On the state of carotenoids bound to reaction centers of photosynthetic bacteria: A resonance raman study

The carotenoids bound to reaction centers of wild, Ga and GlC strains of Rhodopseudomonas spheroides, of Rhodospirillum rubrum, strain S1 and of Rhodopseudomonas viridis, yield very similar, but unusual resonance Raman spectra. Through a comparison with resonance Raman spectra of 15,15′-cis-β-carotene, these carotenoids are shown to assume cis conformations, while the corresponding chromatophores contain all-trans forms only. These cis conformations likely are identical for all the carotenoids studied. They remain unaffected by variations of temperature from 20 to 300 K as well as by the redox state of $\text{P-870}$. They are unstable, being rapidly isomerised towards the all-trans forms when extracted from the reaction centers. The possible nature of these conformers is discussed on the basis of their electronic and vibrational spectra.     

Effect of 13-cis violaxanthin on organization of light harvesting complex II in monomolecular layers

Lutein, neoxanthin and violaxanthin are the main xanthophyll pigment constituents of the largest light-harvesting pigment-protein complex of photosystem II (LHCII). High performance liquid chromatography analysis revealed photoisomerization of LHCII-bound violaxanthin from the conformation all-trans to the conformation 13-cis and 9-cis. Maximally, the conversion of 15% of all-trans violaxanthin to a cis form could be achieved owing to the light-driven reactions. The reactions were dark-reversible. The all-trans to cis isomerization was found to be driven by blue light, absorbed by chlorophylls and carotenoids, as well as by red light, absorbed exclusively by chlorophyll pigments. This suggests that the photoisomerization is a carotenoid triplet-sensitized reaction. The monomolecular layer technique was applied to study the effect of the 13-cis conformer of violaxanthin and its de-epoxidized form, zeaxanthin, on the organization of LHCII as compared to the all-trans stereoisomers. The specific molecular areas of LHCII in the two-component system composed of protein and exogenous 13-cis violaxanthin or 13-cis zeaxanthin show overadditivity, which is an indication of the xanthophyll-induced disassembly of the aggregated forms of the protein. Such an effect was not observed in the monomolecular layers of LHCII containing all-trans conformers of violaxanthin and zeaxanthin. 77 K chlorophyll a fluorescence emission spectra recorded from the Langmuir-Blodgett (L-B) films deposited to quartz from monomolecular layers formed with LHCII and LHCII in the two-component systems with all-trans and 13-cis isomers of violaxanthin and zeaxanthin revealed opposite effects of both conformers on the aggregation of the protein. The cis isomers of both xanthophylls were found to decrease the aggregation level of LHCII and the all-trans isomers increased the aggregation level. The calculated efficiency of excitation energy transfer to chlorophyll a from violaxanthin assumed to remain in two steric conformations was analyzed on the basis of the chlorophyll a fluorescence excitation spectra and the mean orientation of violaxanthin molecules in LHCII (71 degrees with respect to the normal to the membrane), determined recently in the linear dichroism experiments [Gruszecki et al., Biochim. Biophys. Acta 1412 (1999) 173-183]. The calculated efficiency of excitation energy transfer from the violaxanthin pool assumed to remain in conformation all-trans was found to be almost independent on the orientation angle within a variability range. In contrast the calculated efficiency of energy transfer from the form cis was found to be strongly dependent on the orientation and varied between 1.0 (at 67.48 degrees ) and 0 (at 70.89 degrees ). This is consistent with two essentially different, possible functions of the cis forms of violaxanthin: as a highly efficient excitation donor (and possibly energy transmitter between other chromophores) or purely as a LHCII structure modifier.     

Oxidation of the two beta-carotene molecules in the photosystem II reaction center

We present a spectroscopic characterization of the two nonequivalent beta-carotene molecules in the photosystem II reaction center. Their electronic and vibrational properties exhibit significant differences, reflecting a somewhat different configuration for these two cofactors. Both carotenoid molecules are redox-active and can be oxidized by illumination of the reaction centers in the presence of an electron acceptor. The radical cation species show similar differences in their spectroscopic properties. The results are discussed in terms of the structure and unusual function of these carotenoids. In addition, the attribution of resonance Raman spectra of photosystem II preparations excited in the range 800-900 nm is discussed. Although contributions of chlorophyll cations cannot be formally ruled out, our results demonstrate that these spectra mainly arise from the cation radical species of the two carotenoids present in photosystem II reaction centers.     

Resonance Raman scattering of bacteriochlorophyll, bacteriopheophytin and spheroidene in reaction centers of Rhodopseudomonas speroides.

We report and discuss Raman spectra of bacteriochlorophyll $\text{a}$ and of bacteriopheophytin $\text{a}$ obtained $\text{in vitro}$ by resonance effect in their Q_X and Soret electronic bands. Selective excitation of spectra of either of these molecules in reaction centers of $\text{Rhodopseudomonas spheroides}$, strains Y and R 26, was achieved by illumination in their respective Q_X bands. Preliminary interpretation of the spectra yields information about the interactions assumed by these molecules in the reaction centers. Spheroidene bound to reaction centers of strain Y probably affects a conformation different from that assumed by the bulk spheroidene of the chromatophore.     

Binding of carotenoids on reaction centers from Rhodopseudomonas sphaeroides R 26

The carotenoid-less reaction centers isolated from Rhodopseudomonas sphaeroides (strain R 26) bind pure all-trans spheroidene as well as spheroidenone in a nearly 1:1 molar ratio with respect to P-870. Neither β-carotene nor spirilloxanthin, both absent from wild-type Rps. sphaeroides, could be bound in appreciable amounts. Resonance Raman spectra of the carotenoidreaction center complex indicate that the carotenoid is bound as a cis isomer, its conformation being very close, although probably not identical, to that assumed by the carotenoid in the wild-type reaction centers. The electronic absorption spectra of the carotenoid-reaction center complexes are in good agreement with such a interpretation. When bound to the R 26 reaction centers, spheroidene displays light-induced absorbance changes identical in peak wavelengths and comparable in amplitudes to those observed in the wild-type reaction centers. Thus the binding of the carotenoid to the R 26 reaction centers most likely occurs at the same proteic site as in the wild-type reaction centers. This site shows selectivity towards the nature of carotenoids, and has the same sterical requirement as in the wild type, leading to the observed all-trans to cis isomerisation.     

Geometrical isomers of retinene

Five crystalline retinenes have been isolated, which have every appearance of being cis-trans isomers of one another. They are all-trans retinene; three apparently mono-cis isomers: neoretinenes a and b and isoretinene a; and isoretinene b, an apparently di-cis isomer. The absorption spectra of these substances display the relations expected of cis-trans isomers. The main absorption band is displaced 5.5 to 7 mmicro toward shorter wave lengths for each presumptive cis linkage. Some of the presumptive cis isomers also display a cis peak at 255 to 260 mmicro. All five substances yield an identical blue product on mixing with antimony chloride. All of them are converted by light to what appears to be an identical mixture of stereoisomers. Heat isomerizes them very slowly; only neoretinene b exhibits large changes on heating at 70 degrees C. for 3 hours. The various isomers have been extensively interconverted by gentle procedures, and all of them have been converted to all-trans retinene. The present theory of cis-trans isomerism in carotenoids predicts the existence of four stable isomers of retinene. Instead we seem to have five-specifically three mono-cis forms where two are expected. There is no doubt that all these substances are closely related isomers of one another. The only point in question is whether they differ in part by something other than cis-trans configuration. One possibility, as yet little supported by evidence, is that isomerization between beta- and alpha-ionone rings may be involved. If, however, as seems more likely, all these substances are geometrical isomers of one another, some modification is needed in the present theory of configurational relationships in this class of compounds.     

The stereoisomerism of bacterial, reaction-center-bound carotenoids revisited: An electronic absorption, resonance Raman and H-NMR study

In order to solve discrepancies between earlier assignments we have reinvestigated the stereoisomerism of the spheroidene molecule bound to reaction centers (RC) of Rhodobacter sphaeroides. A stable cis isomer could be extracted and purified from the reaction centres by working at very low ambient light. Resonance Raman spectroscopy showed that this cis isomer assumed the same configuration as that of the RC-bound molecule. Proton-NMR spectroscopy of the extracted isomer permitted to assign it the 15–15′ mono cis configuration. Comparisons between resonance Raman spectra of the native form and of the 15 cis extract showed that, in the reaction center, 15 cis spheroidene is in addition twisted into a non-planar conformation. Comparisons of extraction-induced changes in relative intensities of Raman bands of the 760–1060 cm⁻¹ regions, which largely correspond to out-of-plane modes, further indicated that the out-of-plane twist of RC-bound spheroidene should predominantly affect C₈–C₁₂ and/or C_8′–C_12′ regions of the molecule rather than the central region. Comparisons between difference electronic absorption spectra of RC-bound spheroidene and of RC-bound methoxyneurosporene showed that the out-of-plane twisting of both these native forms results in a drastic weakening of their ¹C ← ¹A electronic transitions, compared with those of the planar, 15 cis forms. Finally, it is proposed, on the basis of their resonance Raman spectra, that spirilloxanthin bound to RCs of Rhodospirillum rubrum as well as dihydroneurosporene or dihydrolycopene bound to RCs of Rhodopseudomonas viridis shares 15 cis configurations and out-of-plane twisting with carotenoids bound to RCs of various strains of Rb. sphaeroides.     

Surface-enhanced resonance raman scattering spectroscopy of bacterial photosynthetic membranes: orientation of the carotenoids of Rhodobacter sphaeroides 2.4.1

Surface-enhanced resonance Raman scattering (SERRS) spectra were obtained from carotenoids, in the all-trans configuration, located on the antenna complexes of Rhodobacter sphaeroides 2.4.1 membranes. Since resonance Raman (RR) spectra are barely detectable at the concentration that SERRS signals saturate, SERRS represents a very sensitive means of detecting pigments in biological systems. Prominent SERRS spectra of sphaeroidenone were detected in chromatophores (cytoplasmic side out) but not in spheroplast-derived vesicles (periplasmic side out), demonstrating that the carotenoid is asymmetrically located on the cytoplasmic side of the cell membrane. Comparison of peak frequencies from SERRS and RR spectral data suggests that the carotenoids are oriented into the membrane with the methoxy end of the isoprenoid chains located closest to the cytoplasmic side of the intracytoplasmic membrane. This work not only shows that SERRS spectroscopy can provide information on the location of a chromophore in a biological membrane but also for the first time demonstrates that SERRS data can be used to ascertain the orientation of a chromophore within the membrane. This observation greatly increases the potential of this technique for structural analysis of intact membranes at the molecular level.     

Resonance Raman spectroscopy of carotenoids in Photosystem I particles

Low-temperature resonance Raman (RR) spectroscopy was used for the first time to study the spectral properties, binding sites and composition of major carotenoids in spinach Photosystem I (PSI) particles. Excitation was provided by an argon ion laser at 457.9, 476.5, 488, 496.5, 502 and 514.5 nm. Raman spectra contained the four known groups of bands characteristic for carotenoids (called from nu(1) to nu4). Upon 514.5, 496.5 and 476.5 nm excitations, the nu(1)-nu(3) frequencies coincided with those established for lutein. Spectrum upon 502-nm excitation could be assigned to originate from violaxanthin, at 488 nm to 9-cis neoxanthin, and at 457.9 nm to beta-carotene and 9-cis neoxanthin. The overall configuration and composition of these bound carotenoid molecules in Photosystem I particles were compared with the composition of pigment extracts from the same PSI particles dissolved in pyridine, as well as to configuration in the main chlorophyll a/b light-harvesting protein complex of photosystem II. The absorption transitions for lutein, violaxanthin and 9-cis neoxanthin in spinach photosystem I particles are characterized, and the binding sites of lutein and neoxanthin are discussed. Resonance Raman data suggest that beta-carotene molecules are also present in all-trans and, probably, in 9-cis configurations.     

Vibrational spectroscopy of excited electronic states in carotenoids in vivo. Picosecond time-resolved resonance Raman scattering.

The vibrational spectroscopy and population dynamics of excited singlet (2(1)Ag), excited triplet (3B u), and the ground (1Ag) electronic states of carotenoids in chromatophores of Chromatium vinosum (mainly spirilloxanthin and rhodopin) and of the same carotenoids in benzene solutions are examined by picosecond time-resolved resonance Raman scattering. Coherent Stokes Raman scattering from the ground states of carotenoids in chromatophores also is observed. Resonance Raman spectra of in vitro rhodopin and spirilloxanthin when compared with in vivo data demonstrate that scattering from spirilloxanthin dominates the in vivo spectrum. Comparisons of the time-dependent intensities of 2(1)Ag and 1Ag resonance Raman bands from both in vitro and in vivo carotenoids suggest that vibrationally excited levels in 1Ag are populated directly by the decay of the 2(1)Ag state and that these levels relax into a thermalized distribution in less than 50 ps. The appearance of asymmetrically broadened, ground-state resonance Raman bands supports this conclusion. Formation of the 3Bu state is observed for carotenoids in chromatophores, but not for in vitro spirilloxanthin indicating that the 3Bu state is formed by fission processes originating from the spatial organization of pigments within chromatophores. The rate at which the intensities of 2(1)Ag resonance Raman bands decay is faster for the carotenoids in vivo than for those in vitro thereby indicating that additional relaxation channels (e.g., energy transfer to bacteriochlorophylls) are present in the chromatophore. The similarity of the in vivo and in vitro 2(1)Ag resonance Raman spectra shows that no significant modifications in the vibronic coupling has been caused by the chromatophore environment.     

Identification of a gene required for cis-to-trans carotene isomerization in carotenogenesis of the cyanobacterium Synechocystis sp. PCC 6803.

A disruptive mutant of the sll0033 gene of the cyanobacterium Synechocystis sp. PCC 6803 produced primarily cis carotenes and small amounts of all-trans carotenes, but no xanthophylls, under dark conditions. Under light conditions, however, it produced normal carotenoids, that were the same as those produced by wild-type cells grown under both light and dark conditions. When the mutant cells cultured under dark conditions were irradiated, cis-isomers of carotenes were converted to all-trans lycopene. These findings demonstrate that this gene, designated crtH, is involved in the isomerization of cis-carotenes to all-trans forms in dark conditions, and that cis-carotenes were also converted to all-trans forms under light conditions by photoisomerization.     

Resonance Raman studies of bovine metarhodopsin I and metarhodopsin II

The resonance Raman spectra of bovine metarhodopsin I and metarhodopsin II have been measured. The spectra are compared with model chromophore resonance Raman data. It was found that metarhodopsin I is linked to opsin via a protonated Schiff base linkage, whereas metarhodopsin II is linked by an unprotonated Schiff base. A recent suggestion that the chromophore of metarhodopsin II is retinal is explicitly disproved. The chromophores of both metarhodopsins are found to have an essentially all-trans conformation. The basic mechanism for color regulation in both forms appears to be electron delocalization. The data tend to support the model of cis-trans isomerization as the primary mechanism for vision. Also, the conclusions and inferences of this work on energy uses and storage by rhodopsin in neural generation are discussed.     

Light-induced conformational change at low temperature in the coordination of heme c-556 in the reaction center of Rhodopseudomonas viridis

Isolated reaction centers of Rhodopseudomonas viridis with the two high-potential hemes reduced were illuminated at 5 K. Difference spectra show a bleaching of the heme c-556 alpha bands and a red shift of the Soret band. These effects are reversed by warming to around 80 K. They are not induced by near infra-red light absorbed by the chlorine pigments of the reaction centers and they are not associated with electron transfer from P to Q_A. It is concluded that, following direct excitation, heme c-556 becomes five-coordinated. We find no evidence of a significant photooxidation of heme c-559 under the same conditions.     

Processing of vegetable-borne carotenoids in the human stomach and duodenum

Carotenoids are thought to diminish the incidence of certain degenerative diseases, but the mechanisms involved in their intestinal absorption are poorly understood. Our aim was to obtain basic data on the fate of carotenoids in the human stomach and duodenum. Ten healthy men were intragastrically fed three liquid test meals differing only in the vegetable added 3 wk apart and in a random order. They contained 40 g sunflower oil and mashed vegetables as the sole source of carotenoids. Tomato purée provided 10 mg lycopene as the main carotenoid, chopped spinach (10 mg lutein), and carrot purée (10 mg beta-carotene). Samples of stomach and duodenal contents and blood samples were collected at regular time intervals after meal intake. all-trans and cis carotenoids were assayed in stomach and duodenal contents, in the fat and aqueous phases of those contents, and in chylomicrons. The cis-trans beta-carotene and lycopene ratios did not significantly vary in the stomach during digestion. Carotenoids were recovered in the fat phase present in the stomach during digestion. The proportion of all-trans carotenoids found in the micellar phase of the duodenum was as follows (means +/- SE): lutein (5.6 +/- 0.4%), beta-carotene (4.7 +/- 0.3%), lycopene (2.0 +/- 0.2%). The proportion of 13-cis beta-carotene in the micellar phase was significantly higher (14.8 +/- 1.6%) than that of the all-trans isomer (4.7 +/- 0.3%). There was no significant variation in chylomicron lycopene after the tomato meal, whereas there was significant increase in chylomicron beta-carotene and lutein after the carrot and the spinach meals, respectively. There is no significant cis-trans isomerization of beta-carotene and lycopene in the human stomach. The stomach initiates the transfer of carotenoids from the vegetable matrix to the fat phase of the meal. Lycopene is less efficiently transferred to micelles than beta-carotene and lutein. The very small transfer of carotenoids from their vegetable matrices to micelles explains the poor bioavailability of these phytomicroconstituents.     

Absorption and structure of an LM unit isolated with sodium dodecyl sulfate from reaction centers of Rhodopseudomonas sphaeroides

Low-temperature absorption, circular dichroism and resonance Raman spectra of the LM units isolated with sodium dodecyl sulfate from wild-type Rhodopseudomonas sphaeroides reaction centers (Agalidis, I. and Reiss-Husson, F. (1983) Biochim. Biophys. Acta 724, 340–351) are described in comparison with those of intact reaction centers. In LM unit, the Q_y absorption band of P-870 at 77 K shifted from 890 nm (in reaction center) to 870 nm and was broadened by about 30%. In contrast, the 800 nm bacteriochlorophyll absorption band including the 810 species remained unmodified. It was concluded that the 810 nm transition is not the higher excitonic component of P-870. The Q_x band of P-870 shifted from 602 nm (in reaction center) to 598 nm in LM, whereas the Q_x band of the other bacteriochlorophylls was the same in reaction center and LM and had two components at about 605 and 598 nm. The Q_xII band of bacteriopheophytin was upshifted to 538 nm and a slight blue shift of the Q_y band of bacteriopheophytin was observed. Resonance Raman spectra of spheroidene in LM showed that its native cis-conformation was preserved. Resonance Raman spectroscopy also demonstrated that in LM the molecular interactions assumed by the conjugated carbonyls of bacteriochlorophyll molecules were altered, but not those assumed by the bacteriopheophytins carbonyls. In particular at least one Keto group of bacteriochlorophyll free in reaction center, becomes intermolecularly bounded in LM (possibly with extraneous water). This group may belong to the primary donor molecules.     

Conformational nonequivalence of chains 1 and 2 of dipalmitoyl phosphatidylcholine as observed by Raman spectroscopy.

Raman spectroscopic data indicate that the conformations of the two hydrocarbon chains of dipalmitoyl phosphatidylcholine in aqueous dispersions of the lipid differ signficantly. The compounds 1-palmitoyl, 2-palmitoyl-d31-3-sn-phosphatidylcholine and 1-palmitoyl-d31, 2-palmitoyl-3-sn-phosphatidylcholine were synthesized. Aqueous dispersions of these phospholipids display very similar phase behavior, with both premelting and melting transitions at nearly identical temperatures, midway between the comparable transition temperatures of undeuterated and completely deuterated dipalmitoyl phosphatidylcholine. We have monitored the state of chains 1 and 2 of these molecules simultaneously and independently by Raman spectroscopy. Raman difference spectra taken between samples of the two compounds under identical conditions show significant features. We attribute these spectral differences to nonequivalent conformations of the fatty acyl chains attached at positions 1 and 2 on the glycerol backbone. Below the pretransition the conformation of chain 2 is, on average, slightly less all-trans than is the chain at position 1. There is some evidence that the conformations of the terminal methyl group of the two chains are significantly different at low temperatures.     

In vivo states and functions of carotenoids in an aerobic photosynthetic bacterium,Erythrobacter longus

In vivo states and functions of carotenoids in the membranes and the isolated RC-B865 pigment-protein complexes from an aerobic photosynthetic bacterium, Erythrobacter longus, are investigated by means of fluorescence excitation and resonance Raman (RR) spectra. Erythroxanthin sulfate, a dominant carotenoid species in the membranes (>70%), is found not to transfer the absorbed light energy to bacteriochlorophyll (Bchl), and its RR spectra are similar between the in vivo and in vitro states. These observations indicate that erythroxanthin sulfate does not interact with either Bchl or proteins in the membranes, and suggest that its function may be limited to photoprotection by quenching the harmful singlet oxygen. On the other hand, two other carotenoid species contained in the isolated RC-B865 complexes, zeaxanthin and bacteriorubixanthinal, have a high efficiency of energy transfer to Bchl (88±5%). The RR spectra of these two carotenoids, each of which can be selectively obtained by choosing the excitation wavelength, show some characteristics of interactions with proteins or Bchl.Abbreviations Bchl bacteriochlorophyll a - FWHM full width at half maximum - PAGE polyacrylamide gel electrophoresis - RC reaction center - RR resonance Raman - SDS sodium dodecyl sulfate     

Properties of photochemical reaction centers purified from Rhodopseudomonas gelatinosa

Reaction centers were isolated from a carotenoidless mutant of Rhodopseudomonas gelatinosa by hydroxyapatite chromatography of purified chromatophores treated with lauryl dimethyl amine oxide. Absorption spectra and spectra of light-induced absorbance changes are similar to those of reaction centers from Rhodopseudomonas sphaeroides. The ratio of absorbance at 280 nm to that at 799 nm was 1.8 in the purest preparations. The extinction coefficient at the 799 nm absorption maximum was estimated to be 305 +/- 20 mM--1 . CM--1. The molecular weight based on protein and chromophore assays was found to be 1.5 . 10(5); the reaction center protein accounted for 6% of the total membrane protein. These reaction centers contained no cytochrome and showed just two components of apparent molecular weights 33 000 and 25 000 in polyacrylamide gel electrophoresis. The chromatophores contained 42 molecules of antenna bacteriochlorophyll for each reaction center.     

Electron nuclear double resonance of semiquinones in reaction centers of Rhodopseudomonas sphaeroides

Replacement of Fe2+ by Zn2+ in reaction centers of Rhodopseudomonas sphaeroides enabled us to perform ENDOR (electron nuclear double resonance) experiments on the anion radicals of the primary and secondary ubiquinone acceptors (QA- and QB-. Differences between the QA and QB sites, hydrogen bonding to the oxygens, interactions with the protons of the proteins and some symmetry properties of the binding sites were deduced from an analysis of the ENDOR spectra.     

Linear polyenes: models for the spectroscopy and photophysics of carotenoids

We have developed procedures for synthesizing dimethyl polyenes using living polymerization techniques and have initiated investigations of the spectroscopic properties of these molecules. Purification using high-performance liquid chromatography (HPLC) of the polyene mixtures resulting from the syntheses promises to provide all-trans polyenes with a wide range in the number of conjugated double bonds. Low temperature optical measurements on these model systems, both in glasses and in n-alkane mixed crystals, yield absorption and fluorescence spectra with considerably higher vibronic resolution than the spectra currently available for carotenoids with comparable conjugation lengths. The dimethyl polyenes thus allow a more precise exploration of the electronic properties of long, linearly conjugated systems. These studies can be used to verify the existence of low-lying singlet states predicted by theory and recently invoked to explain low-resolution fluorescence, Raman excitation spectra, and the transient absorption spectroscopy of carotenoids. Steady state and time-resolved optical studies of the dimethyl series will be used to better understand the energies and dynamics of the low energy electronic states relevant to the photochemistry and photobiology of all linearly conjugated systems.