Fluorescence properties of the allenic carotenoid fucoxanthin: Implication for energy transfer in photosynthetic pigment systems

The fluorescence spectrum of an allenic carotenoid, all-trans-fucoxanthin isolated from a brown alga, has been reported for the first time. This carotenoid is known to function efficiently as a primary photosynthetic antenna pigment in marine algae. The emission bands were located around 630, 685 and 750 nm in CS₂ at 20°C, absorption bands being located at 448, 476 and 505 nm. The energy difference between the 0-0 bands of absorption and emission spectra was about 3900 cm^-1 and location of the emission maximum was less sensitive to the polarizability of solvents than that of the absorption maximum. These clearly indicate that the emission originates from the optically forbidden singlet state (2A_g). This is in contrast to other carotenoids whose emission is assigned to 1B_u state, probably due to the symmetric structure of the conjugated double bond responsible for the absorption in the visible region. A rapid internal conversion from 1B_u to 2A_g state might be facilitated by distorted structure of the conjugated double bond of fucoxanthin. The energy level responsible for the emission is almost identical to the Q_y level of the acceptor molecule (Chl a), thus we propose an energy transfer pathway from the optically forbidden 2A_g state of the carotenoid to the Q_y transition of Chl a in algal pigment systems.     

Key cofactors of Photosystem II cores from four organisms identified by 1.7-K absorption, CD and MCD

Active Photosystem II (PS II) cores were prepared from spinach, pea, Synechocystis PCC 6803, and Thermosynechococcus vulcanus, the latter of which has been structurally determined [Kamiya and Shen (2003) Proc Natl Acad Sci USA 100: 98–103]. Electrochromic shifts resulting from Q_A reduction by 1.7-K illumination were recorded, and the Q_x and Q_y absorption bands of the redox-active pheophytin a thus identified in the different organisms. The Q_x transition is ∼3 nm (100 cm⁻¹) to higher energy in cyanobacteria than in the plants. The predominant Q_y shift appears in the range 683–686 nm depending on species, and does not appear to have a systematic shift. Low-temperature absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of the chlorophyll Q_y region are very similar in spinach and pea, but vary in cyanobacteria. We assigned CP43 and CP47 trap-chlorophyll absorption features in all species, as well as a P680 transition. Each absorption identified has an area of one chlorophyll a. The MCD deficit, introduced previously for spinach as an indicator of P680 activity, occurs in the same spectral region and has the same area in all species, pointing to a robustness of this as a signature for P680. MCD and CD characteristics point towards a significant variance in P680 structure between cyanobacteria, thermophilic cyanobacteria, and higher plants.     

Excitation dynamics of two spectral forms of the core complexes from photosynthetic bacterium Thermochromatium tepidum

The intact core antenna-reaction center (LH1-RC) core complex of thermophilic photosynthetic bacterium Thermochromatium (Tch.) tepidum is peculiar in its long-wavelength LH1-Q_y absorption (915 nm). We have attempted comparative studies on the excitation dynamics of bacteriochlorophyll (BChl) and carotenoid (Car) between the intact core complex and the EDTA-treated one with the Q_y absorption at 889 nm. For both spectral forms, the overall Car-to-BChl excitation energy transfer efficiency is determined to be ∼20%, which is considerably lower than the reported values, e.g., ∼35%, for other photosynthetic purple bacteria containing the same kind of Car (spirilloxanthin). The RC trapping time constants are found to be 50∼60 ps (170∼200 ps) for RC in open (closed) state irrespective to the spectral forms and the wavelengths of Q_y excitation. Despite the low-energy LH1-Q_y absorption, the RC trapping time are comparable to those reported for other photosynthetic bacteria with normal LH1-Q_y absorption at 880 nm. Selective excitation to Car results in distinct differences in the Q_y-bleaching dynamics between the two different spectral forms. This, together with the Car band-shift signals in response to Q_y excitation, reveals the presence of two major groups of BChls in the LH1 of Tch. tepidum with a spectral heterogeneity of ∼240 cm⁻¹, as well as an alteration in BChl-Car geometry in the 889-nm preparation with respect to the native one.     

Examination of the putative Ca<Superscript>2+</Superscript>-binding site in the light-harvesting complex 1 of thermophilic purple sulfur bacterium <Emphasis Type="Italic">Thermochromatium</Emphasis><Emphasis Type="Italic">tepidum</Emphasis>

The core light-harvesting complex (LH1) of purple sulfur photosynthetic bacterium Thermochromatium tepidum exhibits an unusual absorption maximum at 915 nm for the Q _y transition, and is highly stable when copurified with reaction center (RC) in a LH1–RC complex form. In previous studies, we demonstrated that the calcium ions are involved in both the large red shift and the enhanced thermal stability, and possible Ca²⁺-binding sites were proposed. In this study, we further examine the putative binding sites in the LH1 polypeptides using purified chromatophores. Incubation of the chromatophores in the presence of EDTA revealed no substantial change in the absorption maximum of LH1 Q _y transition, whereas further addition of detergents to the chromatophores-EDTA solution resulted in a blue-shift for the LH1 Q _y peak with the final position at 892 nm. The change of the LH1 Q _y peak to shorter wavelengths was relatively slow compared to that of the purified LH1–RC complex. The blue-shifted LH1 Q _y transition in chromatophores can be restored to its original position by addition of Ca²⁺ ions. The results suggest that the Ca²⁺-binding site is exposed on the inner surface of chromatophores, corresponding to the C-terminal region of LH1. An Asp-rich fragment in the LH1 α-polypeptide is considered to form a crucial part of the binding network. The slow response of LH1 Q _y transition upon exposure to EDTA is discussed in terms of the membrane environment in the chromatophores.     

Pigment organization and energy transfer in the green photosynthetic bacterium Chloroflexus aurantiacus

The transfer of excitation energy and the pigment arrangement in isolated chlorosomes of the thermophilic green bacterium Chloroflexus aurantiacus were studied by means of absorption, fluorescence and linear dichroism spectroscopy, both at room temperature and at 4 K. The low temperature absorption spectrum shows bands of the main antenna pigments BChl c and carotenoid, in addition to which bands of BChl a are present at 798 and 613 nm. Fluorescence measurements showed that excitation energy from BChl c and carotenoid is transferred to BChl a, which presumably functions as an intermediate in energy transfer from the chlorosome to the cytoplasmic membrane. Measurements of fluorescence polarization and the use of two different orientation techniques for linear dichroism experiments enabled us to determine the orientation of several transition dipole moments with respect to each other and to the three principal axes of the chlorosome. The Q_y transition of BChl a is oriented almost perfectly perpendicular to the long axis of the chlorosome. The Q_y transition of BChl c and the -carotene transition dipole are almost parallel to each other. They make an angle of about 40° with the long axis and of about 70° with the short axis of the chlorosome; the angle between these transitions and the BChl a Q_y transition is close to the magic angle (55°).Abbreviations BChl bacteriochlorophyll - CD circular dichroism - LD linear dichroism Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.     

Excitation delocalization in the bacteriochlorophyll c antenna of the green bacterium Chloroflexus aurantiacus as revealed by ultrafast pump-probe spectroscopy

Room temperature absorption difference spectra were measured on the femtosecond through picosecond time scales for chlorosomes isolated from the green bacterium Chloroflexus aurantiacus. Anomalously high values of photoinduced absorption changes were revealed in the BChl c Q_y transition band. Photoinduced absorption changes at the bleaching peak in the BChl c band were found to be 7–8 times greater than those at the bleaching peak in the BChl a band of the chlorosome. This appears to be the first direct experimental proof of excitation delocalization over many BChl c antenna molecules in the chlorosome.     

Antenna organization in the purple sulfur bacteria Chromatium tepidum and Chromatium vinosum

Structural aspects of the core antenna in the purple sulfur bacteria Chromatium tepidum and Chromatium vinosum were studied by means of fluorescence emission and singlet-singlet annihilation measurements. In both species the number of bacteriochlorophylls of the core antenna between which energy transfer can occur corresponds to one core-reaction center complex only. From measurements of variable fluorescence we conclude that in C. tepidum excitation energy can be transferred back from the core antenna (B920) to the peripheral B800–850 complex in spite of the relatively large energy gap, and on basis of annihilation measurements a model of separate core-reaction center units accompanied by their own peripheral antenna is suggested. C. vinosum contains besides a core antenna, B890, two peripheral antennae, B800–820 and B800–850. Energy transfer was found to occur from the core to B800–850, but not to B800–820, and it was concluded that in C. vinosum each core-reaction center complex has its own complement of B800–850. The results reported here are compared to those obtained earlier with various strains and species of purple non-sulfur bacteria.Abbreviations BChl- bacteriochlorophyll - B800–820 and B800–850- antenna complexes with Q_y-band absorption maxima near 800 nm and 820 or 850 nm, respectively - B890 and B920- antenna complexes with Q_y-band absorption maxima near 890 and 920 nm, respectively - LH1- light harvesting 1 or core antenna - LH2- light harvesting 2 or peripheral antenna     

Tuning antenna function through hydrogen bonds to chlorophyll a

We describe a molecular mechanism tuning the functional properties of chlorophyll a (Chl-a) molecules in photosynthetic antenna proteins. Light-harvesting complexes from photosystem II in higher plants – specifically LHCII purified with α- or β-dodecyl-maltoside, along with CP29 – were probed by low-temperature absorption and resonance Raman spectroscopies. We show that hydrogen bonding to the conjugated keto carbonyl group of protein-bound Chl-a tunes the energy of its Soret and Q_y absorption transitions, inducing red-shifts that are proportional to the strength of the hydrogen bond involved. Chls-a with non-H-bonded keto C13¹ groups exhibit the blue-most absorption bands, while both transitions are progressively red-shifted with increasing hydrogen-bonding strength – by up 382 & 605 cm⁻¹ in the Q_y and Soret band, respectively. These hydrogen bonds thus tune the site energy of Chl-a in light-harvesting proteins, determining (at least in part) the cascade of energy transfer events in these complexes.     

Probing binding site of bacteriochlorophyll a and carotenoid in the reconstituted LH1 complex from Rhodospirillum rubrum S1 by Stark spectroscopy

Stark spectroscopy is a powerful technique to investigate the electrostatic interactions between pigments as well as between the pigments and the proteins in photosynthetic pigment–protein complexes. In this study, Stark spectroscopy has been used to determine two nonlinear optical parameters (polarizability change Tr(Δα) and static dipole-moment change |Δμ| upon photoexcitation) of isolated and of reconstituted LH1 complexes from the purple photosynthetic bacterium, Rhodospirillum (Rs.) rubrum. The integral LH1 complex was prepared from Rs. rubrum S1, while the reconstituted complex was assembled by addition of purified carotenoid (all-trans-spirilloxanthin) to the monomeric subunit of LH1 from Rs. rubrum S1. The reconstituted LH1 complex has its Q_y absorption maximum at 878 nm. This is shifted to the blue by 3 nm in comparison to the isolated LH1 complex. The energy transfer efficiency from carotenoid to bacteriochlorophyll a (BChl a), which was determined by fluorescence excitation spectroscopy of the reconstituted LH1 complex, is increased to 40%, while the efficiency in the isolated LH1 complex is only 28%. Based on the differences in the values of Tr(Δα) and |Δμ|, between these two preparations, we can calculate the change in the electric field around the BChl a molecules in the two situations to be E _Δ ≈ 3.4 × 10⁵ [V/cm]. This change can explain the 3 nm wavelength shift of the Q_y absorption band in the reconstituted LH1 complex.     

Excitation energy transfer in chlorosomes of Chlorobium phaeobacteroides strain CL1401: the role of carotenoids

The role of carotenoids in chlorosomes of the green sulfur bacterium Chlorobium phaeobacteroides, containing bacteriochlorophyll (BChl) e and the carotenoid (Car) isorenieratene as main pigments, was studied by steady-state fluorescence excitation, picosecond single-photon timing and femtosecond transient absorption (TA) spectroscopy. In order to obtain information about energy transfer from Cars in this photosynthetic light-harvesting antenna with high spectral overlap between Cars and BChls, Car-depleted chlorosomes, obtained by inhibition of Car biosynthesis by 2-hydroxybiphenyl, were employed in a comparative study with control chlorosomes. Excitation spectra measured at room temperature give an efficiency of 60–70% for the excitation energy transfer from Cars to BChls in control chlorosomes. Femtosecond TA measurements enabled an identification of the excited state absorption band of Cars and the lifetime of their S₁ state was determined to be 10 ps. Based on this lifetime, we concluded that the involvement of this state in energy transfer is unlikely. Furthermore, evidence was obtained for the presence of an ultrafast (>100 fs) energy transfer process from the S₂ state of Cars to BChls in control chlorosomes. Using two time-resolved techniques, we further found that the absence of Cars leads to overall slower decay kinetics probed within the Q_y band of BChl e aggregates, and that two time constants are generally required to describe energy transfer from aggregated BChl e to baseplate BChl a.This revised version was published online in October 2005 with corrections to the Cover Date.     

Absorption and linear dichroism spectroscopy at room and low temperatures of single crystals of the B800–850 light-harvesting complex from Rhodopseudomonas acidophila strain 10050

The absorbance, polarized absorbance and linear dichroism spectra of single crystals of the B800–850 light-harvesting complex from Rhodopseudomonas acidophila strain 10050 taken at room (298 K) and low (85 K) temperatures are presented. The spectra are compared and contrasted with random phase solution spectra from the same complex. The single crystal spectra display a spectral narrowing at low temperatures in the BChl Q_x (550–650 nm) and carotenoid (450–550 nm) regions similar to that observed from the random phase solution. The single crystal absorption spectra in the BChl Q_y (750–900 nm) region are broader than the solution spectra and remain broad as the temperature is lowered. It is suggested that this broadening is the result of specific exciton interactions between the BChl chromophore Q_y transition dipoles and is a molecular feature which occurs only in the crystalline complex.     

The search for an optimal orientational ordering of <Emphasis Type="Italic">Q</Emphasis><Subscript>y</Subscript> transition dipoles of subantenna molecules in the superantenna of photosynthetic green bacteria: Model calculations

This work continues the series of our studies on the basic principles in the organization of natural light-harvesting antennae, which we theoretically predicted for the optimal model light-harvesting systems, initiated by our concept of the rigorous optimization of photosynthetic apparatus structure by functional criterion. This approach allowed us to determine several main principles in the organization of the natural systems. This work deals with the problem of the structure optimization of heterogeneous superantennae of photosynthetic green bacteria, consisting of several uniform subantennae, which raises the problem of their optimal coordination. Here we used mathematical modeling of the functioning of these natural superantennae to consider a possible optimization of this process via optimizing the mutual spatial orientation of Q _y transition dipole vectors of the light-harvesting molecules in neighboring subantennae. This allowed us to determine possible modes for optimal orientational ordering of Q _y transition dipoles of subantenna molecules in the model of green bacterium Chloroflexus aurantiacus superantenna. The calculations have demonstrated that the optimal mutual spatial orientation of the of Q _y dipoles in subantenna pigments leads to a stable minimization of the energy transfer time within superantennae and, consequently, a decrease in the energy losses, thereby ensuring a high efficiency and stability of the overall superantenna function.     

Effects of detergent on the excited state structure and relaxation dynamics of the photosystem II reaction center: A high resolution hole burning study

Low temperature (4.2 K) absorption and hole burned spectra are reported for a stabilized preparation (no excess detergent) of the photosystem II reaction center complex. The complex was studied in glasses to which detergent had and had not been added. Triton X-100 (but not dodecyl maltoside) detergent was found to significantly affect the absorption and persistent hole spectra and to disrupt energy transfer from the accessory chlorophyll a to the active pheophytin a. However, Triton X-100 does not significantly affect the transient hole spectrum and lifetime (1.9 ps at 4.2 K) of the primary donor state, P680^*. Data are presented which indicate that the disruptive effects of Triton X-100 are not due to extraction of pigments from the reaction center, leaving structural perturbations as the most plausible explanation. In the absence of detergent the high resolution persistent hole spectra yield an energy transfer decay time for the accessory Chl a Q_Y-state at 1.6 K of 12 ps, which is about three orders of magnitude longer than the corresponding time for the bacterial RC. In the presence of Triton X-100 the Chl a Q_Y-state decay time is increased by at least a factor of 50.Abbreviations PS I photosystem I - PS II photosystem II - RC reaction center - P680, P870, P960 the primary electron donor absorption bands of photosystem II, Rhodobacter sphaeroides, Rhodopseudomonas viridis - NPHB nonphotochemical hole burning - TX Triton X-100 - DM Dodecyl Maltoside - Chl chlorophyll - Pheo pheophytin - ZPH ero phonon hole     

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.     

Pigment organization and energy transfer in the green photosynthetic bacterium Chloroflexus aurantiacus

We have studied the pigment arrangement in purified cytoplasmic membranes of the thermophilic green bacterium Chloroflexus aurantiacus. The membranes contain 30–35 antenna bacteriochlorophyll a molecules per reaction center; these are organized in the B808–866 light-harvesting complex, together with carotenoids in a 2:1 molar ratio. Measurements of linear dichroism in a pressed polyacrylamide gel permitted the accurate determination of the orientation of the optical transition dipole moments with respect to the membrane plane. Combination of linear dichroism and low temperature fluorescence polarization data shows that the Q_y transitions of the BChl 866 molecules all lie almost perfectly parallel to the membrane plane, but have no preferred orientation within the plane. The BChl 808 Q_y transitions make an average angle of about 44° with this plane. This demonstrates that there are clear structural differences between the B808–866 complex of C. aurantiacus and the B800–850 complex of purple bacteria. Excitation energy transfer from carotenoid to BChl a proceeds with about 40% efficiency, while the efficiency of energy transfer from BChl 808 to BChl 866 approaches 100%. From the minimal energy transfer rate between the two spectral forms of BChl a, obtained by analysis of low temperature fluorescence emission spectra, a maximal distance between BChl 808 and BChl 866 of 23 was derived.Abbreviations BChl bacteriochlorophyll - BPheo bacteriopheophytin - CD circular dichroism - LD linear dichroism - Tris Tris(hydroxymethyl)aminomethane     

Linear dichroism of electric field oriented bacteriochlorophyll a-protein from green photosynthetic bacteria

Bacteriochlorophyll a-protein from Prosthecochloris aestuarii strain 2K was oriented in a pulsed electric field. The room temperature linear dichroism spectrum of the oriented protein in the Q_y region of the bacteriochlorophyll a absorption exhibits a single asymmetrical peak at 813 nm with a shoulder extending to the blue. The ≈12 nm fullwidth of the linear dichroism peak is only about half that of the 300 K absorption spectrum. The linear dichroism at 813 nm was not saturated at field strengths of up to 15 kV/cm. The time dependence of the linear dichroism suggests that the orienting particles are aggregates of at least some tens of bacteriochlorophyll a-protein trimers. The linear dichroism peak coincides in wavelength with the 813-nm peak of the 300 K, 4th derivative absorption spectrum of the protein and is therefore attributed to the bacteriochlorophyll a Q_y exciton transition observed in absorption at the same wavelength.     

Fluorescence properties of the light-harvesting bacteriochlorophyll protein from Rhodopseudomonas sphaeroides R-26

The light-harvesting bacteriochlorophyll-protein (BChl-protein) from Rhodopseudomonas sphaeroides, R-26 mutant, exhibits a strong optical absorption peak near 850 nm (Q_y band) and a weaker peak at 590 nm (Q_x band). This pigment-protein appears to contain two BChl molecules per subunit, and previous circular dichroism studies indicated the presence of excitonic interactions between the BChl molecules. The complex exhibits a fluorescence maximum near 870 nm at room temperature. Excitation in the Q_y region results in polarization p values that vary only from +0.12 at 820 nm to +0.14 near 900 nm. These values are appreciably smaller than that for monomeric BChl in viscous solvents (p > 0.4). By contrast, using Q_x excitation the p value is ?0.25 for the BChl-protein complex, which is close to that observed for the BChl monomer. For the BChl-protein these polarization values do not change greatly at a temperature of 90 K; however, the Stokes' shift of the fluorescence emission increases significantly over that at room temperature.     

Low-temperature optical properties and pigment organization of the B875 light-harvesting bacteriochlorophyll-protein complex of purple photosynthetic bacteria

Optical and structural properties of the B875 light-harvesting complex of purple bacteria were examined by measurements of low-temperature circular dichroism (CD) and excitation spectra of fluorescence polarization. In the B875 complex isolated from wild-type Rhodopseudomonas sphaeroides, fluorescence polarization increased steeply across the long-wavelength Q_y bacteriochlorophyll a (BChl) absorption band at both 4 and approx. 300 K. With the native complex in the photosynthetic membranes of Rhodospirillum rubrum and Rps. sphaeroides wild-type and R26-carotenoidless strains, this significant increase in polarization from 0.12 to 0.40 was only observed at low temperature. A polarization of ?0.2 was observed upon excitation in the Q_x BChl band. The results indicate that about 15% of the BChl molecules in the complex absorb at wavelengths about 12 nm longer than the other BChls. All BChls have approximately the same orientation with their Q_y transition dipoles essentially parallel and their Q_x transitions perpendicular to the plane of the membrane. At low temperature, energy transfer to the long-wavelength BChls is irreversible, yielding a high degree of polarization upon direct excitation, whereas at room temperature a partial depolarization of fluorescence by energy transfer between different subunits occurs in the membrane, but not in the isolated complex. CD spectra appear to reflect the two spectral forms of B875 BChl in Rps. sphaeroides membranes. They also reveal structural differences between the complexes of Rps. sphaeroides and Rhs. rubrum, in both BChl and carotenoid regions. The CD spectrum of isolated B875 indicates that the interactions between the BChls but not the carotenoids are altered upon isolation.     

The role of chromophore coupling in tuning the spectral properties of peripheral light-harvesting protein of purple bacteria

The publication of a structure for the peripheral light-harvesting complex of a purple photosynthetic bacterium (McDermott et al. (1995), Nature 374: 517–521) provides a framework within which we can begin to understand various functional aspects of these complexes, in particular the relationship between the structure and the red-shift of the bacteriochlorophyll Q_y transition. In this article we describe calculations of some of the spectral properties expected for an array of chromophores with the observed geometry. We report the stability of the calculated absorption spectrum to minor structural alterations, and deduce that the observed red shift of the 850 nm Q_y transition in the B800–850 antenna complexes is about equally attributable to chromophore-chromophore and chromophore-protein interactions, while chromophore-chromophore interactions predominate in generating the red-shift of the 820 nm Q_y transition in B800–820 type peripheral liggt-harvesting complexes. Finally we suggest that the red shift in the absorbance of the monomeric Bchl a found in antenna complexes to 800 nm, from 770 nm as observed in most solvents, is largely attributable to a hydrogen bond with the 2-acetyl group of this chromophore.