Role of B800 in carotenoid-bacteriochlorophyll energy and electron transfer in LH2 complexes from the purple bacterium Rhodobacter sphaeroides

The role of the B800 in energy and electron transfer in LH2 complexes has been studied using femtosecond time-resolved transient absorption spectroscopy. The B800 site was perturbed by application of lithium dodecyl sulfate (LDS), and comparison of treated and untreated LH2 complexes from Rhodobacter sphaeroides incorporating carotenoids neurosporene, spheroidene, and spheroidenone was used to explore the role of B800 in carotenoid to bacteriochlorophyll-a (BChla) energy transfer and carotenoid radical formation. Efficiencies of the S1-mediated energy transfer in the LDS-treated complexes were 86, 61, and 57% in the LH2 complexes containing neurosporene, spheroidene, and spheroidenone, respectively. Analysis of the carotenoid S1 lifetimes in solution, LDS-treated, and untreated LH2 complexes allowed determination of B800/B850 branching ratio in the S1-mediated energy transfer. It is shown that B800 is a major acceptor, as approximately 60% of the energy from the carotenoid S1 state is accepted by B800. This value is nearly independent of conjugation length of the carotenoid. In addition to its role in energy transfer, the B800 BChla is the only electron acceptor in the event of charge separation between carotenoid and BChla in LH2 complexes, which is demonstrated by prevention of carotenoid radical formation in the LDS-treated LH2 complexes. In the untreated complexes containing neurosporene and spheroidene, the carotenoid radical is formed with a time constant of 300-400 fs. Application of different excitation wavelengths and intensity dependence of the carotenoid radical formation showed that the carotenoid radical can be formed only after excitation of the S2 state of carotenoid, although the S2 state itself is not a precursor of the charge-separated state. Instead, either a hot S1 state or a charge-transfer state lying between S2 and S1 states of the carotenoid are discussed as potential precursors of the charge-separated state.     

Triplet Energy Transfer between the Primary Donor and Carotenoids in Rhodobacter sphaeroides R-26.1 Reaction Centers Incorporated with Spheroidene Analogs Having Different Extents of π-Electron Conjugation

Abstract— Three carotenoids, spheroidene, 3,4-dihydrospheroidene and 3,4,5,6-tetrahydrospheroidene, having 8, 9 and 10 conjugated carbon-carbon double bonds, respectively, were incorporated into Rhodobacter (Rb.) sphaeroides R-26.1 reaction centers. The extents of binding were found to be 95±5% for spheroidene, 65±5% for 3,4-dihydrospheroidene and 60±10% for 3,4,5,6-tetrahydrospheroidene. The dynamics of the triplet states of the primary donor and carotenoid were measured at room temperature by flash absorption spectroscopy. The carotenoid, spheroidene, was observed to quench the primary donor triplet state. The triplet state of spheroidene that was formed subsequently decayed to the ground state with a lifetime of 7.0±0.5 μs. The primary donor triplet lifetime in the Rb. sphaeroides R-26.1 reaction centers lacking carotenoids was 60±5 μs. Quenching of the primary donor triplet state by the carotenoid was not observed in the Rb. sphaeroides R-26.1 reaction centers containing 3,4-dihydrospheroidene nor in the R-26.1 reaction centers containing 3,4,5,6-tetrahydrospheroidene. Triplet-state electron paramagnetic resonance was also carried out on the samples. The experiments revealed carotenoid triple-state signals in the Rb. sphaeroides R-26.1 reaction centers incorporated with spheroidene, indicating that the primary donor triplet is quenched by the carotenoid. No carotenoid signals were observed from Rb. sphaeroides R-26.1 reaction centers incorporating 3,4-dihydrospheroidene nor in reaction centers incorporating 3,4,5,6-tetrahydrospheroidene. Circular dichroism, steady-state absorbance band shifts accompanying the primary photochemistry in the reaction center and singlet energy transfer from the carotenoid to the primary donor confirm that the carotenoids are bound in the reaction centers and interacting with the primary donor. These studies provide a systematic approach to exploring the effects of carotenoid structure and excited state energy on triplet transfer between the primary donor and carotenoids in reaction centers from photosynthetic bacteria.     

Ultrafast time-resolved carotenoid to-bacteriochlorophyll energy transfer in LH2 complexes from photosynthetic bacteria

Steady-state and ultrafast time-resolved optical spectroscopic investigations have been carried out at 293 and 10 K on LH2 pigment-protein complexes isolated from three different strains of photosynthetic bacteria: Rhodobacter (Rb.) sphaeroides G1C, Rb. sphaeroides 2.4.1 (anaerobically and aerobically grown), and Rps. acidophila 10050. The LH2 complexes obtained from these strains contain the carotenoids, neurosporene, spheroidene, spheroidenone, and rhodopin glucoside, respectively. These molecules have a systematically increasing number of pi-electron conjugated carbon-carbon double bonds. Steady-state absorption and fluorescence excitation experiments have revealed that the total efficiency of energy transfer from the carotenoids to bacteriochlorophyll is independent of temperature and nearly constant at approximately 90% for the LH2 complexes containing neurosporene, spheroidene, spheroidenone, but drops to approximately 53% for the complex containing rhodopin glucoside. Ultrafast transient absorption spectra in the near-infrared (NIR) region of the purified carotenoids in solution have revealed the energies of the S1 (2(1)Ag-)-->S2 (1(1)Bu+) excited-state transitions which, when subtracted from the energies of the S0 (1(1)Ag-)-->S2 (1(1)Bu+) transitions determined by steady-state absorption measurements, give precise values for the positions of the S1 (2(1)Ag-) states of the carotenoids. Global fitting of the ultrafast spectral and temporal data sets have revealed the dynamics of the pathways of de-excitation of the carotenoid excited states. The pathways include energy transfer to bacteriochlorophyll, population of the so-called S* state of the carotenoids, and formation of carotenoid radical cations (Car*+). The investigation has found that excitation energy transfer to bacteriochlorophyll is partitioned through the S1 (1(1)Ag-), S2 (1(1)Bu+), and S* states of the different carotenoids to varying degrees. This is understood through a consideration of the energies of the states and the spectral profiles of the molecules. A significant finding is that, due to the low S1 (2(1)Ag-) energy of rhodopin glucoside, energy transfer from this state to the bacteriochlorophylls is significantly less probable compared to the other complexes. This work resolves a long-standing question regarding the cause of the precipitous drop in energy transfer efficiency when the extent of pi-electron conjugation of the carotenoid is extended from ten to eleven conjugated carbon-carbon double bonds in LH2 complexes from purple photosynthetic bacteria.     

Carotenoid radical cation formation in LH2 of purple bacteria: a quantum chemical study

In LH2 complexes of Rhodobacter sphaeroides the formation of a carotenoid radical cation has recently been observed upon photoexcitation of the carotenoid S2 state. To shed more light onto the yet unknown molecular mechanism leading to carotenoid radical formation in LH2, the interactions between carotenoid and bacteriochlorophyll in LH2 are investigated by means of quantum chemical calculations for three different carotenoids--neurosporene, spheroidene, and spheroidenone--using time-dependent density functional theory. Crossings of the calculated potential energy curve of the electron transfer state with the bacteriochlorophyll Qx state and the carotenoid S1 and S2 states occur along an intermolecular distance coordinate for neurosporene and spheroidene, but for spheroidenone no crossing of the electron transfer state with the carotenoid S1 state could be found. By comparison with recent experiments where no formation of a spheroidenone radical cation has been observed, a molecular mechanism for carotenoid radical cation formation is proposed in which it is formed via a vibrationally excited carotenoid S1 or S*state. Arguments are given why the formation of the carotenoid radical cation does not proceed via the Qx, S2, or higher excited electron transfer states.     

Mechanism of carotenoid singlet excited state energy transfer in modified bacterial reaction centers

Ultrafast transient laser spectroscopy has been used to investigate carotenoid singlet excited state energy transfer in various Rhodobacter (Rb.) sphaeroides reaction centers (RCs) modified either genetically or chemically. The pathway and efficiency of energy transfer were examined as a function of the structures and energies of the donor and acceptor molecules. On the donor side, carotenoids with various extents of pi-electron conjugation were examined. RCs studied include those from the anaerobically grown wild-type strain containing the carotenoid spheroidene, which has 10 conjugated carbon-carbon double bonds; the GA strain containing neurosporene, which has nine conjugated double bonds; and aerobically grown wild-type cells, as well as aerobically grown H(M182)L mutant, both containing the carbonyl-containing carotenoid spheroidenone, which has 11 conjugated double bonds. By varying the structure of the carotenoid, we observed the effect of altering the energies of the carotenoid excited states on the rate of energy transfer. Both S(1)- and S(2)-mediated carotenoid-to-bacteriochlorophyll energy transfer processes were observed. The highest transfer efficiency, from both the S(1) and S(2) states, was observed using the carotenoid with the shortest chain. The S(1)-mediated carotenoid-to- bacteriochlorophyll energy transfer efficiencies were determined to be 96%, 84%, and 73% for neurosporene, spheroidene, and spheroidenone, respectively. The S(2)-mediated energy transfer efficiencies follow the same trend but could not be determined quantitatively because of limitations in the time resolution of the instrumentation. The dependence of the energy transfer rate on the energetics of the energy transfer acceptor was verified by performing measurements with RCs from the H(M182)L mutant. In this mutant, the bacteriochlorophyll (denoted B(B)) located between the carotenoid and the RC special pair (P) is replaced by a bacteriopheophytin (denoted phi(B)), where the Q(X) and Q(Y) bands of phi(B) are 1830 and 1290 cm(-1), respectively, higher in energy than those of B(B). These band shifts associated with phi(B) in the H(M182)L mutant significantly alter the spectral overlap between the carotenoid and phi(B), resulting in a significant decrease of the transfer efficiency from the carotenoid S(1) state to phi(B). This leaves energy transfer from the carotenoid S(2) state to phi(B) as the dominant channel. Largely because of this change in mechanism, the overall efficiency of energy transfer from the carotenoid to P decreases to less than 50% in this mutant. Because the spectral signature of phi(B) is different from that of B(A) in this mutant, we were able to demonstrate clearly that the carotenoid-to-P energy transfer is via phi(B). This finding supports the concept that, in wild-type RCs, the carotenoid-to-P energy transfer occurs through the cofactor located at the B(B) position.     

CAROTENOID-TO-BACTERIOCHLOROPHYLL SINGLET ENERGY TRANSFER IN CAROTENOID-INCORPORATED B850 LIGHT-HARVESTING COMPLEXES OF Rhodobacter sphaeroides R-26.1

Four carotenoids, 3,4,7,8-tetrahydrospheroidene, 3,4,5,6-tetrahydrospheroidene, 3,4-dihydrospheroidene and spheroidene, have been incorporated into the B850 light-harvesting complex of the carotenoidless mutant, photosynthetic bacterium, Rhodobacter sphaeroides R-26.1. The extent of π-electron conjugation in these molecules increases from 7 to 10 carbon-carbon double bonds. Carotenoid-to-bacteriochlorophyll singlet state energy transfer efficiencies were measured using steady-state fluorescence excitation spectroscopy to be 54 ± 2%, 66 ± 4%, 71 ± 6% and 56 ± 3% for the carotenoid series. These results are discussed with respect to the position of the energy levels and the magnitude of spectral overlap between the S, (2′AJ state emission from the isolated carotenoids and the bacteriochlorophyll absorption of the native complex. These studies provide a systematic approach to exploring the effect of excited state energies, spectral overlap and excited state lifetimes on the efficiencies of carotenoid-to-bacteriochlorophyll singlet energy transfer in photosynthetic systems.     

Triplet State Energy Transfer Between the Primary Donor and the Carotenoid in Rhodobacter sphaeroides R-26.1 Reaction Centers Exchanged with Modified Bacteriochlorophyll Pigments and Reconstituted with Spheroidene

Abstract— The dynamics of triplet energy transfer between the primary donor and the carotenoid were measured on several photosynthetic bacterial reaction center preparations from Rhodobacter sphaeroides : (a) wild-type strain 2.4.1, (b) strain R-26.1, (c) strain R-26.1 exchanged with 13²-hy-droxy-[Zn]-bacteriochlorophyll at the accessory bacteriochlorophyll (BChl) sites and reconstituted with spheroidene and (d) strain R-26.1 exchanged with P-vinyl]-13²-hydroxy-bacteriochlorophyll at the accessory BChl sites and reconstituted with spheroidene. The rise and decay times of the primary donor and carotenoid triplet-triplet absorption signals were monitored in the visible wavelength region between 538 and 555 run as a function of temperature from 4 to 300 K. For the samples containing carotenoids, all of the decay times correspond well to the previously observed times for spheroidene (5 ± 2 us). The rise times of the carotenoid triplets were found in all cases to be biexponen-tial and comprised of a strongly temperature-dependent component and a temperature-independent component. From a comparison of the behavior of the carotenoid-con-taining samples with that from the reaction center of the carotenoidless mutant Rb. sphaeroides R-26.1, the temperature-independent component has been assigned to the buildup of the primary donor triplet state resulting from charge recombination in the reaction center. Arrhenius plots of the buildup of the carotenoid triplet states were used to determine the activation energies for triplet energy transfer from the primary donor to the carotenoid. A model for the process of triplet energy transfer that is consistent with the data suggests that the activation barrier is strongly dependent on the triplet state energy of the accessory BChl pigment, BChl_B.     

TRIPLET STATES OF CAROTENOIDS BOUND TO REACTION CENTERS OF PHOTOSYNTHETIC BACTERIA: TIME-RESOLVED RESONANCE RAMAN SPECTROSCOPY

Time-resolved, low-temperature resonance Raman spectra of triplet states of the carotenoids specifically present in bacterial reaction centers in a strained cis conformation have been obtained, thus demonstrating the possibility of studying intermediate transient states of these structures using resonance Raman spectroscopy. Resonance Raman spectra of triplet cis spheroidene and cis methoxyneurosporene present in reaction centers of Rhodopseudomonas spheroides, (strains 2.4.1. and Ga, respectively) exhibit marked differences with those of triplet, all- trans carotenoids previously studied in vitro. These differences, together with the frequency shifts measured for the v ₁ modes, indicate that triplet carotenoids bound to reaction centers retain a cis conformation, and that probably no isomerization occurs to all- trans carotenoids upon T ← S₀ excitation. P_i electron distributions along the polyene backbone are probably less regular in the triplet state than in the singlet ground state, although probably not to the extent suggested by previous theoretical calculations. The apparently anomalous behaviour of the v ₂ bands of all- trans carotenoids upon T ← S₀ excitation is shown to result largely from the actual complexity of this region of the Raman spectra, together with a weak participation of the v _c—–c internal coordinate in the corresponding modes. Finally, the Raman scattering efficiency of triplet spheroidene bound to reaction centers is lower than that of the singlet, ground state form, under equivalent excitation conditions.     

TRIPLET STATES OF CAROTENOIDS FROM PHOTOSYNTHETIC BACTERIA STUDIED BY NANOSECOND ULTRAVIOLET AND ELECTRON PULSE IRRADIATION

Abstract— Absorptions of the triplet excited states of five carotenoids (15,15'-ds phytoene, all- trans phytoene, C-carotene, spheroidene and spirilloxanthin), extracted from the photosynthetic bacteria Rhodopseudomonas spheroides and Rhodospirillum rubrum, have been detected in solution using pulse radiolysis and laser flash photolysis. Triplet lifetimes, extinction coefficients, lowest energy levels and quantum efficiencies of formation have been determined. Comparison of the carotenoid triplet energy levels with that of O₂('Δ_g) suggests that spirilloxanthin, spheroidene and possibly alsoζ-carotene, would be expected to protect against photodynamic action caused by O₂ ('Δ_g), but not cis or trans phytoene. The S → T intersystem crossing efficiences of all five polyenes were found to be low, being a few per cent or less. In their protective role these triplet states can only therefore be effectively reached via energy transfer from another triplet, except in the case of O₂ ('Δ_g). The low crossover efficiencies also mean that light absorbed by such carotenoids in their possible role as accessory pigments would not be wasted in crossing over to the triplet state.     

Probing the effect of the binding site on the electrostatic behavior of a series of carotenoids reconstituted into the light-harvesting 1 complex from purple photosynthetic bacterium Rhodospirillum rubrum detected by stark spectroscopy

Reconstitutions of the LH1 complexes from the purple photosynthetic bacterium Rhodospirillum rubrum S1 were performed with a range of carotenoid molecules having different numbers of C=C conjugated double bonds. Since, as we showed previously, some of the added carotenoids tended to aggregate and then to remain with the reconstituted LH1 complexes (Nakagawa, K.; Suzuki, S.; Fujii, R.; Gardiner, A.T.; Cogdell, R.J.; Nango, M.; Hashimoto, H. Photosynth. Res. 2008, 95, 339-344), a further purification step using a sucrose density gradient centrifugation was introduced to improve purity of the final reconstituted sample. The measured absorption, fluorescence-excitation, and Stark spectra of the LH1 complex reconstituted with spirilloxanthin were identical with those obtained with the native, spirilloxanthin-containing, LH1 complex of Rs. rubrum S1. This shows that the electrostatic environments surrounding the carotenoid and bacteriochlorophyll a (BChl a) molecules in both of these LH1 complexes were essentially the same. In the LH1 complexes reconstituted with either rhodopin or spheroidene, however, the wavelength maximum at the BChl a Qy absorption band was slightly different to that of the native LH1 complexes. These differences in the transition energy of the BChl a Qy absorption band can be explained using the values of the nonlinear optical parameters of this absorption band, i.e., the polarizability change Tr(Deltaalpha) and the static dipole-moment change |Deltamu| upon photoexcitation, as determined using Stark spectroscopy. The local electric field around the BChl a in the native LH1 complex (ES) was determined to be approximately 3.0x10(6) V/cm. Furthermore, on the basis of the values of the nonlinear optical parameters of the carotenoids in the reconstituted LH1 complexes, it is possible to suggest that the conformations of carotenoids, anhydrorhodovibrin and spheroidene, in the LH1 complex were similar to that of rhodopin glucoside in crystal structure of the LH2 complex from Rhodopseudomonas acidophila 10050.     

HL-LH2中色素分子间的单重激发态能量传递

采用飞秒时间分辨吸收光谱手段观测了在500和800 nm激发下高光培养的紫色光合细菌Rhodopseu-domonas(Rps). palustris外周捕光天线LH2(HL-LH2)中不同共轭链长类胡萝卜素(Carotenoid, 简称Car)和细菌叶绿素a(Bacteriachlorophyll a, 简称BChl a)的特征吸收光谱. 光谱动力学分析结果表明, HL-LH2中不同Car分子间可能存在复杂的单重激发态能量平衡过程, Car分子同时向BChl a分子发生多途径的单重激发态能量传递, B800主要接受来自Car的S2和S1态能量; B850则主要接受来自长共轭链Car(共轭双键数目n=13)的S1态和B800的激发态能量, 整个能量传递过程在3~5 ps内完成.     

CAROTENOID TRIPLET STATES IN PHOTOSYNTHETIC BACTERIA

Abstract— The triplet state spectra of anaerobically grown cells of Rhodopseudomonas sphaeroides GIC and R. sphaeroides wild type and aerobically grown cells of R. sphaeroides wild type were examined under reducing and non-reducing conditions at 100 K. by electron paramagnetic resonance (EPR). Large differences in the zero field splitting parameters, |D| and |E| of the triplets are seen to arise in the different preparations. The spectra are assigned to the triplet states of carotenoids, some of which are closely associated with the reaction center and others are contained in the light harvesting complex. Taken together with the triplet state data on Rhodospirillum rubrum wild type previously published the trends in the zero field splitting parameters can be understood in terms of the extent of electron derealization in carotenoids of varying length.     

LINEAR AND CIRCULAR DICHROISM AND FLUORESCENCE POLARIZATION OF THE B875 LIGHT-HARVESTING BACTERIOCHLOROPHYLL-PROTEIN COMPLEX FROM RHODOPSEUDOMONAS SPHAEROIDES

Abstract— The orientation of the chromophores in the B875 light-harvesting bacteriochlorophyll complex isolated from Rhodopseudomonas sphaeroides by lithium dodecyl sulfate/polyacrylamide gel electrophoresis was examined by linear and circular dichroism and fluorescence polarization procedures. The circular dichroism in the near-IR was weaker than that of the B800–850 light-harvesting complex and had a distinctly different shape. This suggested a different geometry for the two bacteriochlorophylls of B875 and less interactive association between their transition moments. The magnitude of the circular dichroism in the carotenoid region of B875 was similar to that of B800–850 but gave more negative values between approx. 430–485 nm; this may reflect a difference in the asymmetric binding of carotenoids to the B875 protein. The fluorescence polarization increased sharply across the near-IR region of B875 and achieved very high values at long wavelengths. This confirmed that more than one transition contributed to this absorption band. The linear dichroism of B875 did not show a significant change in this near-IR band like that observed for the longest wavelength band of B800–850. Thus, the transition moments for each bacteriochlorophyll within B875 appear to be tilted to approximately the same extent with respect to the protein axis. These results distinguish B875 from all other light-harvesting complexes and suggest that the antennae of Rhodospirillaceae which contain a single near-IR absorption band cannot be classified into a single group.     

THE TRIPLET EXTINCTION COEFFICIENTS OF SOME BACTERIAL CAROTENOIDS

The extinction coefficients of the triplet states of the bacterial carotenoids, neurosporenc (Λ_max 489 nm). sphcroidene (Λ_max 510 nm). spheroidenone (Λ_max 550 nm) and spirilloxanthin (Λ_max 550 nm) in cyclohexane have been determined to be 27.4. 30.9. 6.06 and 9.20 × 10⁴ dm³ mol^?1 cm¹, respectively. These values were obtained by an energy transfer technique using a range of carotenoid concentrations. For the three that had been studied earlier, the extinctions now obtained are suhstantially higher than prcviously reported.     

Ab inito study on triplet excitation energy transfer in photosynthetic light-harvesting complexes

We have studied the triplet energy transfer (TET) for photosynthetic light-harvesting complexes, the bacterial light-harvesting complex II (LH2) of Rhodospirillum molischianum and Rhodopseudomonas acidophila, and the peridinin-chlorophyll a protein (PCP) from Amphidinium carterae. The electronic coupling factor was calculated with the recently developed fragment spin difference scheme (You and Hsu, J. Chem. Phys. 2010, 133, 074105), which is a general computational scheme that yields the overall coupling under the Hamiltonian employed. The TET rates were estimated based on the couplings obtained. For all light-harvesting complexes studied, there exist nanosecond triplet energy transfer from the chlorophylls to the carotenoids. This result supports a direct triplet quenching mechanism for the photoprotection function of carotenoids. The TET rates are similar for a broad range of carotenoid triplet state energy, which implies a general and robust TET quenching role for carotenoids in photosynthesis. This result is also consistent with the weak dependence of TET kinetics on the type or the number of π conjugation lengths in the carotenoids and their analogues reported in the literature. We have also explored the possibility of forming triplet excitons in these complexes. In B850 of LH2 or the peridinin cluster in PCP, it is unlikely to have triplet exciton since the energy differences of any two neighboring molecules are likely to be much larger than their TET couplings. Our results provide theoretical limits to the possible photophysics in the light-harvesting complexes.     

Carotenoid triplet states associated with the long-wavelength-emitting chlorophyll forms of photosystem I in isolated thylakoid membranes

The carotenoid triplet populations associated with the long-wavelength-emitting chlorophyll forms of photosystem I (PS I)(dagger) have been investigated in isolated spinach thylakoids by means of fluorescence-detected magnetic resonance in zero field. The spectra collected in the 730-800 nm emission range can be globally fitted assuming the presence of four different carotenoid triplet states coupled to long-wavelength-emitting forms of PS I, having zero-field-splitting parameters /D/ = 0.0359 cm(-1) and /E/ = 0.00371 cm(-1), /D/ = 0.0382 cm(-1) and /E/ = 0.00388 cm(-1), /D/ = 0.0395 cm(-1) and /E/ = 0.00397 cm(-1), and /D/ = 0.0405 cm(-1) and /E/ = 0.00411 cm(-1). On the basis of the triplet-associated fluorescence emission profile, it is suggested that those triplets are associated with light-harvesting complex I, the peripheral antenna complex of PS I.     

Transitory forms of carotenoids: triplet state and radical cation

Abstract— Reactions between toluidine blue (a thiazine dye) and carotenoids (β-carotene and zeaxanthine) were studied by flash photolysis, in ethanolic solutions. Two types of reactions were evidenced:

• 1 a triplet-triplet energy transfer from the triplet state of monoprotonated toluidine blue to the carotenoid whose triplet state decays rapidly (t_1/2= 5μsec).
• 2 an electron transfer from the carotenoid to the mono- and bi-protonated triplet states of toluidine blue. This produces a radical-cation of the carotenoid (Car⁺) which decays slowly (t_1/2? 200 μsec) and has a strong absorption band around 900 nm.

     

CAROTENOID PIGMENTS AND THE IN VIVO SPECTRUM OF BACTERIOCHLOROPHYLL

Abstract— The isolation of a mutant, strain PM-9, of Rhodopseudonionus spheroides with an abnormal complement of carotenoid pigments is described.
PM-9 accumulates phytoene, phytofluene, ζ-carotene and neurosporene. Semi-aerobic cultures form more ζ-carotene and neurosporene relative to total carotenoids than do photo-synthetic cultures.
PM-9 is killed on exposure to light and oxygen.
By making use of the effect of oxygen on the nature of the carotenoids in PM-9, we have shown that these pigments do not directly influence the in vivo spectrum of bacteriochlorophyll.
Diphenylamine inhibits the synthesis of coloured carotenoids in Rhodopseudonionos gelatinosa but does not change the bacteriochlorophyll spectrum.     

POLARIZED SPECTRA OF ORIENTED PIGMENT-LIPOPROTEIN COMPLEXES FROM THE PURPLE BACTERIUM: Chromatium minutissimum

Abstract— Polarized absorption, fluorescence and photoacoustic spectra of bacteriochlorophyll (BChl)-lipoprotein complexes from the purple bacterium Chromatium minutissimum oriented in stretched polyvinylalcohol films were measured at room temperature and 85 K. The preparations contain large amounts of the B800-820 antenna complexes. From polarized absorption spectra taken under various light beam incidence angles with respect to the film plane, conclusions concerning arrangement of pigment molecules in B800-820 complex are obtained. The transition moments of the BChl Q_y band are not exactly parallel to the membrane plane. It seems that there are pools of differently oriented BChl chromophores absorbing in both 800 nm and 820 nm regions. Change in temperature strongly influences linear dichroism of carotenoids and BChl Q_y bands. The reversible changes in absorption, linear dichroism and photoacoustic spectra caused by the variation in sample temperature suggest strongly the reversible twisting of carotenoid molecules, related probably to modification of the interactions between carotenoids and proteins. Various carotenoids exhibit different yield of thermal deactivation and this yield is also temperature dependent.     

Ultrafast dynamics and excited state spectra of open-chain carotenoids at room and low temperatures

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 (2(1)Ag-) state, the polyene unravels and flattens into a more planar geometry with comparable populations of 6-s-trans and 6-s-cis conformations.