Observation of the energy-level structure of the low-light adapted B800 LH4 complex by single-molecule spectroscopy

Low-light adapted B800 light-harvesting complex 4 (LH4) from Rhodopseudomonas palustris is a complex in which the arrangement of the bacteriochloropyll a pigments is very different from the well-known B800-850 LH2 complex. For bulk samples, the main spectroscopic feature in the near-infrared is the occurrence of a single absorption band at 802 nm. Single-molecule spectroscopy can resolve the narrow bands that are associated with the exciton states of the individual complexes. The low temperature (1.2 K) fluorescence excitation spectra of individual LH4 complexes are very heterogeneous and display unique features. It is shown that an exciton model can adequately reproduce the polarization behavior of the complex, the experimental distributions of the number of observed peaks per complex, and the widths of the absorption bands. The results indicate that the excited states are mainly localized on one or a few subunits of the complex and provide further evidence supporting the recently proposed structure model.     

Circular symmetry of the light-harvesting 1 complex from Rhodospirillum rubrum is not perturbed by interaction with the reaction center

The effect of the interaction of the reaction center (RC) upon the geometrical arrangement of the bacteriochlorophyll a (BChla) pigments in the light-harvesting 1 complex (LH1) from Rhodospirillum rubrum has been examined using single molecule spectroscopy. Fluorescence excitation spectra at 1.8 K obtained from single detergent-solubilized as well as single membrane-reconstituted LH1-RC complexes showed predominantly (>70%) a single broad absorption maximum at 880-900 nm corresponding to the Q(y) transition of the LH1 complex. This absorption band was independent of the polarization direction of the excitation light. The remaining complexes showed two mutually orthogonal absorption bands in the same wavelength region with moderate splittings in the range of DeltaE = 30-85 cm(-1). Our observations are in agreement with simulated spectra of an array of 32 strongly coupled BChla dipoles arranged in perfect circular symmetry possessing only a diagonal disorder of 相似文献   

Reconstitution of photosynthetic reaction centers and core antenna-reaction center complexes in liposomes and their thermal stability

Photosynthetic reaction centers (RCs) and their core light-harvesting complexes (LH1-RCs), purified from a thermophile, Thermochromatium (T.) tepidum, and a mesophile, Allochromatium (A.) vinosum, were reconstituted into liposomes. The RC and the LH1-RC in the reconstituted liposomes were found intact from the absorption spectra at about 4 and 40 degrees C respectively. The thermal stability of the RCs of T. tepidum in the liposome was dependent on whether they were surrounded directly by lipids or by the core light-harvesting complexes. The results show that the RC of T. tepidum gains its thermostability through interactions with the LH1. These results are consistent with the result that the thermal stability of the LH1 in T. tepidum is similar in both the reconstituted LH1-RC liposome and ICM. This is clearly different from the mesophilic bacterium, A. vinosum. The thermal stability of RC was also affected by its subunit constitution: the RC containing a cytochrome subunit was more thermostable than the cytochrome-detached RC. This suggests that the cytochrome subunit might play a role in protecting the special pair pigments from denaturation. The thermal denaturation showed a second-order reaction dependence on time. The interaction of the pigments with proteins and/or lipids might be the cause of the second-order reaction profile.     

Self-assembled monolayer of light-harvesting core complexes of photosynthetic bacteria on an amino-terminated ITO electrode

Light-harvesting antenna core (LH1-RC) complexes isolated from Rhodospirillum rubrum and Rhodopseudomonas palustris were successfully self-assembled on an ITO electrode modified with 3-aminopropyltriethoxysilane. Near infra-red (NIR) absorption, fluorescence, and IR spectra of these LH1-RC complexes indicated that these LH1-RC complexes on the electrode were stable on the electrode. An efficient energy transfer and photocurrent responses of these LH1-RC complexes on the electrode were observed upon illumination of the LH1 complex at 880 nm.     

Formation of a subunit form of the core light-harvesting complex from sulfur purple bacteria <Emphasis Type="Italic">Ectothiorhodospira haloalkaliphila</Emphasis> with different carotenoid composition

B820 subunits from a purple sulfur bacterium Ectothiorhodospira haloalkaliphila strain ATCC 51935^T were obtained by treatment of carotenoid free LH1-RC complexes of this bacterium with ß-octylglucopyranoside (ß-OG). The same complexes with 100% carotenoid content were unable to dißsociate to B820 subunits, but disintegrated to monomeric bacteriochlorophyll (BChl) regardless of their carotenoid composition. The degree of dissociation of the LH1-RC complexes with an intermediate content of carotenoids (the B820 formation) was directly dependent on the quantity of carotenoids in the samples. The resulting B820 subunits did not contain carotenoids. B820 subunits easily aggregated to form a complex with an absorption peak at 880 nm at decreased ß-OG concentration. Analysis of the spectra of the LH1-RC complexes isolated from the cells with different levels of carotenogenesis inhibition led to the conclusion of the heterogeneity of the samples with a predominance of them in (a) the fraction with 100% of carotenoids and (b) the fraction of carotenoid-free complexes.     

Spirilloxanthin incorporation into the LH2 and LH1-RC pigment-protein complexes from a purple sulfur bacterium <Emphasis Type="Italic">Allochromatium minutissimum</Emphasis>

Incorporation of spirilloxanthin into carotenoidless LH2 and LH1-RC complexes from a purple sulfur bacterium Allochromatium (Alc.) minutissimum was studied. Carotenoidless cells of Alc. minutissimum were obtained using diphenylamine, a carotenoid biosynthesis inhibitor. In the course of incorporation of the carotenoid mixture, the composition of which corresponded to that of Alc. minutissimum control photosynthetic membranes, no selective incorporation of spirilloxanthin into the LH1-RC complex was detected. It is assumed that in vivo carotenoids are not incorporated into the LH2 and LH1-RC complexes from a common pool. Pure spirilloxanthin destroys both the LH2 and LH1-RC complexes. Within the concentration range of spirilloxanthin in the incorporated mixture from 27% to 52%, it was found to be incorporated into the LH2 and LH1-RC complexes with the efficiency of 13% and 33%, respectively. The possible existence of different sites of assembly for the LH2 and LH1-RC complexes is discussed, as well as of two fractions of LH2 complexes, in one of which rhodopin may be integrated, and in the other (minor) one, spirilloxanthin.     

Metal cations modulate the bacteriochlorophyll-protein interaction in the light-harvesting 1 core complex from Thermochromatium tepidum

The light-harvesting 1 reaction center (LH1-RC) complex from Thermochromatium (Tch.) tepidum exhibits unusual Q(y) absorption by LH1 bacteriochlorophyll-a (BChl-a) molecules at 915nm, and the transition energy is finely modulated by the binding of metal cations to the LH1 polypeptides. Here, we demonstrate the metal-dependent interactions between BChl-a and the polypeptides within the intact LH1-RC complexes by near-infrared Raman spectroscopy. The wild-type LH1-RC (B915) exhibited Raman bands for the C3-acetyl and C13-keto CO stretching modes at 1637 and 1675cm(-1), respectively. The corresponding bands appeared at 1643 and 1673cm(-1) when Ca(2+) was biosynthetically replaced with Sr(2+) (B888) or at 1647 and 1669cm(-1) in the mesophilic counterpart, Allochromatium vinosum. These results indicate the significant difference in the BChl-polypeptide interactions between B915 and B888 and between B915 and the mesophilic counterpart. The removal of the original metal cations from B915 and B888 resulted in marked band shifts of the C3-acetyl/C13-carbonyl νCO modes to ~1645/~1670cm(-1), supporting a model in which the metal cations are involved in the fine-tuning of the hydrogen bonding between the BChl-a and LH1-polypeptides. Interestingly, the interaction modes were almost identical between the Ca(2+)-depleted B915 and Sr(2+)-depleted B888 and between B915 and Ca(2+)-substituted B888, despite the significant differences in their LH1 Q(y) peak positions and the denaturing temperatures, as revealed by differential scanning calorimetry. These results suggest that not only the BChl-polypeptide interactions but some structural origin may be involved in the unusual Q(y) red-shift and the enhanced thermal stability of the LH1-RC complexes from Tch. tepidum.     

Photocurrent and electronic activities of oriented-His-tagged photosynthetic light-harvesting/reaction center core complexes assembled onto a gold electrode

A polyhistidine (His) tag was fused to the C- or N-terminus of the light-harvesting (LH1)-α chain of the photosynthetic antenna core complex (LH1-RC) from Rhodobacter sphaeroides to allow immobilization of the complex on a solid substrate with defined orientation. His-tagged LH1-RCs were adsorbed onto a gold electrode modified with Ni-NTA. The LH1-RC with the C-terminal His-tag (C-His LH1-RC) on the modified electrode produced a photovoltaic response upon illumination. Electron transfer is unidirectional within the RC and starts when the bacteriochlorophyll a dimer in the RC is activated by light absorbed by LH1. The LH1-RC with the N-terminal His-tag (N-His LH1-RC) produced very little or no photocurrent upon illumination at any wavelength. The conductivity of the His-tagged LH1-RC was measured with point-contact current imaging atomic force microscopy, indicating that 60% of the C-His LH1-RC are correctly oriented (N-His 63%). The oriented C-His LH1-RC or N-His LH1-RC showed semiconductive behavior, that is, had the opposite orientation. These results indicate that the His-tag successfully controlled the orientation of the RC on the solid substrate, and that the RC produced photocurrent depending upon the orientation on the electrode.     

Comparative study of spectral flexibilities of bacterial light-harvesting complexes: structural implications

This work presents a comparative study of the frequencies of spectral jumping of individual light-harvesting complexes of six different types: LH2 of Rhodopseudomonas acidophila, Rhodobacter sphaeroides, and Rhodospirillum molischianum; LH1 of Rhodobacter sphaeroides; and two "domain swap mutants" of LH2 of Rhodobacter sphaeroides: PACLH1 and PACLH2mol, in which the alpha-polypeptide C-terminus is exchanged with the corresponding sequence from LH1 of Rhodobacter sphaeroides or LH2 of Rhodospirillum molischianum, respectively. The quasistable states of fluorescence peak wavelength that were previously observed for the LH2 of Rps. acidophila were confirmed for other species. We also observed occurrences of extremely blue-shifted spectra, which were associated with reversible bleaching of one of the chromophore rings. Different jumping behavior is observed for single complexes of different types investigated with the same equivalent excitation intensity. The differences in spectral diffusion are associated with subtle differences of the binding pocket of B850 pigments and the structural flexibility of the different types of complexes.     

The two light-harvesting membrane chromoproteins of Thermochromatium tepidum expose distinct robustness against temperature and pressure

An increased robustness against high temperature and the much red-shifted near-infrared absorption spectrum of excitons in the LH1-RC core pigment-protein complex from the thermophilic photosynthetic purple sulfur bacterium Thermochromatium tepidum has recently attracted much interest. In the present work, thermal and hydrostatic pressure stability of the peripheral LH2 and core LH1-RC complexes from this bacterium were in parallel investigated by various optical spectroscopy techniques applied over a wide spectral range from far-ultraviolet to near-infrared. In contrast to expectations, very distinct robustness of the complexes was established, while the sturdiness of LH2 surpassed that of LH1-RC both with respect to temperatures between 288 and 360 K, and pressures between 1 bar and 14 kbar. Subtle structural variances related to the hydrogen bond network are likely responsible for the extra stability of LH2.     

Construction and structural analysis of tethered lipid bilayer containing photosynthetic antenna proteins for functional analysis

The construction and structural analysis of a tethered planar lipid bilayer containing bacterial photosynthetic membrane proteins, light-harvesting complex 2 (LH2), and light-harvesting core complex (LH1-RC) is described and establishes this system as an experimental platform for their functional analysis. The planar lipid bilayer containing LH2 and/or LH1-RC complexes was successfully formed on an avidin-immobilized coverglass via an avidin-biotin linkage. Atomic force microscopy (AFM) showed that a smooth continuous membrane was formed there. Lateral diffusion of these membrane proteins, observed by a fluorescence recovery after photobleaching (FRAP), is discussed in terms of the membrane architecture. Energy transfer from LH2 to LH1-RC within the tethered membrane was observed by steady-state fluorescence spectroscopy, indicating that the tethered membrane can mimic the natural situation.     

Polarization angle dependence of stark absorption spectra of spirilloxanthin bound to the reconstituted LH1 complexes using LH1-subunits isolated from the purple photosynthetic bacterium Rhodospirillum rubrum

Reconstituted LH1 complexes were prepared using the LH1 subunit-type complexes, isolated from the purple photosynthetic bacterium Rhodospirillum (Rs.) rubrum, and purified all-trans spirilloxanthin. Stark absorption spectra of spirilloxanthin bound to both the native and reconstituted LH1 complexes were compared in different polarization angles (χ) against the external electric field. From the polarization angle dependence of the Stark absorption spectra, two angles were determined in reference to the direction of transition dipole moment (m) of spirilloxanthin: one is the change in polarizability upon photoexcitation (Δα), θ(Δα) and the other is the change in static dipole moment upon photoexcitation (Δμ), θ(Δμ). Despite the symmetric molecular structure of all-trans spirilloxanthin, its Stark absorption spectra show pronounced values of Δμ. This large Δμ values essentially caused by the effect of induced dipole moment through Δα both in the cases for native and reconstituted LH1 complexes. However, slightly different values of θ(Δα) and θ(Δμ) observed for the native LH1 complex suggest that spirilloxanthin is asymmetrically distorted when bound to the native LH1 complex and gives rise to intrinsic Δμ value.     

The spirilloxanthine-pathway carotenoid incorporation into light-harvesting complexes of Ectothiorhodospira haloalkaliphila in vitro

Carotenoidless light-harvesting complexes (DPA-complexes) LH1-RC and LH2 were isolated from the purple sulfur bacterium Ectothiorhodospira haloalkaliphila in which carotenoid biosynthesis was suppressed with diphenylamine (DPA). Carotenoids of the spirilloxanthine series, which were isolated from the same bacterium, were incorporated into the DPA-complexes in vitro with an efficiency of 95–100%. The comparison of characteristics of the complexes with the incorporated carotenoids and the control complexes showed that the LH2 complexes with the incorporated carotenoids restored their absorption spectra, circular dichroism signals, and energy transfer from carotenoids to bacteriochlorophyll, which indicates that carotenoids were correctly incorporated into the structure of this complex.     

Self-assembled monolayer of light-harvesting core complexes from photosynthetic bacteria on a gold electrode modified with alkanethiols

Light-harvesting antenna core (LH1-RC) complexes isolated from Rhodoseudomonas palustris were self-assembled on a gold electrode modified with self-assembled monolayers (SAMs) of the alkanethiols NH2(CH2)nSH, n = 2, 6, 8, 11; HOOC(CH2)7SH; and CH3(CH2)7SH, respectively. Adsorption of the LH1-RC complexes on the SAMs depended on the terminating group of the alkanethiols, where the adsoption increased in the following order for the terminating groups: amino groups > carboxylic acid groups > methyl groups. Further, the adsorption on a gold electrode modified with SAMs of NH2(CH2)nSH, n = 2, 6, 8, 11, depended on the methylene chain length, where the adsorption increased with increasing the methylene chain length. The presence of the well-known light-harvesting and reaction center peaks of the near infrared (NIR) absorption spectra of the LH1-RC complexes indicated that these complexes were only fully stable on the SAM gold electrodes modified with the amino group. In the case of modification with the carboxyl group, the complexes were partially stable, while in the presence of the terminal methyl group the complexes were extensively denatured. An efficient photocurrent response of these complexes on the SAMs of NH2(CH2)nSH, n = 2, 6, 8, 11, was observed upon illumination at 880 nm. The photocurrent depended on the methylene chain length (n), where the maximum photocurrent response was observed at n = 6, which corresponds to a distance between the amino terminal group in NH2(CH2)6SH and the gold surface of 1.0 nm.     

Heterogeneite des variations spectrales photoinduites vers 700 nm observees sur les membranes chlorophylliennes et les complexes chlorophylle-proteines isoles de divers organismes photosynthetiques

A heterogeneity of photoinduced spectral variations near 700 nm is observed at −196°C, by comparison of difference spectra obtained from several materials (two blue-green algae, Spirulina and Fremyella; a red alga Porphyridium and a higher plant, Nicotiana tabacum). The maximum of complexity is seen with Spirulina chlorophyllous membranes where three bands are detected at 718, 705 and 695 nm. These bands are recovered in relatively different amounts in two chlorophyll-protein complexes prepared after sodium dodecyl sulfate electrophoresis of Spirulina membranes.

The photoinduced changes near 700 nm with Spirulina membranes are reversible and seem to be synchronous and related to the bleaching of three different pigments. This spectral diversity for photosensizable pigments is comparable with the spectral heterogeneity of the major forms of the bulk chlorophyll. It is assumed that these pigments may play the same functional role. This assumption may be related to a possible heterogeneity of Photosystem I centres recently detected by Breton using linear dichroism method (Breton, J. (1977) Biochim. Biophys. Acta 459, 66–70).     

Spectral diffusion of the lowest exciton component in the core complex from Rhodopseudomonas palustris studied by single-molecule spectroscopy

We have recorded fluorescence-excitation spectra from individual RC–LH1 complexes from Rhodopseudomonas palustris. The spectra feature a few broad bands accompanied by a sharp line at the low-energy side of the spectrum which is ascribed to the lowest exciton state of the BChl a assembly. Recording several fluorescence-excitation spectra from the same individual complex in rapid succession reveals that the linewidth of the lowest exciton transition is determined by spectral diffusion which increases for higher excitation energies.     

C-terminal cleavage of the LH1 <Emphasis Type="Italic">α</Emphasis>-polypeptide in the Sr<Superscript>2+</Superscript>-cultured <Emphasis Type="Italic">Thermochromatium tepidum</Emphasis>

The light-harvesting 1 reaction center (LH1-RC) complex in the thermophilic purple sulfur bacterium Thermochromatium (Tch.) tepidum binds Ca ions as cofactors, and Ca-binding is largely involved in its characteristic Q_y absorption at 915 nm and enhanced thermostability. Ca²⁺ can be biosynthetically replaced by Sr²⁺ in growing cultures of Tch. tepidum. However, the resulting Sr²⁺-substituted LH1-RC complexes in such cells do not display the absorption maximum and thermostability of those from Ca²⁺-grown cells, signaling that inherent structural differences exist in the LH1 complexes between the Ca²⁺- and Sr²⁺-cultured cells. In this study, we examined the effects of the biosynthetic Sr²⁺-substitution and limited proteolysis on the spectral properties and thermostability of the Tch. tepidum LH1-RC complex. Preferential truncation of two consecutive, positively charged Lys residues at the C-terminus of the LH1 α-polypeptide was observed for the Sr²⁺-cultured cells. A proportion of the truncated LH1 α-polypeptide increased during repeated subculturing in the Sr²⁺-substituted medium. This result suggests that the truncation is a biochemical adaptation to reduce the electrostatic interactions and/or steric repulsion at the C-terminus when Sr²⁺ substitutes for Ca²⁺ in the LH1 complex. Limited proteolysis of the native Ca²⁺-LH1 complex with lysyl protease revealed selective truncations at the Lys residues in both C- and N-terminal extensions of the α- and β-polypeptides. The spectral properties and thermostability of the partially digested native LH1-RC complexes were similar to those of the biosynthetically Sr²⁺-substituted LH1-RC complexes in their Ca²⁺-bound forms. Based on these findings, we propose that the C-terminal domain of the LH1 α-polypeptide plays important roles in retaining proper structure and function of the LH1-RC complex in Tch. tepidum.     

Construction of hybrid photosynthetic units using peripheral and core antennae from two different species of photosynthetic bacteria: detection of the energy transfer from bacteriochlorophyll a in LH2 to bacteriochlorophyll b in LH1

Typical purple bacterial photosynthetic units consist of supra-molecular arrays of peripheral (LH2) and core (LH1-RC) antenna complexes. Recent atomic force microscopy pictures of photosynthetic units in intact membranes have revealed that the architecture of these units is variable (Scheuring et al. (2005) Biochim Bhiophys Acta 1712:109–127). In this study, we describe methods for the construction of heterologous photosynthetic units in lipid-bilayers from mixtures of purified LH2 (from Rhodopseudomonas acidophila) and LH1-RC (from Rhodopseudomonas viridis) core complexes. The architecture of these reconstituted photosynthetic units can be varied by controlling ratio of added LH2 to core complexes. The arrangement of the complexes was visualized by electron-microscopy in combination with Fourier analysis. The regular trigonal array of the core complexes seen in the native photosynthetic membrane could be regenerated in the reconstituted membranes by temperature cycling. In the presence of added LH2 complexes, this trigonal symmetry was replaced with orthorhombic symmetry. The small lattice lengths for the latter suggest that the constituent unit of the orthorhombic lattice is the LH2. Fluorescence and fluorescence-excitation spectroscopy was applied to the set of the reconstituted membranes prepared with various proportions of LH2 to core complexes. Remarkably, even though the LH2 complexes contain bacteriochlorophyll a, and the core complexes contain bacteriochlorophyll b, it was possible to demonstrate energy transfer from LH2 to the core complexes. These experiments provide a first step along the path toward investigating how changing the architecture of purple bacterial photosynthetic units affects the overall efficiency of light-harvesting.     

Molecular arrangement of pigment-protein complex of photosystem 1

The circular dichroism (CD) method was applied to study the molecular organization of P700, antenna chlorophyll and protein of photosystem 1 complexes (CP1), isolated from chloroplasts under mild treatment with Triton X-100. Analysis of CD spectra and protein: chlorophyll: P700 ratios for CP1 complexes that were different in their chlorophyll content indicate that CP1 preparations can be considered as a mixture of CP1-RC, containing P700 (10–20%), and CP1-LH without P700 (80–90%). Both types of complexes contain approximately 25 chlorophyll molecules, and the destruction of their spatial organization with detergents represents a cooperative transition. The rate of chlorophyll destruction in CP1-LH is much higher than that in CP1-RC. In both complexes a 65 kDa polypeptide predominates, whose secondary structure (typical for / proteins) is stable to Triton X-100 and does not depends on the chlorophyll content. Chlorophyll seems to be grouped in clusters (5–7 molecules) in the hydrophobic cores of 2–3 parallel / domains of the 65 kDa protein. Only one of the clusters in CP1-RC includes P700; on P700 photooxidation the change of its interaction with the nearest pigment environment results in a complicated shape of the light-induced CD spectra.Abbreviations PS1 photosystem 1 - CP1 pigment-protein complex of PS1 - Chl chlorophyll a - CP1-140 CP1 with ratio Ch1:P700 140 - RC reaction center - LH light-harvesting pigment - CP1-RC CP1, containing P700 - CP1-LH CP1 without P700 (containing LH) - CD circular dichroism - SDS sodium dodecyl sulfate Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement