously assembles from its pigment and protein components in detergent solution. The formation of functional LHCIIb can be detected in time-resolved experiments by monitoring the establishment of excitation energy transfer from protein-bound chlorophyll b to chlorophyll a. To detect the possible initial steps of chlorophyll binding that may not yet give rise to chlorophyll b-to-a energy transfer, we have monitored LHCIIb assembly by measuring excitation energy transfer from a fluorescent dye, covalently bound to the protein, to the chlorophylls. In order to exclude interference of the dye with protein folding or pigment binding, the experiments were repeated with the dye bound to four different positions in the protein. Initial chlorophyll binding occurs at roughly the same rate as the establishment of chlorophyll b-to-a energy transfer, in the range of 10 s. However, under limiting chlorophyll concentrations, the binding of chlorophyll a clearly precedes that of chlorophyll b. The complex containing the apoprotein, carotenoids, and chlorophyll a but no chlorophyll b is biochemically unstable and therefore cannot be isolated. However, chlorophyll a binding into this weak complex is specific, as it does not occur with a C-terminal deletion mutant of Lhcb1 which still contains most chlorophyll-ligating amino acids but is unable to fold and assemble into functional LHCIIb. As a scenario for LHCIIb assembly in the thylakoid, we propose the initial formation of a labile Lhcb1-chlorophyll a-carotenoid complex that then becomes stabilized by the binding (or formation in situ) of chlorophyll b.

Porphyrins and their metal derivatives are strong nucleic acids binders. Some of these compounds have been used for radiation sensitization therapy of cancer and are targeted to interact with cellular DNA. Chlorophyll (Chl) binds DNA via guanine N-7 atom (major groove) and the backbone phosphate group (Neault and Tajmir-Riahi. Biophys. J. 76, 2177, 1999), whereas chlorophyllin (Chln) intercalates into A-T and G-C regions (Neault and Tajmir-Riahi. J. Phys. Chem. B. 102, 1610, 1998). This study was designed to examine the interaction of RNA with chlorophyll a and chlorophyllin in aqueous solution at physiological pH with pigment/RNA(phosphate) ratios (r) of 1/80 to 1/2. Fourier transform infrared (FTIR) and UV-visible difference spectroscopic methods were used to characterize the nature of pigment-RNA interaction and to establish correlation between spectral changes and the pigment binding mode, binding constant, RNA secondary structure and structural variations of pigment-RNA complexes in aqueous solution. Spectroscopic results showed that Chl and Chln bind RNA through G-C and A-U bases and the backbone phosphate group with overall binding constants of KChl = 1.95 x 10(5) M(-1) and KChln = 1.61 x 10(5) M(-1). The larger K value obtained for Chl-RNA complexes is attributed to the formation of more stable five or six-coordinate Mg cation in the RNA adducts, while the four-coordination Cu(II) in Chln can be more stable than that of the five or six-coordinated copper ion in the Chln-RNA complexes. Aggregation of pigment-RNA complexes occurs at high metalloporphyrin concentrations. No biopolymer secondary structural changes were observed upon pigment interaction and RNA remains in the A-family structure in these pigment complexes.

Reversible phosphorylation of chl a/b protein complex II (LHCII), the mobile light-harvesting antenna, regulates its association and energy transfer/dissipation to photosystem (PS) II or I (state transition). Excitation of LHCII induces conformational changes affecting the exposure of the phosphorylation site at the N-terminal domain to protein kinase(s) [Zer, H., et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 8277-8282; Zer, H., et al. (2003) Biochemistry 42, 728-738]. Thus, it was of interest to examine whether the pigment composition of LHCII affects the light-induced modulation of LHCII phosphorylation and state transition. To this end, we have used thylakoids of wild-type Chlamydomonas reinhardtii and xanthophyll deficient mutants npq1, lor1, npq2, npq1 lor1, and npq2 lor1. Phosphorylated protein bands P11, P13, and P17 are considered components of the mobile C. reinhardtii LHCII complex. The protein composition of these bands has been analyzed by mass spectrometry using Qtof-2 with a nanospray attachment. P11 and P13 contain C. reinhardtii light-harvesting chlorophyll a/b binding protein LhcII type I. P17 contains C. reinhardtii LhcII types III and IV. Illumination of isolated thylakoids inhibits the redox-controlled phosphorylation of polypeptide bands P13 and P17 and to a lower extent that of P11. The light-induced inhibition of LHCII phosphorylation and the state transition process are not influenced by extensive differences in the xanthophyll composition of the mutants. Thus, LHCII can be visualized as possessing two functionally distinct, independent domains: (i) the pigment binding transmembrane domain regulating the extent of energy transfer/dissipation and (ii) the surface-exposed phosphorylation site regulating the association of LHCII with PSII or PSI.

Reconstitution is one of the most fundamental and powerful tools to investigate pigment—protein complexes, for example, light-harvesting complexes and reaction center complexes. Two reconstitution methods, in vitro and in vivo, have been applied to complexes. In vitro reconstitution methods were first developed using isolated proteins and pigments, and recently using over-expressed proteins. This method enables analysis of pigment binding, pigment stoichiometry, and protein flexibility when accepting extrinsic pigments, however, it has not yet been successfully applied to the core antenna system. In vivo reconstitution, which was developed using genetic modifications, is applicable even on core systems. In this Review, the in vivo reconstitution is mainly considered on the basis of the in vitro reconstitution, because the former was a recent development and will be expanded to many systems. When genes for a new pigment are acquired and expressed, the new pigment is incorporated into a pre-exiting complex(es) and becomes functional when it is accepted by this complex(es), or abandoned if it is not. This process is postulated to occur during the evolutionary process(es) of antenna and reaction centers, and it is now possible to reproduce this evolutionary developmental pathway(s). Several examples of in vivo reconstitution are given and considered from the viewpoint of evolutionary implication with regards to the antenna and reaction centers.

The purple bacterium Roseospirillum(Rss.)parvum 930I, like most other purple bacteria, contains bacteriochlorophyll (BChl)-a as a LH chromophore, but exhibits an extremely red-shifted Qy absorption maximum centered at 909 nm. The puf operon encoding the LH1 and other pigment-binding proteins was cloned and sequenced, revealing the gene structure pufBALMC. Comparative analysis of predicted amino acid sequences of the α- and β-core LH polypeptides (PufA and PufB) revealed five amino-acid substitutions at positions that are highly conserved in other purple bacteria. In the primary structure, these residues are in close proximity to the BChl-a ligating histidine residue (α+3, α+5, α+6 and β−4, β+9, with respect to the central histidine, His0). The nature of the enormous red-shifts to the near-infrared region of the light were attributed to those residues, with special emphasis to cysteine residues rarely present in other purple bacterial antenna polypeptides. Three-dimensional structural models of the Rss. parvum LH1-α and -β polypeptides suggest that the thiol groups of αCys+3 and βCys−4 are oriented toward the BChl-a macrocycle in the native antenna complex. Because of their predicted close proximity to BChl, these thiol groups are likely to modulate the spectral properties of the LH1 complex. Phylogenetic comparisons of LH1 polypeptides indicate that Rss. parvum-like amino-acid substitutions in proteobacterial LH1 complexes arose independently during evolution.

Modern water-borne paints are widely used in different areas of applications ranging from high-gloss lacquers to flat, scrub-resistant interior paints. From this point of view, the pigment volume concentration (PVC) is one key-parameter adjusting the desired application properties. In high-gloss paints, for example, a low PVC is required to accommodate the proper surface roughness to achieve a high gloss. Consequently, a high concentration of TiO 2 is needed to obtain a good hiding power at the same time. Flat paints nonetheless are highly filled due to cost reasons preferentially by CaCO 3 and the pigment binding capacity of the binder is crucial. In this work, paint formulations differing in PVC, and the type of binder or dispersing agent were investigated by various techniques concerning the distribution and aggregation of pigment particles, e.g. TiO 2 . To get a detailed insight into the structure of the liquid paints and the corresponding dried paint films, suitable analytical tools were applied for characterization. The structure of the liquid paints was analyzed by remission light spectroscopy (RLS), disc centrifugation, cryogenic-replication transmission electron microscopy (Cryo-replica TEM) and cryogenic-scanning electron microscopy (Cryo-SEM). The pigment distribution in the corresponding dried paint films was examined by means of atomic force microscopy (AFM), TEM and RLS. The tendency of the TiO 2 -pigments to form aggregates was found to depend on both: first on the type of binder used in the formulation and second on the employed dispersing agent. It is shown that only by adjusting the properties of the binder in combination with common dispersants, it is possible to get well-distributed TiO 2 particles within the paint. Correlation of application properties, e.g. gloss and blocking to the microscopic structure is presented.

Chlorophyll fluorescence quenching can be stimulated in vitro in purified photosystem II antenna complexes. It has been shown to resemble nonphotochemical quenching observed in isolated chloroplasts and leaves in several important respects, providing a model system for study of the mechanism of photoprotective energy dissipation. The effect of temperature on the rate of quenching in trimeric and monomeric antenna complexes revealed the presence of two temperature-dependent processes with different activation energies, one between approximately 15 and 35 degrees C and another between approximately 40 and 60 degrees C. The temperature of the transition between the two phases was higher for trimers than for monomers. Throughout this temperature range, the quenching was almost completely reversible, the protein CD was unchanged, and pigment binding was maintained. The activation energy for the low temperature phase was consistent with local rearrangements of pigments within some of the protein domains, whereas the higher temperature phase seemed to arise from large scale conformational transitions. For both phases, there was a strong linear correlation between the quenching rate and the appearance of an absorption band at 685 nm. In addition, quenching was correlated with a loss of CD at approximately 495 nm from Lutein 1 and at 680 nm from chlorophylls a1 and a2, the terminal emitters. The results obtained indicate that quenching of chlorophyll fluorescence in antenna complexes is brought about by perturbation of the lutein 1/chlorophyll a1/chlorophyll a2 locus, forming a poorly fluorescing chlorophyll associate, either a dimer or an excimer.

The biochemical properties of PsbS protein, a nuclear-encoded Photosystem II subunit involved in the high energy quenching of chlorophyll fluorescence, have been studied using preparations purified from chloroplasts or obtained by overexpression in bacteria. Despite the homology with chlorophyll a/b/xanthophyll-binding proteins of the Lhc family, native PsbS protein does not show any detectable ability to bind chlorophylls or carotenoids in conditions in which Lhc proteins maintain full pigment binding. The recombinant protein, when refolded in vitro in the presence of purified pigments, neither binds chlorophylls nor xanthophylls, differently from the homologous proteins LHCII, CP26, and CP29 that refold into stable pigment-binding complexes. Thus, it is concluded that if PsbS is a pigment-binding protein in vivo, the binding mechanism must be different from that present in other Lhc proteins. Primary sequence analysis provides evidence for homology of PsbS helices I and III with the central 2-fold symmetric core of chlorophyll a/b-binding proteins. Moreover, a structural homology owed to the presence of acidic residues in each of the two lumen-exposed loops is found with the dicyclohexylcarbodiimide/Ca(2+)-binding domain of CP29. Consistently, both native and recombinant PsbS proteins showed [(14)C]dicyclohexylcarbodiimide binding, thus supporting a functional basis for its homology with CP29 on the lumen-exposed loops. This domain is suggested to be involved in sensing low luminal pH.

Beta-Amyloid peptide (Abeta) is the main protein components of neuritic plaques and its neurotoxicity would be exposed by formation of aggregate. The aggregation inhibitors composed of an Abeta recognition element (KLVFF) and a flexible hydrophilic disrupting element (aminoethoxy ethoxy acetate and aspartate) are designed and chemically synthesized. The inhibitory effects are examined by a pigment binding assay using Congo red or thioflavin T. The present compounds suppress the formation of aggregate, and the compound DDX3 is an especially effective inhibitor. In addition, the synthesized compounds efficiently suppress the cytotoxicity of Abeta against IMR-32 neuroblastoma cells in vitro.

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 1987(1), who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

The functions of the helix E (W97-F105), an amphiphilic lumenal 310 helix of the major antenna of photosystem II (LHCII), are still unidentified. To elucidate the roles of individual amino acid residue of the helix E, alanine scanning mutagenesis has been performed to mutate every residue of this domain to alanine. The influence of every alanine substitution on the structure and function of LHCII has been investigated biochemically and spectroscopically. The results show that all mutations have little impact on the pigment binding and configuration. However, many mutants presented decreased thermo- or photo-stability compared with the wild type, highlighting the significance of this helix to the stability of LHCII. The most critical residue for stability is W97. The mutant W97A yielded very fragile trimeric pigment protein complexes. The structural analysis revealed that the hydrogen bonding and aromatic interactions between W97, F195, F194 and a water molecule contributed greatly to the stability of LHCII. Moreover, Q103A and F105A have been identified to be able to reinforce the tendency of aggregation in vitro. The structural analysis suggested that the enhancement in aggregation formation for Q103A and F105A might be attributed to the changing hydrophobicity of the region.

The membrane proteins of peripheral light-harvesting complexes (LHCs) bind chlorophylls and carotenoids and transfer energy to the reaction centers for photosynthesis. LHCs of chlorophytes, chromophytes, dinophytes, and rhodophytes are similar in that they have three transmembrane regions and several highly conserved Chl-binding residues. All LHCs bind Chl a, but in specific taxa certain characteristic pigments accompany Chl a : Chl b and lutein in chlorophytes, Chl c and fucoxanthin in chromophytes, Chl c and peridinin in dinophytes, and zeaxanthin in rhodophytes. The specificity of pigment binding was examined by in vitro reconstitution of various pigments with a simple light-harvesting protein (LHCaR1), from a red alga ( Porphyridium cruentum ), that normally has eight Chl a and four zeaxanthin molecules. The pigments typical of a chlorophyte ( Spinacea oleracea ), a chromophyte ( Thallasiosira fluviatilis ), and a dinophyte ( Prorocentrum micans ) were found to functionally bind to this protein as evidenced by their participation in energy transfer to Chl a, the terminal pigment. This is a demonstration of a functional relatedness of rhodophyte and higher plant LHCs. The results suggest that eight Chl-binding sites per polypeptide are an ancestral trait, and that the flexibility to bind various Chl and carotenoid pigments may have been retained throughout the evolution of LHCs.

The major light-harvesting complex of photosystem II can be reconstituted in vitro from its bacterially expressed apoprotein with chlorophylls a and b and neoxanthin, violaxanthin, lutein, or zeaxanthin as the only xanthophyll. Reconstitution of these one-carotenoid complexes requires low-stringency conditions during complex formation and isolation. Neoxanthin complexes (containing 30–50% of the all-trans isomer) disintegrate during electrophoresis, exhibit a largely reduced resistance against proteolytic attack; in addition, energy transfer from Chl b to Chl a is easily disrupted at elevated temperature. Complexes reconstituted in the presence of either zeaxanthin or lutein contain nearly two xanthophylls per 12 chlorophylls and are more resistant against trypsin. Lutein–LHCIIb also exhibits an intermediate maintenance of energy transfer at higher temperature. Violaxanthin complexes approach a xanthophyll/12 chlorophyll ratio of 3, similar to the ratio in recombinant LHCIIb containing all xanthophylls. On the other hand, violaxanthin–LHCIIb exhibits a low thermal stability like neoxanthin complexes, but an intermediate accessibility towards trypsin, similar to lutein–LHCIIb and zeaxanthin–LHCIIb. Binary competition experiments were performed with two xanthophylls at varying ratios in the reconstitution. Analysis of the xanthophyll contents in the reconstitution products yielded information about relative carotenoid affinities of three assumed binding sites. In lutein/neoxanthin competition experiments, two binding sites showed a strong preference (> 200-fold) for lutein, whereas the third binding site had a higher affinity (25-fold) to neoxanthin. Competition between lutein and violaxanthin gave a similar result, although the specificities were lower: two binding sites have a 36-fold preference for lutein and one has a fivefold preference for violaxanthin. The lowest selectivity was between lutein and zeaxanthin: two binding sites had a fivefold higher affinity for lutein and one has a threefold higher affinity to zeaxanthin.

The peripheral light-harvesting complex of Photosystem I consists of two subpopulations, LHC I-680 and LHC I-730. The latter is composed of the two apoproteins Lhca1 and Lhca4. Recently, reconstitution of monomeric LHC I using bacterially overexpressed Lhca1 or Lhca4 was achieved. In order to obtain insight into the structure requirements for formation of monomeric light-harvesting complexes, we produced a series of N- and C-terminal deletion mutants and used the overexpressed proteins for reconstitution experiments. We found the entire extrinsic N-terminal region dispensable for monomer formation in Lhca1 and Lhca4. Also at the C-terminus, both subunits revealed similarity since all amino acids up to the end of the fourth helix could be removed without abolishing monomer formation. In connection with former corresponding results for Lhcb1, the dispensability of these regions appears to be a general feature in LHC-formation. In LHC I, however, a stabilising effect can be ascribed to these regions since the yield of complexes was decreased. In the majority of the mutant LHC I versions no effect on pigment binding was detected. However, in the LHC with the most extensively N-terminally truncated mutant of Lhca4 a dramatic shift in the 77 K fluorescence emission to shorter wavelengths was observed. This suggests that chlorophylls involved in long wavelength fluorescence emission are located in the chlorophyll array located towards the stromal face of the thylakoid membrane assuming a pigment arrangement corresponding to that in LHC II and CP29.

Four amino acids in the major light-harvesting chlorophyll (Chl) a/b complex (LHCII) that are thought to coordinate Chl molecules have been exchanged with amino acids that presumably cannot bind Chl. Amino acids H68, Q131, Q197, and H212 are positioned in helixes B, C, A, and D, respectively, and, according to the LHCII crystal structure [Kühlbrandt, W., et al. (1994) Nature 367, 614-621], coordinate the Chl molecules named a(5), b(6), a(3), and b(3). Moreover, a double mutant was analyzed carrying exchanges at positions E65 and H68, presumably affecting Chls a(4) and a(5). All mutant proteins could be reconstituted in vitro with pigments, although the thermal stability of the resulting mutant versions of recombinant LHCII varied significantly. All complexes reconstituted with the mutant proteins contained fewer chlorophyll molecules per two lutein molecules than complexes reconstituted with the wild-type protein. However, the chlorophyll-binding amino acids could not be unambiguously assigned to binding either chlorophyll a or b, as in most cases more than one chlorophyll molecule was lost due to the mutation. The changes in Chl stoichiometries suggest that in LHCII some chlorophyll positions can be filled with either Chl a or b. Only some of the point mutations in LHCII affected the ability of the apoprotein to assemble into trimeric LHCII upon insertion into isolated thylakoid membranes. Among these were exchanges of H68 with either F or L, suggesting that the stability of the LHCII trimer significantly depends on this amino acid or the Chl molecule named a(5) that is attached to it and is located close to the center of the trimeric complex. The ion pair bridge between E65 and R185 in LHCII does not appear to be essential for the proper folding of the protein.

The chromophore binding properties of the higher plant light-harvesting complex II have been studied by site-directed mutagenesis of pigment-binding residues. Mutant apoproteins were overexpressed in Escherichia coli and then refolded in vitro with purified chromophores to yield holoproteins selectively affected in chlorophyll-binding sites. Biochemical and spectroscopic characterization showed a specific loss of pigments and absorption spectral forms for each mutant, thus allowing identification of the chromophores bound to most of the binding sites. On these bases a map for the occupancy of individual sites by chlorophyll a and chlorophyll b is proposed. In some cases a single mutation led to the loss of more than one chromophore indicating that four chlorophylls and one xanthophyll could be bound by pigment-pigment interactions. Differential absorption spectroscopy allowed identification of the Q(y) transition energy level for each chlorophyll within the complex. It is shown that not only site selectivity is largely conserved between light-harvesting complex II and CP29 but also the distribution of absorption forms among different protein domains, suggesting conservation of energy transfer pathways within the protein and outward to neighbor subunits of the photosystem.

The chromophore-binding properties of the higher plant light-harvesting protein CP29 have been studied by using site-directed mutagenesis of pigment-binding residues. Overexpression of the apoproteins in bacteria was followed by reconstitution in vitro with purified pigments, thus obtaining a family of mutant CP29 proteins lacking individual chromophore-binding sites. Biochemical characterization allowed identification of the eight porphyrins and two xanthophyll-binding sites. It is shown that the four porphyrin-binding sites (A1, A2, A4, and A5) situated in the central, twofold-symmetrical domain of the protein are selective for Chl-a, whereas the four peripheral sites (A3, B3, B5, and B6) have mixed Chl-a–Chl-b specificity. Within a site, porphyrin coordination by glutamine increases affinity for Chl-b as compared with glutamate. Xanthophyll site L1 is occupied by lutein, whereas site L2 can bind violaxanthin or neoxanthin. The protein is relatively stable when site L2 site is empty, suggesting that xanthophylls can be exchanged during operation of xanthophyll cycle-dependent photoprotection mechanism. Differential absorption spectroscopy allowed determination of transition energy levels for individual chromophores, thus opening the way to calculation of energy-transfer rates between Chl in higher plant antenna proteins.

This paper presents results on the influence of the nonionic surfactant on the properties of vinyl acetate/VeoVa10 ® and vinyl acetate/ethylene emulsions and paints made thereof. Emulsions were prepared in which the concentration of the nonionic surfactant and its degree of ethoxylation were varied. An increase of the nonionic emulsifier concentration and of the length of the ethylene oxide chain leads to dispersions with smaller particles and higher viscosities. Using these emulsions as binders in high pigmented paints, it was observed that the pigment binding capacity of the interior paints goes through a maximum which is located at a emulsifier concentration of about 2–3% and at a degree of ethoxylation of 17–28 mol ethylene oxide (EO). In (semi-) gloss paints, the gloss of the paint films improves with an increase of the emulsifier concentration and reaches a constant value at around 4% or a degree of ethoxylation of ca. 17 EO-moieties per molecule. The blocking of the films shows a drastic increase at a concentration above 4% and at a chain length of greater than 17 EO-moieties.

In the cyanobacterium Synechocystis sp. PCC 6803 five genes were identified with significant sequence similarity to regions of members of the eukaryotic chlorophyll a/b binding gene family (Cab family) and to hliA, a gene coding for a small high-light-induced protein in Synechococcus sp. PCC 7942. Four of these five genes are 174-213 bp in length and code for small proteins predicted to have a single transmembrane helix. The fifth Cab-like gene in Synechocystis sp. PCC 6803 is much longer and codes for a protein of which the N-terminal 80% resemble ferrochelatase but the C-terminal domain has similarity to Cab regions. The small genes were expressed preferentially in the absence of photosystem I, but gene expression was not significantly enhanced at moderately high light intensity. Therefore they were not designated as hli (high-light-induced) as was done for the Synechococcus sp. PCC 7942 homolog. Instead, the genes have been named scp, as the corresponding polypeptides of Synechocystis sp. PCC 6803 are small Cab-like proteins (SCP). The scpA gene, which codes for ferrochelatase with a C-terminal Cab-like extension, was interrupted by the insertion of a kanamycin-resistance cassette between the ferrochelatase and Cab-like gene domains. In the PS I-less background, interruption of scpA was found to lead to increased tolerance to high light intensity and to the requirement of a slightly higher light intensity to drive photosystem II electron transfer, suggestive of decreased light-harvesting efficiency in the absence of the C-terminal extension of ScpA. Immunodetection of ScpC and ScpD indicated that either or both accumulated in PS I-less strains. These proteins were also detected in bands of more than 45 kDa on denaturing gels, raising the possibility that they may occur as stable oligomers. The SCPs represent a new group of cyanobacterial proteins that, in view of their primary structure and response to deletion of photosystem I, are likely to be involved in transient pigment binding.

The results of low temperature absorption, fluorescence and hole burning spectroscopy of pigment binding proteins in normal-grown and iron-stressed thermophilic Synecococcus sp. are reported. These experiments revealed that the growing of Synecococcus sp. under the iron-limited condition affects spectral characteristics and excited energy transfer (EET) in pigment proteins. The comparison of low temperature absorption spectra of normal-grown and iron-stressed thermophilic cyanobacteria well documents major changes in composition of the antenna systems and in composition of the core of photosystem II. The absorption of membrane chlorophyll in particular, is blue-shifted from 679 nm in normal cells to 673 nm which is caused by absorption of chlorophyll binding protein CP 34 in stressed sample. The presence of CP 34 in the photosystem II (PS 1 II) has also been seen in the low temperature fluorescence spectra where the increased luminescence at 685 nm has been observed. This implies the decreased efficiency of photosynthesis in iron-limited sample. We studied the energy transfer in PS II by the means of fluorescence and absorption hole-burning spectroscopy which enabled to study the influence of iron-stressed condition on energy transfer and pigment–protein interaction (Debye–Waller factor). The stressed cells exhibit the broadened spectral hole width at 682 and 685.5 nm.

The early light-inducible proteins (ELIPs) in chloroplasts possess a high sequence homology with the chlorophyll a/b-binding proteins but differ from those proteins by their substoichiometric and transient appearance. In the present study ELIPs of pea were isolated by a two-step purification strategy: perfusion chromatography in combination with preparative isoelectric focussing. Two heterogeneous populations of ELIPs were obtained after chromatographic separation of solubilized thylakoid membranes using a weak anion exchange column. One of these populations contained ELIPs in a free form providing the first isolation of these proteins. To prove whether the isolated and pure forms of ELIP bind pigments, spectroscopic and chromatographic analysis were performed. Absorption spectra and TLC revealed the presence of chlorophyll a and lutein. Measurements of steady-state fluorescence emission spectra at 77 K exhibited a major peak at 674 nm typical for chlorophyll a bound to the protein matrix. The action spectrum of the fluorescence emission measured at 674 nm showed several peaks originating mainly from chlorophyll a. It is proposed that ELIPs are transient chlorophyll-binding proteins not involved in light-harvesting but functioning as scavengers for chlorophyll molecules during turnover of pigment-binding proteins.

We quantified the expression of the puf and puc operons, which encode pigment binding proteins, in a number of strains of Rhodobacter capsulatus that have defects in genes affecting different steps of bacteriochlorophyll biosynthesis. Our results show that these mutations have a very similar effect on puf and puc expression. This suggests that the reduced expression of genes encoding pigment-binding proteins is due neither to the accumulation of a specific intermediate of bacteriochlorophyll synthesis nor to the lack of an early intermediate, but is rather the result of the lack of a very late intermediate or the bacteriochlorophyll molecule itself.

The formation of the photosynthetic apparatus in facultatively photosynthetic bacteria is mainly regulated by oxygen partial pressure in the environment. Rhodobacter capsulatus cells contain low levels of pigment protein complexes under aerobic conditions, and energy is generated by aerobic respiration. A reduction of the oxygen partial pressure strongly induces the formation of pigments and pigment binding proteins, and an intracytoplasmic membrane system develops (for a review, see reference 12). Many investigations have addressed the effect of oxygen on transcription of genes encoding pigment binding proteins and the enzymes that catalyze pigment synthesis. Several trans-acting factors have been identified that activate the transcription of photosynthesis genes under low oxygen tension or function as repressors under high oxygen tension (reviewed in references 3 and 4). The puf operon (Fig. 1A) encodes proteins of light-harvesting complex I (LHI complex) (genes pufB and pufA) and of the reaction center (genes pufL and pufM) and the proteins PufQ, which is involved in regulation of bacteriochlorophyll synthesis (1), and PufX, which most likely assists in organizing the bacterial photosystem for efficient transduction of light energy (15). The non-pigment binding protein of the reaction center is encoded by the puhA gene. The puc operon encodes proteins involved in the formation of the LHII antenna complex. The levels of puf and puc mRNA are determined not only by the rate of transcription but also by the rate of mRNA decay. Individual segments of the polycistronic puf operon mRNA exhibit different stabilities, leading to differential expression of puf-encoded genes (5, 22). The pufBALMX segment decays at a higher rate under high oxygen tension than under low oxygen tension (23). The molecular mechanism responsible for this oxygen effect has not yet been elucidated. In order to systematically study the effect of oxygen on posttranscriptional steps in gene expression, we have compared the puf and puc mRNA levels and the rate of synthesis of the corresponding proteins. Correlation of mRNA levels and rates of protein synthesis and incorporation. If the expression of puf and puc mRNAs were solely regulated on the levels of transcription and mRNA stability, the rates of synthesis of individual puf and puc proteins would strictly correlate with the mRNA amounts. We quantified the levels of puf and puc mRNAs after the transition of R. capsulatus 37b4 (DSM936) grown in minimal malate salt medium (10) from growth under high oxygen (20% [monitored by an Ag-Pt electrode]) to that under low oxygen (1 to 2%) with Northern blots obtained by using a phosphoimager. Cultures (100 ml) were grown in baffled flasks under vigorous agitation to an optical density (at 660 nm) of 0.4 to 0.6. Aliquots (4 to 5 ml) of these cultures were then transferred into a small vessel and further incubated at 32°C. Due to the respiration of the bacteria, the oxygen tension dropped to 1 to 2% within 3 to 5 min. The oxygen tension was then adjusted to 1 to 2% by supplying air and stirring the culture and was monitored throughout the experiment. In order to determine the rates of protein synthesis, the cultures that had been grown with low oxygen for different times after the shift were pulse-labeled with 40 mCi of L-[ 35 S]methionine (Amersham) for 3 min.

The puf and puc operons of Rhodobacter capsulatus, which encode pigment binding proteins, are regulated by oxygen tension. Strain IR4 which carries a point mutation in the himA gene for integration host factor (IHF) shows a delayed increase in the amount of photosynthetic complexes after reduction of oxygen tension when compared to the isogenic wild-type strain. This delay in the formation of photosynthetic complexes correlates with a delay in increasing the level of transcription of the puf and puc operons, whereas the transcription of the bchD genes is not affected by IHF. Several IHF binding sites can be identified upstream of the puf and puc transcriptional starts by in vitro footprint analysis. Our results suggest that IHF facilitates the binding of other trans-acting factors, e.g. RegA, which in turn stimulate transcription of the puf and puc genes under low oxygen tension.

In our previous study by means of circular dichroism (CD) spectroscopy we have shown that the pigment organisation in the chlorophyll a/c light harvesting complex (Chl a/c LHC) isolated from Pleurochloris meiringensis significantly differs from the architecture of the main Chl a/b light harvesting antenna complexes of higher plants (Buchel and Garab. J. Photochem. Photobiol., B: Biol., 37 (1997) 118–124). In this work we measured the CD spectra in the far-UV, between 190 nm and 240 nm. and investigated the variations of the secondary structure of the protein upon treatments which affect the binding of the long wavelength Chl a. We found that low concentrations (

In the silkworm Bombyx mori, pigment mutants with diverse body colors have been maintained throughout domestication for about 5000 years. The silkworm larval body color is formed through the mutual interaction of melanin, ommochromes, pteridines and uric acid. These pigments/compounds are synthesized by the cooperative action of various genes and enzymes. Previous reports showed that melanin, ommochrome and pteridine are increased in silkworm quail (q) mutants. To understand the pigment increase and alterations in pigment synthesis in q mutant, transcriptome profiles of the silkworm integument were investigated at 16 h after head capsule slippage in the fourth molt in q mutants and wild-type (Dazao). Compared to the wild-type, 1161 genes were differentially expressed in the q mutant. Of these modulated genes, 62.4% (725 genes) were upregulated and 37.6% (436 genes) were downregulated in the q mutant. The molecular function of differently expressed genes was analyzed by Blast2GO. The results showed that upregulated genes were mainly involved in protein binding, small molecule binding, transferase activity, nucleic acid binding, specific DNA-binding transcription factor activity and chromatin binding, while exclusively down-expressed genes functioned in oxidoreductase activity, cofactor binding, tetrapyrrole binding, peroxidase activity and pigment binding. We focused on genes related to melanin, pteridine and ommochrome biosynthesis; transport of uric acid; and juvenile hormone metabolism because of their importance in integument coloration during molting. This study identified differently expressed genes implicated in silkworm integument formation and pigmentation using silkworm q mutant. The results estimated the number and types of genes that drive new integument formation.

Comparison of the absorption maxima with the hydrogen bonding of the C2 acetyl groups for various mutants and wild-type bacterial antenna complexes has demonstrated a role for this molecular interaction in the tuning of the absorption properties of these complexes. There is a consistent linear relationship, between the downshift in the bacteriochlorophyll a C2 acetyl stretching mode and the red shift in the absorption maximum, in both core and peripheral antenna complexes. This linear relationship allows us to estimate the contribution of H-bonding to the red shifts of these complexes and the sensitivity of the absorption to changes in the C2 acetyl group environment. Investigation of changes in the Raman spectra caused by altering the excitation conditions reveal either no or only minor changes in the positions of the methine bridge, the C2 acetyl, and the C9 keto stretching modes. We interpret the lack of sensitivity of these bands to the resonance condition as indicating that neither differences in rin...

Summary The pigment composition of the chlorophyll binding proteins of Photosystem II (PS II) of spinach ( Spinacea oleracea L.) has been determined using sucrose gradient ultracentrifugation, anion exchange chromatography and HPLC based pigment analysis. The xanthophyll cycle pigments violaxanthin, antheraxanthin and zeaxanthin were exclusively found in the proteins of the outer PS II antenna, with the highest amounts being present in the minor chlorophyll alb binding proteins CP 29 and CP 26. PS II core particles containing the reaction centre proteins D1, D2, cytochrome b 559 and the proteins of the inner antenna CP 47 and CP 43 bind β-carotene as the only carotenoid. The presence of the xanthophyll cycle pigments in the PS II antenna proteins gives further indication to their proposed role in the dissipation of excess excitation energy. The purification procedure presented in this study differs from previous isolation methods (i. e. isoelectric focusing) used to determine the pigment composition of the PS II proteins. The pigment stoichiometries shown here therefore provide further insight in the pigmentation of the PS II chlorophyll binding complexes.

This book is another valuable contribution to the growing library of volumes in the Springer series ‘Advances in Photosynthesis and Respiration’ (almost 40 volumes and counting). While much of the present material has been covered in previous volumes, The Chloroplast seeks to take a unique approach. In the words of the editors, ‘this volume on the chloroplast is meant to provide the basis for chloroplast bioengineering and foster closer cooperation between scientists [in multiple disciplines] … and further the implementation of more efficient photosynthesis’ (Preface, p. xxviii). In doing so, the volume focuses on the bioengineering of improved photosynthetic efficiency as a method to increase agricultural plant productivity, improve biofuel production, and even support extra-terrestrial space colonization. The volume's contents can be arranged into several categories, as follows. Chapter 1, as befitting its place, lays out the possible sources for the mismatch between the theoretical photosynthetic efficiency of 12 % and the actual measured efficiency of less than half a percent. It is that gap to which the volume editors seek to direct the attention of the reader and the researcher. Chapters 2–5 cover chlorophyll biosynthetic pathways, including synthesis of the vinyl side-chain, tetrapyrrole synthesis, interconversion of Chl a and b, and Mg2+ insertion. Chapter 6, an interesting, four-page piece titled, ‘The enigmatic chlorophyll a molecule in the cytochrome b6/f complex’, finds a home in this section. Chapters 7–10 focus on isoprenoids: their biosynthesis and connection to the isoprenoid-derived pigments (Chapter 7); the regulatory role of the MEP pathway in chloroplast isoprenoid production (Chapter 8); the role of protein geranylgeranylation in cellular metabolic control (Chapter 9); and the emission of a wide range of volatile terpenoid compounds from flowers and leaves of numerous plants (Chapter 10). A comprehensive treatment of chloroplast lipids follows. Chapter 11 concerns engineering plastids for enhanced tocochromanols (vitamin E) production. The biosynthesis, function and importance of the four major chloroplast structural lipids are next covered in sequence: phosphatidylglycerol and sulfoquinovosylidiacylglycerol (Chapter 12), monogalactosyldiacylglycerol (Chapter 13) and digalactosyldiacylglycerol (Chapter 14). The light-harvesting Chl-protein complex of photosystem II (LHCII) is treated in Chapter 15 (chemistry and biology) and Chapter 16 (folding and pigment binding). Both chapters do a fine job of bringing together structural and functional data on this important pigment–protein complex. The heart of the subject matter that is the most pertinent to the research questions set out in the Preface and Chapter 1 – namely, chloroplast genomics and genetic engineering – is addressed in Chapters 17–20. This core portion of the volume begins with Chapter 17, a technical paper on increasing the resistance of plants and plant products to microbial attack. Up to 25 % of food is lost post-harvest, much of it through the action of food-degrading microbes. Engineering the plastic genome may hold the key to the introduction of disease-resistance genes and add an important layer of protection in the food production supply chain. Chapter 18 also focuses on chloroplast genetic engineering. The authors provide a review of available methods of plastid transformation and give examples of the successful production of chloroplast-derived vaccine antigens, biopharmaceutical proteins and industrially valuable biomaterials. Chapter 19 is a comprehensive case study of transforming tobacco chloroplasts with the sunflower Rubisco large and small subunits. Data are presented ranging from initial plastid transformation to eventual whole-leaf gas exchange measurements in mature Nicotiana leaves. The study identifies both hurdles encountered and achievements possible using this emerging experimental system. Chapter 20 is perhaps the signature chapter of the volume as it speaks directly to genetically engineering photosynthetic enzymes for the enhancement of photosynthetic efficiency. Gene shuffling of Chlamydomonas reinhardtii Rubisco genes was used to drive directed molecular evolution of increased carboxylase activity or CO2/O2 specificity. The experimental technique was extended to A. thaliana and generated several Rubisco variants with increased thermostability. The take-home message from these four chapters that focus on chloroplast genetic engineering is quite encouraging. Finally, Chapters 21–25 expand the volume's coverage to include consideration of the chloroplast response in the face of environmental factors such as elevated CO2, elevated O3 and abiotic stress. While the individual chapters are of a high standard, and the authors of well-earned international reputation, many of the contributions drifted significantly from the volume's stated goal of fully exploring how to bioengineer increased photosynthetic efficiency into chloroplasts in order to enhance food and biofuels production. The book is somewhat heavy on chloroplast basics (information that although well done, is available elsewhere in the literature), at the expense of a rather limited coverage of chloroplast applications. Beginning students will gain much from the review material and advanced researchers will find the chapters on direct applications to be very useful in guiding their thinking and future experiments. In terms of presentation of the material, the inclusion of colour figures in the text, rather than collected at the front of the book as has been the norm with this series, is a definite improvement in the readability of the text.