Single-Molecule Spectroscopy Unmasks the Lowest Exciton State of the B850 Assembly in LH2 from Rps. acidophila

We have recorded fluorescence-excitation and emission spectra from single LH2 complexes from Rhodopseudomonas (Rps.) acidophila. Both types of spectra show strong temporal spectral fluctuations that can be visualized as spectral diffusion plots. Comparison of the excitation and emission spectra reveals that for most of the complexes the lowest exciton transition is not observable in the excitation spectra due to the cutoff of the detection filter characteristics. However, from the spectral diffusion plots we have the full spectral and temporal information at hand and can select those complexes for which the excitation spectra are complete. Correlating the red most spectral feature of the excitation spectrum with the blue most spectral feature of the emission spectrum allows an unambiguous assignment of the lowest exciton state. Hence, application of fluorescence-excitation and emission spectroscopy on the same individual LH2 complex allows us to decipher spectral subtleties that are usually hidden in traditional ensemble spectroscopy.     

Spectroscopy on the B850 band of individual light-harvesting 2 complexes of Rhodopseudomonas acidophila. II. Exciton states of an elliptically deformed ring aggregate

Spectroscopy of individual light-harvesting 2 complexes from purple photosynthetic bacteria revealed a deformation of the circular complex into C(2) symmetry. The present work relates the geometry of the deformed aggregate to its spectroscopic properties. Different models of elliptical deformation are discussed and compared with the experimental findings. It is shown that the model with smaller interpigment distances, where the curvature of the ellipse is small, provides the best agreement with fluorescence excitation spectra of individual complexes.     

Comparison of the fluorescence kinetics of detergent-solubilized and membrane-reconstituted LH2 complexes from Rps. acidophila and Rb. sphaeroides

Picosecond time-resolved fluorescence spectroscopy has been used in order to compare the fluorescence kinetics of detergent-solubilized and membrane-reconstituted light-harvesting 2 (LH2) complexes from the purple bacteria Rhodopseudomonas (Rps.) acidophila and Rhodobacter (Rb.) sphaeroides. LH2 complexes were reconstituted in phospholipid model membranes at different lipid:protein-ratios and all samples were studied exciting with a wide range of excitation densities. While the detergent-solubilized LH2 complexes from Rps. acidophila showed monoexponential decay kinetics (τ_f = 980 ps) for excitation densities of up to 3·10¹³ photons/(pulse·cm²), the membrane-reconstituted LH2 complexes showed multiexponential kinetics even at low excitation densities and high lipid:protein-ratios. The latter finding indicates an efficient clustering of LH2 complexes in the phospholipid membranes. Similar results were obtained for the LH2 complexes from Rb. sphaeroides. Guest editor: Dr. Conrad Mullineaux.     

The structure and thermal motion of the B800-850 LH2 complex from Rps.acidophila at 2.0A resolution and 100K: new structural features and functionally relevant motions

The structure at 100K of integral membrane light-harvesting complex II (LH2) from Rhodopseudomonas acidophila strain 10050 has been refined to 2.0A resolution. The electron density has been significantly improved, compared to the 2.5A resolution map, by high resolution data, cryo-cooling and translation, libration, screw (TLS) refinement. The electron density reveals a second carotenoid molecule, the last five C-terminal residues of the alpha-chain and a carboxy modified alpha-Met1 which forms the ligand of the B800 bacteriochlorophyll. TLS refinement has enabled the characterisation of displacements between molecules in the complex. B850 bacteriochlorophyll molecules are arranged in a ring of 18 pigments composed of nine approximate dimers. These pigments are strongly coupled and at their equilibrium positions the excited state dipole interaction energies, within and between dimers, are approximately 370cm(-1) and 280cm(-1), respectively. This difference in coupling energy is similar in magnitude to changes in interaction energies arising from the pigment displacements described by TLS tensors. The displacements appear to be non-random in nature and appear to be designed to optimise the modulation of pigment energy interactions. This is the first time that LH2 pigment displacements have been quantified experimentally. The calculated energy changes indicate that there may be significant contributions to inter-pigment energy interactions from molecular displacements and these may be of importance to photosynthetic energy transfer.     

Spectroscopy on the B850 band of individual light-harvesting 2 complexes of Rhodopseudomonas acidophila. I. Experiments and Monte Carlo simulations

The electronic structure of the circular aggregate of 18 bacteriochlorophyll a (BChl a) molecules responsible for the B850 absorption band of the light-harvesting 2 (LH2) complex of the photosynthetic purple bacterium Rhodopseudomonas acidophila has been studied by measuring fluorescence-excitation spectra of individual complexes at 1.2 K. The spectra reveal several well-resolved bands that are obscured in the single, broad B850 band observed in conventional absorption measurements on bulk samples. They are interpreted consistently in terms of the exciton model for the circular aggregate of BChl a molecules. From the energy separation between the different exciton transitions a reliable value of the intermolecular interaction is obtained. The spectra of the individual complexes allow for a distinction between the intra- and the intercomplex disorder. In addition to the random disorder, a regular modulation of the interaction has to be assumed to account for all the features of the observed spectra. This modulation has a C(2) symmetry, which strongly suggests a structural deformation of the ring into an ellipse.     

Single-Molecule Spectroscopy Reveals that Individual Low-Light LH2 Complexes from Rhodopseudomonas palustris 2.1.6. Have a Heterogeneous Polypeptide Composition

Rhodopseudomonas palustris belongs to the group of purple bacteria that have the ability to produce LH2 complexes with unusual absorption spectra when they are grown at low-light intensity. This ability is often related to the presence of multiple genes encoding the antenna apoproteins. Here we report, for the first time to our knowledge, direct evidence that individual low-light LH2 complexes have a heterogeneous αβ-apoprotein composition that modulates the site energies of Bchl a molecules, producing absorption bands at 800, 820, and 850 nm. The arrangement of the Bchl a molecules in the “tightly coupled ring” can be modeled by nine αβ-Bchls dimers, such that the Bchls bound to six αβ-pairs have B820-like site energies and the remaining Bchl a molecules have B850-like site energies. Furthermore, the experimental data can only be satisfactorily modeled when these six αβ-pairs with B820 Bchl a molecules are distributed such that the symmetry of the assembly is reduced to C₃. It is also clear from the measured single-molecule spectra that the energies of the electronically excited states in the mixed B820/850 ring are mainly influenced by diagonal disorder.     

Characterisation of hydrophobic peptides by RP-HPLC from different spectral forms of LH2 isolated from Rps. palustris

The separation of light-harvesting peptides by RP-HPLC is notoriously difficult due to the typically strong interaction of peptides with the column matrix, their relatively low solubility in the mobile phase and the tendency for non-specific aggregation during sample preparation. This paper illustrates a reproducible method for investigating the composition of four spectrally different forms of LH2 isolated from Rps. palustris. The method contrasts with previous attempts to isolate peptides from these multi-LH2 complexes and uses the well characterised B800–850 complex from Rps. acidophila as a test of reliability. Three pairs of LH2 peptides, _a, _b and _d, were identified from Rps. palustris grown under high- (7000 lux) or intermediate- (1000 lux) light conditions. At lower light (300 and 90 lux), _b was absent, and the level of _a was significantly reduced. Results show that _a and _b peptides form the high light B800-850 complex, whereas the low light LH2 complex is only composed of _d peptides and resembles the B800–820 complex from Rps. acidophila by sequence homology. The absorption spectrum of this complex has a single peak centred on 800 nm and appears to be a novel LH2 complex. At low light growth conditions, this B800 species is the predominant LH2 complex in Rps. palustris and indicates that peptide expression is a crucial factor in adapting to different light intensities.     

Fluorescence spectral fluctuations of single LH2 complexes from Rhodopseudomonas acidophila strain 10050

We have investigated the energy landscape of the bacterial photosynthetic peripheral light-harvesting complex LH2 of purple bacterium Rhodopseudomonas acidophila by monitoring sequences of fluorescence spectra of single LH2 assemblies, at room temperature, with different excitation intensities as well as at elevated temperatures, utilizing a confocal microscope. The fluorescence peak wavelength of individual LH2 complexes was found to abruptly move between quasi-stable levels differing by up to 30 nm. These spectral shifts either to the blue or to the red were accompanied by a broadening and decrease of the intensity of the fluorescence spectrum. The frequency and size of these fluorescence peak movements were found to increase linearly with excitation intensity. Using the modified Redfield theory, changes in the realization of the static disorder accounted for the observed changes in spectral shape and intensity. Long lifetimes of the quasi-stable states suggest large free energy barriers between the different realizations.     

Crystallization and characterization of two crystal forms of the B800-850 light-harvesting complex from Rhodopseudomonas acidophila strain 10050

Two different crystal forms of the B800-850-antenna complex from Rhodopseudomonas acidophila strain 10050 have been grown. This complex is an integral membrane protein and is isolated as an oligomeric assembly with a molecular weight of approximately 84 kDa. This assembly contains six alpha/beta apoprotein pairs, 18 molecules of bacteriochlorophyll a and nine molecules of carotenoid. The first crystal form has dimensions unit cell a = b = 75.8 A, c = 97.5 A with the space group P4 and diffracts to a resolution of 12.0 A. The second crystal form is rhombohedral with dimensions unit cell a = 121.1 A, alpha = 60 degrees, space group R32 and diffracts to a resolution of 3.5 A. Native data have been processes in both cases, to an Rmerge value of 9.0 to 11.0%. The X-ray data suggest that the asymmetric unit, in both crystal forms, contains one 84 kDa antenna complex.     

Detergent structure in crystals of the integral membrane light-harvesting complex LH2 from Rhodopseudomonas acidophila strain 10050

Integral membrane proteins are solubilized by their incorporation into a detergent micelle. The detergent micelle has a critical influence on the formation of a three-dimensional crystal lattice. The bulk detergent phase is not seen in X-ray crystal structures of integral membrane proteins, due to its disordered character. Here, we describe the detergent structure present in crystals of the peripheral light-harvesting complex of the purple bacteria Rhodopseudomonas acidophila strain 10050 at a maximal resolution of 12A as determined by neutron crystallography. The LH2 molecule has a toroidal shape and spans the membrane completely in vivo. A volume of 16% of the unit cell could be ascribed to detergent tails, localized on both the inner and outer hydrophobic surfaces of the molecule. The detergent tail volumes were found to be associated with individual LH2 molecules and had no direct role in the formation of the crystalline lattice.     

Selective release, removal, and reconstitution of bacteriochlorophyll a molecules into the B800 sites of LH2 complexes from Rhodopseudomonas acidophila 10050.

A method is described which allows the selective release and removal of the Bchla-B800 molecules from the LH2 complex of Rhodopseudomonas acidophila 10050. This procedure also allows reconstitution of approximately 80% of the empty binding sites with native Bchla. As shown by circular dichroism spectroscopy, the overall structures of the B850-only and reconstituted complexes are not affected by the pigment-exchange procedure. The pigments reconstituted into the B800 sites can also efficiently transfer excitation energy to the Bchla-B850 molecules.     

B800-->B850 energy transfer mechanism in bacterial LH2 complexes investigated by B800 pigment exchange

Femtosecond transient absorption measurements were performed on native and a series of reconstituted LH2 complexes from Rhodopseudomonas acidophila 10050 at room temperature. The reconstituted complexes contain chemically modified tetrapyrrole pigments in place of the native bacteriochlorophyll a-B800 molecules. The spectral characteristics of the modified pigments vary significantly, such that within the B800 binding sites the B800 Q(y) absorption maximum can be shifted incrementally from 800 to 670 nm. As the spectral overlap between the B800 and B850 Q(y) bands decreases, the rate of energy transfer (as determined by the time-dependent bleaching of the B850 absorption band) also decreases; the measured time constants range from 0.9 ps (bacteriochlorophyll a in the B800 sites, Q(y) absorption maximum at 800 nm) to 8.3 ps (chlorophyll a in the B800 sites, Q(y) absorption maximum at 670 nm). This correlation between energy transfer rate and spectral blue-shift of the B800 absorption band is in qualitative agreement with the trend predicted from F?rster spectral overlap calculations, although the experimentally determined rates are approximately 5 times faster than those predicted by simulations. This discrepancy is attributed to an underestimation of the electronic coupling between the B800 and B850 molecules.     

The crystallographic structure of the B800-820 LH3 light-harvesting complex from the purple bacteria Rhodopseudomonas acidophila strain 7050

The B800-820, or LH3, complex is a spectroscopic variant of the B800-850 LH2 peripheral light-harvesting complex. LH3 is synthesized by some species and strains of purple bacteria when growing under what are generally classed as "stressed" conditions, such as low intensity illumination and/or low temperature (<30 degrees C). The apoproteins in these complexes modify the absorption properties of the chromophores to ensure that the photosynthetic process is highly efficient. The crystal structure of the B800-820 light-harvesting complex, an integral membrane pigment-protein complex, from the purple bacteria Rhodopseudomonas (Rps.) acidophila strain 7050 has been determined to a resolution of 3.0 A by molecular replacement. The overall structure of the LH3 complex is analogous to that of the LH2 complex from Rps. acidophila strain 10050. LH3 has a nonameric quaternary structure where two concentric cylinders of alpha-helices enclose the pigment molecules bacteriochlorophyll a and carotenoid. The observed spectroscopic differences between LH2 and LH3 can be attributed to differences in the primary structure of the apoproteins. There are changes in hydrogen bonding patterns between the coupled Bchla molecules and the protein that have an effect on the conformation of the C3-acetyl groups of the B820 molecules. The structure of LH3 shows the important role that the protein plays in modulating the characteristics of the light-harvesting system and indicates the mechanisms by which the absorption properties of the complex are altered to produce a more efficient light-harvesting component.     

Crystallization of the B800-820 light-harvesting complex from Rhodopseudomonas acidophila strain 7750.

The B800-820 light-harvesting complex, an integral membrane protein, from Rhodopseudomonas acidophila strain 7750 has been crystallized. The tabular plates have a hexagonal unit cell of a = b = 121.8 A and c = 283.1 A and belong to the space group R32. X-ray diffraction data have been collected to 6 A resolution, using an area detector on a rotating anode source. The B800-820 light-harvesting complex is comprised of four low molecular weight apoproteins (B800-820 alpha 1, B800-820 alpha 2, B800-820 beta 1 and B800-820 beta 2). Polyacrylamide gel electrophoresis shows that the complex exists as an oligomeric assembly, with an apparent molecular weight of 92,000.     

The Origin of the Low-Energy Form of Photosystem I Light-Harvesting Complex Lhca4: Mixing of the Lowest Exciton with a Charge-Transfer State

The peripheral light-harvesting complex of photosystem I contains red chlorophylls (Chls) that, unlike the typical antenna Chls, absorb at lower energy than the primary electron donor P700. It has been shown that the red-most absorption band arises from two excitonically coupled Chls, although this interaction alone cannot explain the extreme red-shifted emission (25 nm, ∼480 cm⁻¹ for Lhca4 at 4 K) that the red Chls present. Here, we report the electric field-induced absorption changes (Stark effect) on the Q_y region of the Lhca4 complex. Two spectral forms, centered around 690 nm and 710 nm, were necessary to describe the absorption and Stark spectra. The analysis of the lowest energy transition yields a high value for the change in dipole moment, Δμ_710nm ≈ 8 Df⁻¹, between the ground and excited states as compared with monomeric, Δμ = 1 D, or dimeric, Δμ = 5 D, Chl a in solution. The high value of the Δμ demonstrates that the origin of the red-shifted emission is the mixing of the lowest exciton state with a charge-transfer state of the dimer. This energetic configuration, an excited state with charge-transfer character, is very favorable for the trapping and dissipation of excitations and could be involved in the photoprotective mechanism(s) of the photosystem I complex.     

Carotenoids in LH2 Complexes from Allochromatium vinosum under Illumination Are Able to Generate Singlet Oxygen Which Oxidizes BChl850

Microbiology - The interaction of singlet oxygen with bacteriochlorophyll (BChl) in the membranes, LH2 light-harvesting complexes and pigment extracts from Allochromatium (Alc.) vinosum strain MSU...     

Spectroscopy of individual light-harvesting 2 complexes of Rhodopseudomonas acidophila: diagonal disorder, intercomplex heterogeneity, spectral diffusion, and energy transfer in the B800 band

This paper reports a detailed spectroscopic study of the B800 absorption band of individual light-harvesting 2 (LH2) complexes of the photosynthetic purple bacterium Rhodopseudomonas acidophila at 1. 2 K. By applying single-molecule detection techniques to this system, details and properties can be revealed that remain obscured in conventional ensemble experiments. For instance, from fluorescence-excitation spectra of the individual complexes a more direct measure of the diagonal disorder could be obtained. Further spectral diffusion phenomena and homogeneous linewidths of individual bacteriochlorophyll a (BChl a) molecules are observed, revealing valuable information on excited-state dynamics. This work demonstrates that it is possible to obtain detailed spectral information on individual pigment-protein complexes, providing direct insight into their electronic structure and into the mechanisms underlying the highly efficient energy transfer processes in these systems.     

5-methylthioadenosine phosphorylase from C.acidophila. 2. Identification and characterization of reaction products]

Evidences for a phosphorolitic cleavage of 5'-MTA by a specific enzyme from C. acidophila are reported. Methylthioribose-1-phosphate and adenine have been identified as reaction products, by several analytical procedures.     

Assembly of the LH2 Light-Harvesting Complexes of Thiorhodospira sibirica with Different Carotenoid Levels

Microbiology - Regardless of cultivation conditions, two types of LH2 complexes are always assembled in photosynthetic membranes of Thiorhodospira (T.) sibirica: short-wave B800?830 and...