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1.
Biochemistry ; 43(51): 16487-96, 2004 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-15610043

RESUMO

A simple reconstitution technique has been developed and then applied to prepare a series of light-harvesting antenna 1 (LH1) complexes with a programmed carotenoid composition, not available from native photosynthetic membranes. The complexes were reconstituted with different C(40) carotenoids, having two structural parameters variable: the functional side groups and the number of conjugated C-C double bonds, systematically increasing from 9 to 13. The complexes, differing only in the type of carotenoid, bound to an otherwise identical bacteriochlorophyll-polypeptide matrix, can serve as a unique model system in which the relationship between the carotenoid character and the functioning of pigment-protein complexes can be investigated. The reconstituted LH1 complexes resemble the native antenna, isolated from wild-type Rhodospirillum rubrum, but their coloration is entirely determined by carotenoid. Along with the increase in its conjugation size, the carotenoid absorption transitions gradually shift to the red. Thus, the extension of the conjugation size of the antenna carotenoids provides a mechanism for the spectral tuning of light harvesting in the visible part of the spectrum. The carotenoids in the reconstitution system promote the LH1 formation and seem to bind and transfer the excitation energy specifically only to a species with characteristically red-shifted absorption and emission maxima, apparently, due to a cooperative effect. Monitoring the LH1 formation by steady-state absorption and fluorescence spectroscopies reveals that in the presence of carotenoids it proceeds without spectrally resolved intermediates, leading directly to B880. The effect of the carotenoid is enhanced when the pigment contains the hydroxy or methoxy side groups, implying that, in parallel to hydrophobic interactions and pi-pi stacking, other interactions are also involved in the formation and stabilization of LH1.


Assuntos
Proteínas de Bactérias/metabolismo , Carotenoides/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Xantofilas/análogos & derivados , Proteínas de Bactérias/química , Dicroísmo Circular , Complexos de Proteínas Captadores de Luz/química , Rhodospirillum rubrum/metabolismo , Espectrometria de Fluorescência , Espectrofotometria , Xantofilas/metabolismo
2.
Biopolymers ; 74(1-2): 2-18, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15137086

RESUMO

This minireview article highlights the energetics and the dynamics of the 1(1)B(u)(-) and 3(1)A(g)(-) states of carotenoids discovered very recently. Those "hidden" covalent states have been revealed by measurements of resonance-Raman excitation profiles of crystalline carotenoids. The dependence of the energies of the low-lying singlet states, including the 1(1)B(u)(+), 3(1)A(g)(-), 1(1)B(u)(-), and 2(1)A(g)(-) states, on the number of conjugated double bonds (n) is in agreement with the extrapolation of those state energies calculated by Tavan and Schulten for shorter polyenes (P. Tavan and K. Schulten, Journal of Chemical Physics, 1986, vol. 85, pp. 6602-6609). It has also been shown that the internal-conversion processes among those singlet states take place in accord with the state ordering, i.e., 1(1)B(u)(+) --> 1(1)B(u)(-) --> 2(1)A(g)(-) --> 1(1)A(g)(-) (the ground state) for carotenoids having n = 9 and 10, whereas 1(1)B(u)(+) --> 3(1)A(g)(-) --> 1(1)B(u) (-) --> 2(1)A(g)(-) --> 1(1)A(g)(-) for carotenoids having n = 11-13. Radiative transitions of 1(1)B(u)(+) --> 2(1)A(g)(-) and 1(1)B(u)(-) --> 2(1)A(g)(-) as well as a branching into the triplet manifold of 1(1)B(u)(-) --> 1(3)A(g) --> 1(3)B(u) have also been found. Those low-lying singlet states of all-trans carotenoids can facilitate multiple channels of singlet-energy transfer to bacteriochlorophyll in the LH2 antenna complexes of purple photosynthetic bacteria. Thus, the newly found 1(1)B(u)(-) and 3(1)A(g)(-) states of carotenoids need to be incorporated into the picture of carotenoid-to-bacteriochlorophyll singlet-energy transfer.


Assuntos
Carotenoides/química , Fotossíntese , Xantofilas/análogos & derivados , Bacterioclorofilas/química , Biopolímeros/química , Carotenoides/farmacologia , Cinética , Luz , Licopeno , Modelos Químicos , Polienos/química , Proteobactérias/metabolismo , Espectrofotometria , Espectrofotometria Infravermelho , Análise Espectral Raman , Fatores de Tempo , Xantofilas/farmacologia
3.
J Biol Chem ; 279(15): 15076-83, 2004 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-14734565

RESUMO

Aerobic photosynthetic bacteria possess the unusual characteristic of producing different classes of carotenoids. In this study, we demonstrate the presence of two distinct crt gene clusters involved in the synthesis of spirilloxanthin and canthaxanthin in a Bradyrhizobium strain. Each cluster contains the genes crtE, crtB, and crtI leading to the common precursor lycopene. We show that spirilloxanthin is associated with the photosynthetic complexes, while canthaxanthin protects the bacteria from oxidative stress. Only the spirilloxanthin crt genes are regulated by light via the control of a bacteriophytochrome. Despite this difference in regulation, the biosyntheses of both carotenoids are strongly interconnected at the level of the common precursors. Phylogenetic analysis suggests that the canthaxanthin crt gene cluster has been acquired by a lateral gene transfer. This acquisition may constitute a major selective advantage for this class of bacteria, which photosynthesize only under conditions where harmful reactive oxygen species are generated.


Assuntos
Bradyrhizobium/genética , Cantaxantina/biossíntese , Carotenoides/genética , Família Multigênica , Xantofilas/análogos & derivados , Xantofilas/biossíntese , Cantaxantina/química , Carotenoides/química , Carotenoides/metabolismo , Membrana Celular/metabolismo , Relação Dose-Resposta a Droga , Luz , Licopeno , Modelos Químicos , Dados de Sequência Molecular , Mutação , Estresse Oxidativo , Paraquat/farmacologia , Filogenia , Pigmentos Biológicos , Espécies Reativas de Oxigênio , Espectrometria de Fluorescência
4.
Arch Biochem Biophys ; 414(1): 51-8, 2003 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-12745254

RESUMO

The carotenoid 1,2-hydratase CrtC from Rubrivivax gelatinosus has been expressed in Escherichia coli in an active form and purified by affinity chromatography. The enzyme catalyzes the conversion of various acyclic carotenes including 1-hydroxy derivatives. This broad substrate specificity reflects the participation of CrtC in 1'-HO-spheroidene and in spirilloxanthin biosynthesis. Enzyme kinetic studies including the determination of substrate specificity constants indicate that among the alternative biosynthetic routes to 1'-HO-spheroidene the one via spheroidene is the dominating pathway. In contrast to CrtC from Rvi. gelatinosus, the equivalent enzyme from Rhodobacter capsulatus, a closely related bacterium which lacks the biosynthetic branch to spirilloxanthin and accumulates spheroidene instead of substantial amounts of 1'-HO-spheroidene, is extremely poor in converting 1-HO-carotenoids. The individual catalytic properties of both carotenoid 1,2-hydratases reflect the in situ carotenogenic pathways in both purple photosynthetic bacteria.


Assuntos
Hidroliases/química , Hidroliases/metabolismo , Proteobactérias/enzimologia , Rhodobacter capsulatus/enzimologia , Xantofilas/análogos & derivados , Carotenoides/biossíntese , Carotenoides/química , Carotenoides/isolamento & purificação , Cromatografia de Afinidade/métodos , Ativação Enzimática , Escherichia coli/química , Escherichia coli/enzimologia , Escherichia coli/genética , Hidroliases/classificação , Hidroliases/isolamento & purificação , Cinética , Proteobactérias/química , Proteobactérias/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Rhodobacter capsulatus/química , Rhodobacter capsulatus/genética , Especificidade da Espécie , Especificidade por Substrato , Xantofilas/biossíntese
5.
Proc Natl Acad Sci U S A ; 98(5): 2364-9, 2001 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-11226245

RESUMO

Carotenoids are important biomolecules that are ubiquitous in nature and find widespread application in medicine. In photosynthesis, they have a large role in light harvesting (LH) and photoprotection. They exert their LH function by donating their excited singlet state to nearby (bacterio)chlorophyll molecules. In photosynthetic bacteria, the efficiency of this energy transfer process can be as low as 30%. Here, we present evidence that an unusual pathway of excited state relaxation in carotenoids underlies this poor LH function, by which carotenoid triplet states are generated directly from carotenoid singlet states. This pathway, operative on a femtosecond and picosecond timescale, involves an intermediate state, which we identify as a new, hitherto uncharacterized carotenoid singlet excited state. In LH complex-bound carotenoids, this state is the precursor on the reaction pathway to the triplet state, whereas in extracted carotenoids in solution, this state returns to the singlet ground state without forming any triplets. We discuss the possible identity of this excited state and argue that fission of the singlet state into a pair of triplet states on individual carotenoid molecules constitutes the mechanism by which the triplets are generated. This is, to our knowledge, the first ever direct observation of a singlet-to-triplet conversion process on an ultrafast timescale in a photosynthetic antenna.


Assuntos
Carotenoides/análogos & derivados , Carotenoides/metabolismo , Fotossíntese , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Xantofilas/análogos & derivados , Cinética , Rhodospirillum rubrum/metabolismo , Análise Espectral/métodos
6.
Int J Syst Evol Microbiol ; 50 Pt 4: 1441-1447, 2000 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10939648

RESUMO

A new phototrophic purple sulfur bacterium, isolated from benthic microbial mats from the White Sea littoral zone, is described. Individual cells were spherical, non-motile and lacked gas vesicles. In pure cultures cells appeared in regular platelet-like arrangements of four, eight or sixteen cells. Cell division occurred inside a common envelope, surrounded by a thick capsule. Internal photosynthetic membranes were of the vesicular type. The colour of cell suspensions was pink to rose-red. Bacteriochlorophyll a and carotenoids of the spirilloxanthin series were found as photosynthetic pigments. Under anoxic conditions in the light, photolithoautotrophic growth occurred with sulfide, thiosulfate, sulfite and elemental sulfur as electron donors. Sulfur globules were stored as an intermediary oxidation product and were visible microscopically inside the cells. In the presence of sulfide and bicarbonate, photomixotrophic growth occurred with a number of organic substrates. Sulfate could serve as sole assimilatory source of sulfur. Chemolithoautotrophic growth in the dark was possible with sulfide and thiosulfate as electron donors. Optimum growth occurred in the presence of 1% NaCl, at pH 6.5 and at 30 degrees C. The DNA base composition of the type strain, BM5T, was 64.0 mol% G+C. According to 16S rDNA sequence information and DNA-DNA hybridization, the new isolate clearly belongs to the genus Thiocapsa, but is sufficiently different from other recognized Thiocapsa species to be described as a new species of this genus for which the name Thiocapsa litoralis sp. nov. is proposed. The type strain is BM5T (= ATCC 700894).


Assuntos
Thiocapsa/classificação , Microbiologia da Água , Xantofilas/análogos & derivados , Bacterioclorofilas/análise , Composição de Bases , Bicarbonatos/química , Carotenoides/análogos & derivados , Carotenoides/análise , Meios de Cultura , Escuridão , Concentração de Íons de Hidrogênio , Luz , Dados de Sequência Molecular , Cloreto de Sódio/química , Sulfetos/química , Sulfitos/química , Temperatura , Thiocapsa/química , Thiocapsa/fisiologia , Tiossulfatos/química
7.
Biochem J ; 349(Pt 2): 635-40, 2000 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-10880364

RESUMO

Carotenoid biosynthesis in the photosynthetic bacterium Rubrivivax gelatinosus leads to the formation of hydroxyspheroidene and spirilloxanthin as the products of a branched pathway. In this study we investigated the role of the desaturase encoded by crtD which catalyses the introduction of C-3,4 double bonds into acyclic carotenoids. The desaturase was expressed in Escherichia coli, and the activity and the substrate specificity of the enzyme were evaluated in vitro by application of structurally different carotenoids. The results indicate that the enzyme is a 3,4-desaturase that converts 1-hydroxy carotenoids. The 3,4-desaturation reaction can only occur with mono-1-hydroxy carotenoids at a psi-end group or with 1,1'-dihydroxy derivatives carrying a 3',4'-double bond. In addition, 1-HO-zeta-carotene could also be converted by the desaturase. Enzyme kinetic studies showed a substrate preference of 1-HO-neurosporene over 1-HO-lycopene. Consequences from the biochemical data for the reaction sequence of hydroxyspheroidene and spirilloxanthin formation and the interconnection of both branches are discussed.


Assuntos
Carotenoides/análogos & derivados , Carotenoides/biossíntese , Oxirredutases/metabolismo , Proteobactérias/enzimologia , Xantofilas/análogos & derivados , Carotenoides/metabolismo , Escherichia coli , Especificidade por Substrato
8.
Biochemistry ; 37(25): 8987-94, 1998 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-9636041

RESUMO

The carotenoids accumulated by a mutant Rhodospirillum rubrum ST4, containing a single Tn5 lesion in the pathway for carotenoid biosynthesis, were analyzed by HPLC, 1H NMR spectroscopy, and field desorption mass spectrometry. The main carotenoid was identified as 3,4,3',4'-tetrahydrospirilloxanthin, and the four minor carotenoids were identified as rhodopin, 3,4-dihydroanhydrorhodovibrin, 3', 4'-dihydrorhodovibrin, and 1,1'-dihydroxylycopene. The C-3,4 and C-3',4' bonds of all 5 carotenoids are saturated, and they have 11 conjugated double bonds. With the exception of rhodopin, which is a normal intermediate of the wild-type pathway, all of the carotenoids are not naturally occurring. The Tn5 lesion was assigned to rhodopin 3,4-desaturase which is proposed to catalyze dehydrogenation at both ends of the symmetrical spirilloxanthin derivative. An unexpected finding was that the enzymes following rhodopin 3,4-desaturase are still able to end-modify the 3,4-, and 3',4'-saturated precursors and that the order of methylation and hydroxylation is not obligatory. It is proposed that the observed nonnatural carotenoids can be explained by the inclusion of a cryptic branch, unmasked by the absence of rhodopin 3,4-desaturase, in the established linear pathway for spirilloxanthin biosynthesis. This is the first example of latent branching of the carotenoid biosynthesis pathway exhibited by a carotenoid mutant of a phototrophic bacterium.


Assuntos
Carotenoides/biossíntese , Carotenoides/genética , Oxirredutases/genética , Rhodospirillum rubrum/enzimologia , Xantofilas/análogos & derivados , Carotenoides/análogos & derivados , Carotenoides/análise , Mutagênese , Oxirredutases/metabolismo , Fenótipo , Rhodospirillum rubrum/genética , Especificidade por Substrato
9.
Biophys J ; 61(6): 1462-9, 1992 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-1617133

RESUMO

The apparent quantum yield of singlet-singlet spirilloxanthin-to-bacteriochlorophyll a energy transfer increases linearly with the residual spirilloxanthin content in Rhodospirillum rubrum membrane vesicles from which this carotenoid has been partially removed. Since it has been previously shown that carotenoid-carotenoid interaction is a linear function of the residual spirilloxanthin level in the major pigment-protein complex of those vesicles (Zurdo, J., R. M. Lozano, C. Fernandez-Cabrera, and J. M. Ramirez. 1991. Biochem. J. 274:881-884), it appears that such degenerate interaction enhances singlet energy transfer. Part of the enhancement may be explained if the energy donor is the spirilloxanthin 1Bu----1Ag (S2----S0) transition, because exciton coupling probably brings its energy closer to that of the Qx (S2----S0) transition of bacteriochlorophyll. In contrast, it seems that the possible stabilization of the spirilloxanthin 2Ag (S1) state would hardly improve energy transfer, because this hidden state probably lies below the S1 bacteriochlorophyll state. In any case, the stabilizing effects of carotenoid-carotenoid interactions seem insufficient to explain the enhancement of energy transfer. Direct or indirect effects of carotenoid dimerization on the three-dimensional structure of the pigment cluster appear to be required to account for such enhancement.


Assuntos
Bacterioclorofilas/metabolismo , Carotenoides/análogos & derivados , Carotenoides/metabolismo , Rhodospirillum rubrum/metabolismo , Xantofilas/análogos & derivados , Transferência de Energia
10.
J Biol Chem ; 263(9): 4374-80, 1988 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-3126188

RESUMO

Resonance Raman scattering by the carotenoid, spirilloxanthin (Spx), in a suspension of chromatophores (cytoplasmic side out) isolated from the photosynthetic bacterium, Rhodospirillum rubrum, is greatly enhanced when the membranes are adsorbed onto the surface of an anodized Ag electrode. The phenomenon is the basis for surface-enhanced resonance Raman scattering (SERRS) spectroscopy. The Spx SERRS peaks observed were at 1505-1510, 1150-1155, and 1000-1005 cm-1 with laser excitation wavelengths ranging between 457.9 and 568.2 nm. Similar peaks were not observed with spheroplasts (periplasmic side out) isolated from the same species. The difference in signal detected in chromatophores and spheroplasts is not due to differences in membrane surface charge, presence of residual cell wall on the spheroplast surface, lack of adhesion of spheroplasts to metals, or large differences in pigment content per unit membrane area. Instead, the results indicate an asymmetric distribution of Spx in vivo across the membrane (i.e., it is located on the cytoplasmic side of the membrane). The results also demonstrate that the SERRS effect is extremely distance sensitive, and the thickness of a single bacterial membrane (separating the Ag electrode from the carotenoid) is sufficient to prevent detection of Spx spectra. Studies of chromatophores from the F24 strain (a reaction centerless mutant) have pin-pointed B880 antenna complex as the source of the Spx SERRS spectra, and a schematic model of the minimal structural unit of B880 is presented. This work demonstrates the potential of the SERRS technique as a probe for surface topology of pigmented membranes.


Assuntos
Carotenoides/análogos & derivados , Fotossíntese , Rhodospirillum rubrum/análise , Xantofilas/análogos & derivados , Microscopia Eletrônica , Modelos Moleculares , Análise Espectral Raman
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