spirilloxanthin and spheroidene

To explore the photoprotection role of multicompositional carotenoid (Car) in photosynthetic purple bacteria, we investigated, by means of triplet excitation profile (TEP) combined with steady-state optical spectroscopies, the core light-harvesting complex-reaction center of a mutant strain of Rhodobacter sphaeroides (m-LH1-RC) at room temperature. TEP spectra revealed that spheroidene and derivative (Spe) preferentially protect bacteriochlorophylls (BChls) of relatively lower site energy by quenching the triplet excitation (

Topics: Bacterial Proteins; Carotenoids; Chromatiaceae; Light; Light-Harvesting Protein Complexes; Rhodobacter sphaeroides; Spectrum Analysis; Xanthophylls

The reaction centre-light harvesting 1 (RC-LH1) complex of Thermochromatium (Tch.) tepidum has a unique calcium-ion binding site that enhances thermal stability and red-shifts the absorption of LH1 from 880nm to 915nm in the presence of calcium-ions. The LH1 antenna of mesophilic species of phototrophic bacteria such as Rhodobacter (Rba.) sphaeroides does not possess such properties. We have engineered calcium-ion binding into the LH1 antenna of Rba. sphaeroides by progressively modifying the native LH1 polypeptides with sequences from Tch. tepidum. We show that acquisition of the C-terminal domains from LH1 α and β of Tch. tepidum is sufficient to activate calcium-ion binding and the extent of red-shifting increases with the proportion of Tch. tepidum sequence incorporated. However, full exchange of the LH1 polypeptides with those of Tch. tepidum results in misassembled core complexes. Isolated α and β polypeptides from our most successful mutant were reconstituted in vitro with BChl a to form an LH1-type complex, which was stabilised 3-fold by calcium-ions. Additionally, carotenoid specificity was changed from spheroidene found in Rba. sphaeroides to spirilloxanthin found in Tch. tepidum, with the latter enhancing in vitro formation of LH1. These data show that the C-terminal LH1 α/β domains of Tch. tepidum behave autonomously, and are able to transmit calcium-ion induced conformational changes to BChls bound to the rest of a foreign antenna complex. Thus, elements of foreign antenna complexes, such as calcium-ion binding and blue/red switching of absorption, can be ported into Rhodobacter sphaeroides using careful design processes.

Topics: Amino Acid Sequence; Bacterial Proteins; Binding Sites; Calcium; Carotenoids; Cations, Divalent; Chromatiaceae; Gene Expression; Genetic Engineering; Mutant Chimeric Proteins; Photosynthetic Reaction Center Complex Proteins; Protein Binding; Rhodobacter sphaeroides; Sequence Alignment; Sequence Homology, Amino Acid; Xanthophylls

Effect of illumination intensity and inhibition of carotenoid biosynthesis on assemblage of different spectral types of LH2 complexes in a purple sulfur bacterium Allochromatium (Alc.) vinosum ATCC 17899 was studied. Under illumination of 1200 and 500 lx, the complexes B800-850 and B800-840 and B800-820 were assembled. While rhodopine was the major carotenoid in all spectral types of the LH2 complex, a certain- increase in the content of carotenoids with higher numbers of conjugated double bonds (anhydrorhodovibrin and didehydrorhodovibrin) was observed in the B800-820 complex. At 1200 lx, the cells grew slowly at diphe- nylamine (DPA) concentrations not exceeding 53 .iM, while at illumination intensity decreased to 500 Ix they could grow at 71 jiM DPA (DPA cells). Independent on illumination level, the inhibitor is supposed to impair the functioning of phytoine synthetase (resulting in a decrease in the total carotenoid content) and of phyto- ine desturase, which results in formation of neurosporene hydroxy derivatives and ;-carotene. In the cells grown at 500 lx, small amounts of spheroidene and.OH-spheroidene were detected. These carotenoids were originally found under conditions of carotenoid synthesis inhibition in bacteria with spirilloxanthin as the major carotenoid. Carotenoid content in the LH2 complexes isolated from the DPA cells was -15% of the control (without inhibition) for the B800-850 and -20%of the control for the B800-820 and B800-840 DPA complexes. Compared to the DPA pigment-containing membranes, the DPA complexes were enriched with -carotenoids due to- disintegration of some carotenoid-free complexes in the course of isolation. These results support the supposition that some of the B800-820, B800-840, and B800-850 complexes may be Assembled in the cells of Alc. vinosum ATCC 17899 without carotenoids. Comparison of the characteristics obtained for Alc. vinosum ATCC 17899 and the literature data on strain D of the same bacteria shows that they belong to two different strains, rather than to one as was previously supposed.

Topics: Bacterial Proteins; Carotenoids; Chromatiaceae; Culture Media; Diphenylamine; Dose-Response Relationship, Radiation; Gene Expression; Ligases; Light; Light-Harvesting Protein Complexes; Mixed Function Oxygenases; Xanthophylls; zeta Carotene

The relative energy of carotenoid neutral radicals formed by proton loss from the radical cations of linear carotenoids has been examined as a function of conjugation length from n = 15 to 9. For a maximum conjugation length of n = 15 (bisdehydrolycopene, a symmetrical compound), proton loss can occur from any of the 10 methyl groups, with proton loss from the methyl group at position C1 or C1' being the most favorable. In contrast, the most energetically favorable proton loss from the radical cations of lycopene, neurosporene, spheroidene, spheroidenone, spirilloxanthin, and anhydrorhodovibrin occurs from methylene groups that extend from the conjugated system. For example, decreasing the conjugation length to n = 11 (lycopene) by saturation of the double bonds C3-C4 and at C3'-C4' of bisdehydrolycopene favors proton loss at C4 or C4' methylene groups. Saturation at C7'-C8' in the case of neurosporene, spheroidene, and spheroidenone (n = 9, 10, 11) favors the formation of a neutral radical at the C8' methylene group. Saturation of C1-C2 by addition of a methoxy group to a bisdehydrolycopene-like structure with conjugation of n = 12 or 13 (anhydrorhodovibrin, spirilloxanthin) favors proton loss at the C2 methylene group. As a consequence of deprotonation of the radical cation, the unpaired electron spin distribution changes so that larger β-methyl proton couplings occur for the neutral radicals (13-16 MHz) than for the radical cation (7-10 MHz), providing a means to identify possible carotenoid radicals in biological systems by Mims ENDOR.

Topics: Carotenoids; Electron Spin Resonance Spectroscopy; Free Radicals; Lycopene; Protons; Thermodynamics; Xanthophylls

Purple photosynthetic bacteria synthesize the acyclic carotenoids spheroidene and spirilloxanthin which are ketolated to spheroidenone and 2,2'-diketospirilloxanthin under aerobic growth. For the studies of the catalytic reaction of the ketolating enzyme, the crtA genes from Rubrivivax gelatinosus and Rhodobacter capsulatus encoding acyclic carotenoid 2-ketolases were expressed in Escherichia coli to functional enzymes. With the purified enzyme from the latter, the requirement of molecular oxygen and reduced ferredoxin for the catalytic activity was determined. Furthermore, the putative intermediate 2-HO-spheroidene was in vitro converted to the corresponding 2-keto product. Therefore, a monooxygenase mechanism involving two consecutive hydroxylation steps at C-2 were proposed for this enzyme. By functional pathway complementation studies in E. coli and enzyme kinetic studies, the product specificity of both enzymes were investigated. It appears that the ketolases could catalyze most intermediates and products of the spheroidene and spirilloxanthin pathway. This was also the case for the enzyme from Rba. capsulatus from which spirilloxanthin synthesis is absent. In general, the ketolase of Rvi. gelatinosus had a better specificity for spheroidene, HO-spheroidene and spirilloxanthin as substrates than the ketolase from Rba. capsulatus.

Topics: Betaproteobacteria; Carotenoids; Catalysis; Escherichia coli; Hydro-Lyases; Rhodobacter capsulatus; Substrate Specificity; Xanthophylls

Many of the spectroscopic features and photophysical properties of carotenoids are explained using a three-state model in which the strong visible absorption of the molecules is associated with an S0 (1(1)Ag-) --> S2 (1(1)Bu+) transition, and the lowest lying singlet state, S1 (2(1)Ag-), is a state into which absorption from the ground state is forbidden by symmetry. However, semiempirical and ab initio quantum calculations have suggested additional excited singlet states may lie either between or in the vicinity of S1 (2(1)Ag-) and S2 (1(1)Bu+), and some ultrafast spectroscopic studies have reported evidence for these states. One such state, denoted S*, has been implicated as an intermediate in the depopulation of S2 (1(1)Bu+) and as a pathway for the formation of carotenoid triplet states in light-harvesting complexes. In this work, we present the results of an ultrafast, time-resolved spectroscopic investigation of a series of open-chain carotenoids derived from photosynthetic bacteria and systematically increasing in their number of pi-electron carbon-carbon double bonds (n). The molecules are neurosporene (n = 9), spheroidene (n = 10), rhodopin glucoside (n = 11), rhodovibrin (n = 12), and spirilloxanthin (n = 13). The molecules were studied in acetone and CS2 solvents at room temperature. These experiments explore the effect of solvent polarity and polarizability on the spectroscopic and kinetic behavior of the molecules. The molecules were also studied in ether/isopentane/ethanol (EPA) glasses at 77 K, in which the spectral resolution is greatly enhanced. Analysis of the data using global fitting techniques has revealed the ultrafast dynamics of the excited states and spectral changes associated with their decay, including spectroscopic features not previously reported. The data are consistent with S* being identified with a twisted conformational structure, the yield of which is increased in molecules having longer pi-electron conjugations. In particular, for the longest molecule in the series, spirilloxanthin, the experiments and a detailed quantum computational analysis reveal the presence of two S* states associated with relaxed S1 (2(1)Ag-) conformations involving nearly planar 6-s-cis and 6-s-trans geometries. We propose that in polar solvents, the ground state of spirilloxanthin takes on a corkscrew conformation that generates a net solute dipole moment while decreasing the cavity formation energy. Upon excitation and relaxation into the S1

Topics: Carotenoids; Glucosides; Kinetics; Models, Chemical; Molecular Structure; Quantum Theory; Rhodobacter sphaeroides; Rhodopseudomonas; Sensitivity and Specificity; Spectrum Analysis; Temperature; Vibration; Xanthophylls

Carotenoids have been known for their photoprotective role for about 50 years. However, despite many advances in laser flash photolysis, no photodynamic studies have been so far performed on whole cells to determine the harmful threshold of light. In the present work, we investigate the coupling between energy conversion and energy deactivation, in isolated complexes of RC-LH1 and LH2 increasingly integrated systems up to intact cells of the purple anaerobic photosynthetic bacterium Rubrivivax gelatinosus. A continuous light similar to the mean daily sun irradiance on the surface of the earth is found to saturate the in vivo electron transfer turnover and to give rise to carotenoid triplet formation. This accounts for the widespread use of carotenoids among phototrophic prokaryotes and emphasizes their essential protective role in the natural environment.

Topics: Betaproteobacteria; Carotenoids; Dithionite; Energy Transfer; Ferricyanides; Light; Light-Harvesting Protein Complexes; Oxidation-Reduction; Xanthophylls

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.

Topics: Carotenoids; Chromatography, Affinity; Enzyme Activation; Escherichia coli; Hydro-Lyases; Kinetics; Proteobacteria; Recombinant Proteins; Rhodobacter capsulatus; Species Specificity; Substrate Specificity; Xanthophylls

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.

Topics: Carotenoids; Escherichia coli; Oxidoreductases; Proteobacteria; Substrate Specificity; Xanthophylls

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.

Topics: Carotenoids; Mutagenesis; Oxidoreductases; Phenotype; Rhodospirillum rubrum; Substrate Specificity; Xanthophylls