Light-harvesting Complex Of Rhodobacter Sphaeroides Research Articles

Excited-state dynamics in light-harvesting complex of Rhodobacter sphaeroides

Photodynamics of peripheral antenna complexes, light-harvesting complex (LH2) of Rhodobacter (Rb) Sphaeroides 601, was studied using femtosecond pump-probe technique at different laser wavelengths. The obtained results reveal dramatic dynamical evolutions within B800 and B850 absorption bands of antenna complexes LH2. At excitation wavelength around 835 nm, a sharp photobleaching signal was observed which was assigned to the contribution of the two-exciton state, which was further confirmed by the power dependence measurement. Rate equations with eight-level scheme were used to calculate the population evolution in LH2 and the transient dynamics under femtosecond pulse excitation. The research results prove that not only the transition from ground state to one-exciton state but also that from one-exciton state to two-exciton state contribute to the photodynamics of B850.

On the Effects of PufX on the Absorption Properties of the Light-Harvesting Complexes of Rhodobacter sphaeroides

Some species of purple bacteria as, e.g., Rhodobacter sphaeroides contain the protein PufX. Concurrently, the light harvesting complexes 1 (LH1) form dimers of open rings. In mutants without PufX, the LH1s are closed rings and photosynthesis breaks down, because the ubiquinone exchange at the reaction center is blocked. However, the main purpose of the LH1 is light harvesting. We therefore investigate the effects that the PufX-induced dimerization has on the absorption properties of the core complexes. Calculations with a dipole model, which compare the photosynthetic efficiency of various configurations of monomeric and dimeric core complexes, show that the dimer can absorb photons directly into the reaction centers more efficiently, but that the performance of the more sophisticated dimeric LH1 antenna degrades faster with structural perturbations. The calculations predict an optimal orientation of the reaction centers relative to the LH1 dimer, which agrees well with the experimentally found configuration. Based on experimental observations indicating that the dimeric core complexes are indeed rather rigid, we hypothesize that in PufX + species the association between the LH1 and the reaction centers is enhanced. This mechanical stabilization of the core complexes would lead to the observed quinone blockage, when PufX is missing.

Mechanism of Energy Transfer from Carotenoids to Bacteriochlorophyll: Light-Harvesting by Carotenoids Having Different Extents of π-Electron Conjugation Incorporated into the B850 Antenna Complex from the Carotenoidless Bacterium Rhodobacter sphaeroides R-26.1

Spheroidene and a series of spheroidene analogues with extents of π-electron conjugation ranging from 7 to 13 carbon−carbon double bonds were incorporated into the B850 light-harvesting complex of Rhodobacter sphaeroides R-26.1. The structures and spectroscopic properties of the carotenoids and the dynamics of energy transfer from the carotenoid to bacteriochlorophyll (BChl) in the B850 complex were studied by using steady-state absorption, fluorescence, fluorescence excitation, resonance Raman, and time-resolved absorption spectroscopy. The spheroidene analogues used in this study were 5‘,6‘-dihydro-7‘,8‘-didehydrospheroidene, 7‘,8‘-didehydrospheroidene, and 1‘,2‘-dihydro-3‘,4‘,7‘,8‘-tetradehydrospheroidene. These data, taken together with results from 3,4,7,8-tetrahydrospheroidene, 3,4,5,6-tetrahydrospheroidene, 3,4-dihydrospheroidene, and spheroidene already published (Frank, H. A.; Farhoosh, R.; Aldema, M. L.; DeCoster, B.; Christensen, R. L.; Gebhard, R.; Lugtenburg, J. Photochem. Photobiol. 1993, 57...

Functions of conserved tryptophan residues of the core light-harvesting complex of Rhodobacter sphaeroides.

We have examined mutants in the core light-harvesting complex of Rhodobacter sphaeroides in which the tryptophan residues located at positions alpha+11, beta+6, and beta+9 have been mutated to each of the three other aromatic amino acids, namely tyrosine, phenylalanine, and histidine. We confirm that the alpha+11 residue and show that the beta+9 residue each form a hydrogen bond to a C2-acetyl group of a BChl molecule. Mutation of either of these residues to a phenylalanine results in a breakage of the normal hydrogen bond, whereas a histidine in either of these positions is able to form a hydrogen bond to the BChl. Comparison of the absorption spectra with the hydrogen bonding of the C2-acetyl groups for the various mutants demonstrates a role for this molecular interaction in the tuning of the absorption properties of the complex. We further demonstrate that there is a consistent linear relationship between the downshift in the C2-acetyl stretching mode and the red shift in the absorption maximum, in both core and peripheral antenna complexes. This linear relationship allows us to estimate the contribution of H bonding to the red shifts of these complexes. Though the residue beta+6 is found not to be directly involved in interactions with the pigment molecules, mutation of this residue is shown in some cases to result in both a destabilization of the complex and a decrease in the binding site homogeneity. Finally, a consideration of the amount of antenna complex present in the various mutants shows an important role for the reaction center and/or the pufX gene product in the assembly or stabilization of this membrane protein.

Internal conversion and energy transfer dynamics of spheroidene in solution and in the LH-1 and LH-2 light-harvesting complexes

Femtosecond time resolved fluorescence upconversion was applied to determine directly the lifetime of the 1B u + state of the carotenoid spheroidene in vitro in various polar and nonpolar solvents and in the LH-1 and LH-2 light-harvesting complexes of Rhodobacter sphaeroides at room temperature. The spheroidene 1B u + → 2A g − internal conversion time varies from 150 to 250 fs in the solvents studied and depends on the polarizability of the surrounding environment. The internal conversion time in the protein environment is estimated based on the solution measurements. There is a dramatic shortening for the spheroidene 1B u + fluorescence lifetime seen inside LH-1 and LH-2 (60 fs and 80 fs, respectively) over this expected internal conversion time.

Low-temperature absorption and site-selected fluorescence of the light-harvesting antenna of Rhodopseudomonas viridis. Evidence for heterogeneity

The main infrared absorption band of the antenna of the purple bacterium Rps. uiridis is investigated using low-temperature absorption and site-selected fluorescence spectroscopy. Low-temperature absorption spectra show that at least three bands are present in the near-infrared. Gaussian fits indicate that the maxima are located around 1015, 1030 and 1045 run. Fluorescence measurements using narrow-band laser excitation in the red flank of the absorption band show that the emission is highly polarized ( p > 0.3). This was found for membranes and (isolated) core complexes. In isolated core complexes, the polarization of the emission increases smoothly as a function of the excitation wavelength, starting in the center of the absorption band, whereas in membranes the increase is abrupt and occurs in the extreme red edge. These results differ significantly from similar measurements performed on core light-harvesting complexes from Rhodobacter sphaeroides [Van Mourik, F., Visschers, R.W., Van Grondelle, R. (1992) Energy transfer and aggregate size effect in the inhomogeneously broadened core light-harvesting complex of Rhodobacter sphaeroides, Chem. Phys. Lett. 193, 1–7] and cannot be interpreted by assuming a single inhomogeneously broadened pool of antenna pigments. Moreover, the low-temperature absorption spectrum gives clear evidence for a long-wavelength spectral form. Therefore, it is concluded that the antenna is heterogeneous, with a minor long-wavelength spectral form (131045). The fluorescence measurements show that this 131045 band is inhomogeneously broadened.

Reconstitution of the Bacterial Core Light-harvesting Complexes of Rhodobacter sphaeroides and Rhodospirillum rubrum with Isolated α- and β-Polypeptides, Bacteriochlorophyll a, and Carotenoid

Methodology has been developed to reconstitute carotenoids and bacteriochlorophyll alpha with isolated light-harvesting complex I (LHI) polypeptides of both Rhodobacter sphaeroides and Rhodospirillum rubrum. Reconstitution techniques first developed in this laboratory using the LHI polypeptides of R. rubrum, R. sphaeroides, and Rhodobacter capsulatus reproduced bacteriochlorophyll alpha spectral properties characteristic of LHI complexes lacking carotenoids. In this study, carotenoids are supplied either as organic-solvent extracts of chromatophores or as thin-layer chromatography or high performance liquid chromatography-purified species. The resulting LHI complexes exhibit carotenoid and bacteriochlorophyll a spectral properties characteristic of native LHI complexes of carotenoid-containing bacteria. Absorption and circular dichroism spectra support the attainment of a native-like carotenoid environment in the reconstituted LHI complexes. For both R. sphaeroides- and R. rubrum-reconstituted systems, fluorescence excitation spectra reveal appropriate carotenoid to bacteriochlorophyll alpha energy-transfer efficiencies based on comparisons with the in vivo systems. In the case of R. rubrum reconstitutions, carotenoids afford protection from photodynamic degradation. Thus, carotenoids reconstituted into LHI exhibit spectral and functional characteristics associated with native pigments. Heterologous reconstitutions demonstrate the applicability of the developed assay in dissecting the molecular environment of carotenoids in light-harvesting complexes.

PROTEIN STRUCI'URE MODELLING OF THE BACTERIAL LIGHT‐HARVESTING COMPLEX

Protein structure modelling offers a method of obtaining 3-dimensional information that can be tested and used to plan mutagenesis experiments when a crystallographically determined structure is not available. At its simplest a model may consist of little more than a secondary structure prediction coupled with a determination of the likely regions of transmembrane/membrane surface/globular configuration. These methods can yield an interesting topology map of the protein, which places the residues in their likely positions with respect to, for example, the membrane interface. If it is a member of a large family of related proteins then aligned protein sequences can be used to predict the residues that have an important function as these will be largely conserved in the alignments. Using all these methods a model can be constructed (using for example, the Nicholson Molecular Modelling Kit) to visualize the proposed structure in three dimensions following the premise of good design, that is, avoiding obvious steric clashes, packing of helices in a realistic manner, observing the correct H-bond lengths, etc. In this latter exercise the review of Chothia (Annu. Rev. Biochem. 53, 537-572, 1984) of the principles of protein structure is particularly helpful as it clearly sets out how proteins pack and their preferred configuration. There is a wealth of information about individual amino acid conformational preferences and observed frequencies of occurrence in known protein structures, which can help decide how the residues in the model can be oriented. In this article we have collated the various protein models of the bacterial light-harvesting complexes and present our own model, which is a synthesis of the available biophysical data and theoretical predictions, and show its performance in explaining recent results of site-directed mutants of the LH1 and LH2 light-harvesting complexes of Rhodobacter sphaeroides.

Triplet energy transfer between bacteriochlorophyll and carotenoids in B850 light-harvesting complexes ofRhodobacter sphaeroides R-26.1

The build-up and decay of bacteriochlorophyll (BChl) and carotenoid triplet states were studied by flash absorption spectroscopy in (a) the B800-850 antenna complex ofRhodobacter (Rb.)sphaeroides wild type strain 2.4.1, (b) theRb. sphaeroides R-26.1 B850 light-harvesting complex incorporated with spheroidene, (c) the B850 complex incorporated with 3,4-dihydrospheroidene, (d) the B850 complex incorporated with 3,4,5,6-tetrahydrospheroidene and (e) theRb. sphaeroides R-26.1 B850 complex lacking carotenoids. Steady state absorption and circular dichroism spectroscopy were used to evaluate the structural integrity of the complexes. The transient data were fit according to either single or double exponential rate expressions. The triplet lifetimes of the carotenoids were observed to be 7.0±0.1 μs for the B800-850 complex, 14±2 μs for the B850 complex incorporated with spheroidene, and 19±2 μs for the B850 complex incorporated with 3,4-dihydrospheroidene. The BChl triplet lifetime in the B850 complex was 80±5 μs. No quenching of BChl triplet states was seen in the B850 complex incorporated with 3,4,5,6-tetrahydrospheroidene. For the B850 complex incorporated with spheroidene and with 3,4-dihydrospheroidene, the percentage of BChl quenched by carotenoids was found to be related to the percentage of carotenoid incorporation. The triplet energy transfer efficiencies are compared to the values for singlet energy transfer measured previously (Frank et al. (1993) Photochem. Photobiol. 57: 49-55) on the same samples. These studies provide a systematic approach to exploring the effects of state energies and lifetimes on energy transfer between BChls and carotenoids in vivo.

Exciton dynamics in the light-harvesting complexes of Rhodobacter sphaeroides

We have used ≈ 40 fs pulses from a Ti:Sa laser to investigate the ultrafast energy transfer and relaxation dynamics in the LH1 and LH2 light-harvesting pigment—proteins of photosynthetic purple bacteria. Our results reveal a new feature, not earlier observed in light-harvesting pigments, ultrafast relaxation characterized by a time constant of approximately 20 fs, which we interpret as interexciton state relaxation. The experimental results are compared with computer simulations of transient excited state absorption from the excited states of an excitonic aggregate.

Effects of phospholipase A2 digestion on the carotenoid and bacteriochlorophyll components of the light-harvesting complexes in Rhodobacter sphaeroides chromatophores

The instantaneous electrochromic response of carotenoids associated with the B800-850 light-harvesting complex of Rhodobacter sphaeroides has been used widely as an intrinsic probe of membrane potential. In the present study, the structural basis for this phenomenon was examined by phospholipase A2 digestion of chromatophores from R. sphaeroides strain NF57G, containing B800-850 as the sole pigment-protein complex. The major phospholipase-induced alterations of the overall carotenoid absorption spectrum were characterized by an absorbance loss and a blue shift that were accompanied by a decrease in absorbance at 800 nm and a red shift in the B850 absorbance band. In wild-type chromatophores, the electrochromic carotenoid response induced by both flash illumination and a K+ diffusion potential was diminished by approximately 60% after 1 h of digestion. The initial loss of the carotenoid response was correlated specifically to the hydrolysis of phosphatidylethanolamine, and was shown to arise from effects exerted directly upon the electrochromically active carotenoid pool, possibly by alterations in the spatial relationship between the field-sensitive carotenoids and the polarizing permanent field. In phospholipase A2-digested NF57G preparations in which the B800 band was diminished by nearly half and the carotenoid response was abolished, no significant changes in the efficiency of energy transfer from carotenoids to bacteriochlorophyll were detected at 77 K, suggesting that the electrochromically active carotenoids are not energetically linked to B800 bacteriochlorophyll.

Energy transfer and aggregate size effects in the inhomogeneously broadened core light-harvesting complex of Rhodobacter sphaeroides

The 4 K fluorescence emission spectrum of the core light-harvesting complex of the photosynthetic purple bacterium Rhodobacter sphaeroides is shown to depend strongly on the wavelength of excitation. It is concluded that the dominant factor determining the absorption spectrum is inhomogeneous broadening. The site-selected emission spectra are interpreted in terms of energy transfer in a cluster of weakly coupled pigments. Each individual cluster is supposed to be a random sample from the total inhomogeneously broadened pigment pool. For small clusters, with efficient energy transfer leading to fast thermalization, the effects of site-selection depend on the cluster-size only. It is shown that site-selective excitation can yield information about the number of coupled pigments in such a cluster. For the purified LH-1 of Rb. sphaeroides the cluster size was estimated to be 16 pigments.

Genetically modified photosynthetic antenna complexes with blueshifted absorbance bands.

Light energy for photosynthesis is collected by the antenna system, creating an excited state which migrates energetically 'downhill'. To achieve efficient migration of energy the antenna is populated with a series of pigments absorbing at progressively redshifted wavelengths. This variety in absorbing species in vivo has been created in a biosynthetically economical fashion by modulating the absorbance behaviour of one kind of (bacterio)chlorophyll molecule. This modulation is poorly understood but has been ascribed to pigment-pigment and pigment-protein interactions. We have examined the relationship between aromatic residues in antenna polypeptides and pigment absorption, by studying the effects of site-directed mutagenesis on a bacterial antenna complex. A clear correlation was observed between the absorbance of bacteriochlorophyll a and the presence of two tyrosine residues, alpha Tyr44 and alpha Tyr45, in the alpha subunit of the peripheral light-harvesting complex of Rhodobacter sphaeroides, a purple photosynthetic bacterium that provides a well characterized system for site-specific mutagenesis. By constructing single (alpha Tyr44, alpha Tyr45----PheTyr) and then double (alpha Tyr44, alpha Tyr45----PheLeu) site-specific mutants, the absorbance of bacteriochlorophyll was blueshifted by 11 and 24 nm at 77 K, respectively. The results suggest that there is a close approach of tyrosine residues to bacteriochlorophyll, and that this proximity may promote redshifts in vivo.

Femtosecond energy-transfer processes in the B800–850 light-harvesting complex of Rhodobacter sphaeroides 2.4.1

The B800-to-B850 energy transfer time in the purified B800-850 light-harvesting complex of Rhodobacter sphaeroides 2.4.1 is determined to be 0.7 ps at room temperature. The electronic state dynamics of the principal carotenoid of this species, spheroidene, are examined, both in vivo and in vitro, by direct femtosecond time-resolved experiments and by fluorescence emission yield studies. Evidence is presented which suggests that carotenoid-to-bacteriochlorophyll energy transfer may occur directly from the initially excited carotenoid S2 state, as well as from the carotenoid S1 state. Further support for this conjecture is obtained from calculations of energy transfer rates from the carotenoid S2 state. Previous measurements of in vivo carotenoid and B800 dynamics are discussed in light of the new results, and currently unresolved issues are described.

Cloning, nucleotide sequence and transfer of genes for the B800–850 light harvesting complex of Rhodobacter sphaeroides

The genes that encode the α and β polypeptides of the major light-harvesting complex of Rhodobacter sphaeroides, B800–850, have been cloned and sequenced through the use of oligonucleotides based upon the known polypeptide sequences. These genes, pucA and B, are transcribed in the order B, A, are of 150 and 164 nucleotides respectively, and are separated by a spacer region of 14 nucleotides. Transfer of these genes to mutant M21 lacking the B800–850 complex has been accomplished and absorbance spectra of recombinant strains M2181 and M2184 show that expression of pucA and B is comparable to levels found in the wild type.