Light-harvesting Complex LH2 Research Articles

The long-range organization of a native photosynthetic membrane.

Photosynthesis relies on the delicate interplay between a specific set of membrane-bound pigment-protein complexes that harvest and transport solar energy, execute charge separation, and conserve the energy. We have investigated the organization of the light-harvesting (LH) and reaction-center (RC) complexes in native bacterial photosynthetic membranes of the purple bacterium Rhodobacter sphaeroides by using polarized light spectroscopy, linear dichroism (LD) on oriented membranes. These LD measurements show that in native membranes, which contain LH2 as the major energy absorber, the RC-LH1-PufX complexes are highly organized in a way similar to that which we found previously for a mutant lacking LH2. The relative contribution of LH1 and LH2 light-harvesting complexes to the LD spectrum shows that LH2 preferentially resides in highly curved parts of the membrane. Combining the spectroscopic data with our recent atomic force microscopy (AFM) results, we propose an organization for this photosynthetic membrane that features domains containing linear arrays of RC-LH1-PufX complexes interspersed with LH2 complexes and some LH2 found in separate domains. The study described here allows the simultaneous assessment of both global and local structural information on the organization of intact, untreated membranes.

Protein folding in the periplasm in the absence of primary oxidant DsbA: modulation of redox potential in periplasmic space via OmpL porin

Corrigendum29 September 2004free access Protein folding in the periplasm in the absence of primary oxidant DsbA: modulation of redox potential in periplasmic space via OmpL porin Claire Dartigalongue Claire Dartigalongue Search for more papers by this author Hiroshi Nikaido Hiroshi Nikaido Search for more papers by this author Satish Raina Satish Raina Search for more papers by this author Claire Dartigalongue Claire Dartigalongue Search for more papers by this author Hiroshi Nikaido Hiroshi Nikaido Search for more papers by this author Satish Raina Satish Raina Search for more papers by this author Author Information Claire Dartigalongue, Hiroshi Nikaido and Satish Raina The EMBO Journal (2004)23:3907-3907https://doi.org/10.1038/sj.emboj.7600439 This article corrects the following: Protein folding in the periplasm in the absence of primary oxidant DsbA: modulation of redox potential in periplasmic space via OmpL porin15 November 2000 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Correction to: The EMBO Journal (2000) 19, 5980–5988. doi:10.1093/emboj/19.22.5980 During reconstruction of ompL/dsbA double mutants, we observed that null mutation in ompL suppressed the defects of dsbA mutant bacteria in oxidative folding of secreted proteins only partially (about 20–30%). During our initial series of experiments, we observed a complete suppression of dsbA phenotypes upon introduction of the ompL null allele. The discrepancy was repeatedly observed for Escherichia coli strain SR4444Δ (dsbA ompL). We conclude that this strain likely carries a second mutation, which contributes to the complete restoration of oxidative protein folding. Our conclusions for a role of OmpL in modulating the oxidative environment of E. coli periplasmic space gain further support as we have again cloned the ompL gene, which, when overexpressed from a plasmid, confers resistance to sublethal concentrations of oxidizing agents such as diamide. We would like to bring to the attention of readers that it was MC1000 that was used in our studies and is the parent of SR2262 and SR4444, and that in page 5987 temperature used for motility assay was 30°C and the agar concentration was 0.3%. Next ArticlePrevious Article Read MoreAbout the coverClose modalView large imageVolume 23,Issue 19,September 29, 2004Top view of a model membrane containing all the proteins of the photosynthetic unit in Rhodospirillum photometricum: LH2 light harvesting complexes and core complexes, consisting of LH1 light harvesting complexes surrounding reaction centers. The structures of the individual complexes were assembled following the arrangement observed in the native membrane by atomic force microscopy. The image was composed by Simon Scheuring from the Institut Curie, Paris, France, whose accompanying article will appear in one of the next printed issues of The EMBO Journal. Volume 23Issue 1929 September 2004In this issue RelatedDetailsLoading ...

The native architecture of a photosynthetic membrane.

In photosynthesis, the harvesting of solar energy and its subsequent conversion into a stable charge separation are dependent upon an interconnected macromolecular network of membrane-associated chlorophyll-protein complexes. Although the detailed structure of each complex has been determined, the size and organization of this network are unknown. Here we show the use of atomic force microscopy to directly reveal a native bacterial photosynthetic membrane. This first view of any multi-component membrane shows the relative positions and associations of the photosynthetic complexes and reveals crucial new features of the organization of the network: we found that the membrane is divided into specialized domains each with a different network organization and in which one type of complex predominates. Two types of organization were found for the peripheral light-harvesting LH2 complex. In the first, groups of 10-20 molecules of LH2 form light-capture domains that interconnect linear arrays of dimers of core reaction centre (RC)-light-harvesting 1 (RC-LH1-PufX) complexes; in the second they were found outside these arrays in larger clusters. The LH1 complex is ideally positioned to function as an energy collection hub, temporarily storing it before transfer to the RC where photochemistry occurs: the elegant economy of the photosynthetic membrane is demonstrated by the close packing of these linear arrays, which are often only separated by narrow 'energy conduits' of LH2 just two or three complexes wide.

Multichromophoric Förster resonance energy transfer.

The theory of Förster resonance energy transfer is generalized for multichromophoric (MC) and nonequilibrium situations. For the first time, it is clarified that the far-field linear spectroscopic information is insufficient for the determination of the reaction rate and that distance dependence of the rate can vary with the disorder and temperature. Application to a light harvesting complex LH2 reveals the important consequences of a MC structure.

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.

Determination of the Topological Shape of Integral Membrane Protein Light-Harvesting Complex LH2 from Photosynthetic Bacteria in the Detergent Solution by Small-Angle X-Ray Scattering

The topological shape of the integral membrane protein light-harvesting complex LH2 from photosynthetic bacteria Rhodobacter spheroides 2.4.1 in detergent solution has been determined from synchrotron small-angle X-ray scattering data using direct curve-fitting by the ellipsoid, ab initio shape determination methods of simulated annealing algorithm and multipole expansion, respectively. The results indicate that the LH2 protein in aqueous solution is encapsulated by a monolayered detergent shell. The detergent-stabilized structure has the shape of an oblate plate, with a thickness of 40 Å, a long axis of 110 Å, and a short axis of 85 Å. After correction for the detergent shell, the shape of the LH2 core is also an oblate plate with a height of 40 Å, a long axis of 80 Å, and a short axis of 55 Å. In contrast to the cylindrical crystal structure with a height of 40 Å and a diameter of 68 Å, the molecular shape of the LH2 complex in detergent solution clearly deviates from the ringlike crystal structure, with an eccentricity found to be 0.59—consistent with the result of single molecular spectroscopy study of the isolated single LH2 molecules.

Ultrafast excitation relaxation in light-harvesting complex LH2 from Rb. sphaeroides 601

The energy relaxation and kinetic evolution of transient spectra of bacteriochlorophylls (BChls) in light-harvesting complex LH2 from Rb. sphaeroides 601 were investigated using femtosecond pump-probe technique. Upon 783 nm excitation, the energy at B800 BChls experiences an intramolecular redistribution with 0.35 ps time constant before transferring to B850 BChls. With tuning the excitation wavelength, the dynamical evolution of excited BChls was clearly observed, which indicates an obvious competition between the ground state bleaching and excited state absorption (ESA) of BChls involved and an isosbestic point near 818 nm, and also demonstrates that from the lower electronic excited state of B800 BChls to the higher excitonic state of B850 BChls is an efficient routine for energy transfer. The excitation energy in higher excitonic states of B850 BChls relaxes rapidly to the next lowest excitonic state by interconversion, delocalization to adjacent molecular, populating the lowest excitonic state and the change of molecular conformation.

Exciton dynamics in ring-like photosynthetic light-harvesting complexes: a hopping model

Excitation localization and dynamics in circular molecular aggregates is considered. It is shown that the Anderson localization of the excitons is taking place even in the finite size of the ring-type systems containing tens of pigments in the case of comparable values of the spectral inhomogeneity and of the intermolecular resonance interaction. The second type of localization comes from the dynamical disorder caused by exciton interactions with environment fluctuations. Because of these two reasons the hopping type migration of the small-size excitons is postulated to be responsible for the excitation dynamics in this kind of systems. This process is considered for the ensemble of independent rings and for the array of the interacting rings by means of Monte Carlo simulations. The intra-ring and inter-ring energy disorder with possible correlations is accepted in simulations performed for the cases of high and low temperatures. It is shown that for the typical parameters of the peripheral light-harvesting pigment-protein complexes LH2 of photosynthetic bacteria the excitation population reaches equilibrium within 1 ps in the case of the disconnected rings at nonselective excitation conditions, while equilibration on longer time scale is taking place in the system of connected rings. This nonexponential relaxation kinetics is observed at room temperature and is more pronounced by lowering the temperature. In the case of selective excitation the equilibration process is wavelength dependent for the disconnected rings at room temperature and becomes more pronounced by lowering the temperature. The wavelength dependence is resulted from the interplay between exciton population redistribution among pigments and the population, which stucks in the most red pigments.

Identification of intramembrane hydrogen bonding between 13 1 keto group of bacteriochlorophyll and serine residue α27 in the LH2 light-harvesting complex

Intramembrane hydrogen bonding and its effect on the structural integrity of purple bacterial light-harvesting complex 2, LH2, have been assessed in the native membrane environment. A novel hydrogen bond has been identified by Raman resonance spectroscopy between a serine residue of the membrane-spanning region of LH2 α-subunit, and the C-13 1 keto carbonyl of bacteriochlorophyll (BChl) B850 bound to the β-subunit. Replacement of the serine by alanine disrupts this strong hydrogen bond, but this neither alters the strongly red-shifted absorption nor the structural arrangement of the BChls, as judged from circular dichroism. It also decreases only slightly the thermal stability of the mutated LH2 in the native membrane environment. The possibility is discussed that weak H-bonding between the C-13 1 keto carbonyl and a methyl hydrogen of the alanine replacing serine(−4) or the imidazole group of the nearby histidine maintains structural integrity in this very stable bacterial light-harvesting complex. A more widespread occurrence of H-bonding to C-13 1 not only in BChl, but also in chlorophyll proteins, is indicated by a theoretical analysis of chlorophyll/polypeptide contacts at <3.5 Å in the high-resolution structure of Photosystem I. Nearly half of the 96 chlorophylls have aa residues suitable as hydrogen bond donors to their keto groups.

Low-Intensity Pump-Probe Measurements on the B800 Band of Rhodospirillum molischianum

We have measured low-intensity, polarized one-color pump-probe traces in the B800 band of the light-harvesting complex LH2 of Rhodospirillum molischianum at 77 K. The excitation/detection wavelength was tuned through the B800 band. A single-wavelength and a global target analysis of the data were performed with a model that accounts for excitation energy transfer among the B800 molecules and from B800 to B850. By including the anisotropy of the signals into the fitting procedure, both transfer processes could be separated. It was estimated in the global target analysis that the intra-B800 energy transfer, i.e., the hopping of the excitation from one B800 to another B800 molecule, takes ∼0.5 ps at 77 K. This transfer time increases with the excitation/detection wavelength from 0.3 ps on the blue side of the B800 band to ∼0.8 ps on the red side. The residual B800 anisotropy shows a wavelength dependence as expected for energy transfer within an inhomogeneously broadened cluster of weakly coupled pigments. In the global target analysis, the transfer time from B800 to B850 was determined to be ∼1.7 ps at 77 K. In the single-wavelength analysis, a speeding-up of the B800 → B850 energy transfer rate toward the blue edge of the B800 band was found. This nicely correlates with the proposed position of the suggested high-exciton component of the B850 band acting as an additional decay channel for B800 excitations.

Quantum control of energy flow in light harvesting.

Coherent light sources have been widely used in control schemes that exploit quantum interference effects to direct the outcome of photochemical processes. The adaptive shaping of laser pulses is a particularly powerful tool in this context: experimental output as feedback in an iterative learning loop refines the applied laser field to render it best suited to constraints set by the experimenter. This approach has been experimentally implemented to control a variety of processes, but the extent to which coherent excitation can also be used to direct the dynamics of complex molecular systems in a condensed-phase environment remains unclear. Here we report feedback-optimized coherent control over the energy-flow pathways in the light-harvesting antenna complex LH2 from Rhodopseudomonas acidophila, a photosynthetic purple bacterium. We show that phases imprinted by the light field mediate the branching ratio of energy transfer between intra- and intermolecular channels in the complex's donor acceptor system. This result illustrates that molecular complexity need not prevent coherent control, which can thus be extended to probe and affect biological functions.

Functional assembly of the foreign carotenoid lycopene into the photosynthetic apparatus of Rhodobacter sphaeroides, achieved by replacement of the native 3-step phytoene desaturase with its 4-step counterpart from Erwinia herbicola.

Photosynthetic organisms synthesize a diverse range of carotenoids. These pigments are important for the assembly, function and stability of photosynthetic pigment-protein complexes, and they are used to quench harmful radicals. The photosynthetic bacterium Rhodobacter sphaeroides was used as a model system to explore the origin of carotenoid diversity. Replacing the native 3-step phytoene desaturase (CrtI) with the 4-step enzyme from Erwinia herbicola results in significant flux down the spirilloxanthin pathway for the first time in Rb. sphaeroides. In Rb. sphaeroides, the completion of four desaturations to lycopene by the Erwinia CrtI appears to require the absence of CrtC and, in a crtC background, even the native 3-step enzyme can synthesize a significant amount (13%) of lycopene, in addition to the expected neurosporene. We suggest that the CrtC hydroxylase can intervene in the sequence of reactions catalyzed by phytoene desaturase. We investigated the properties of the lycopene-synthesizing strain of Rb. sphaeroides. In the LH2 light-harvesting complex, lycopene transfers absorbed light energy to the bacteriochlorophylls with an efficiency of 54%, which compares favourably with other LH2 complexes that contain carotenoids with 11 conjugated double bonds. Thus, lycopene can join the assembly pathway for photosynthetic complexes in Rb. sphaeroides, and can perform its role as an energy donor to bacteriochlorophylls.

Dynamics of excitation energy transfer in the LH1 and LH2 light-harvesting complexes of photosynthetic bacteria.

Photosynthetic light harvesting is a unique life process that occurs with amazing efficiency. Since the discovery of the structure of the bacterial peripheral light-harvesting complex (LH2), this process has been studied using a variety of advanced laser spectroscopic methods. We are now in a position to discuss the physical origins of excitation energy transfer and trapping in the LH2 and LH1 antennae of photosynthetic purple bacteria. We demonstrate that the time evolution of the state created by the light is determined by the combined action of excitonic pigment-pitment interactions, energetic disorder, and coupling to nuclear motion in a pigment-protein complex. A quantitative fit of experimental data using Redfield theory allowed us to determine the pathways and time scales of exciton and vibrational relaxation and analyze separately different contributions to the measured transient absorption dynamics. Furthermore, these dynamics were observed to be strongly dependent on the excitation wavelength. A numerical fit of this dependence turns out to be extremely critical to a variation of the structure and disorder parameters and, therefore, can be used as a test for different antenna models (disordered ring, elliptical deformations, correlated disorder, etc.). The calculated equilibration dynamics in the exciton basis allow a visualization of the exciton motion using a density matrix picture in real space.

Microscopic Theory of Exciton Annihilation: Application to the LH2 Antenna System

The multiexciton density matrix theory is utilized to achieve a microscopic description of exciton−exciton annihilation (EEA). We apply the theory to the 18 bacteriochlorophyll (BChl) molecules of the B850 ring of the light-harvesting complex LH2 of Rhodobacter sphaeroides. The simulation of the EEA process reproduces the intensity-dependent transient absorption kinetic experiments very well, and insight is obtained on microscopic parameters such as the internal conversion rate of BChl in LH2. The exciton dynamics and the different relaxation processes are visualized by constructing a multiexciton spectrogram.

Self Diffusion and Spectral Modifications of a Membrane Protein, the Rubrivivax gelatinosus LH2 Complex, Incorporated into a Monoolein Cubic Phase

The light-harvesting complex LH2 from a purple bacterium, Rubrivivax gelatinosus, has been incorporated into the Q230 cubic phase of monoolein. We measured the self-diffusion of LH2 in detergent solution and in the cubic phase by fluorescence recovery after photobleaching. We investigated also the absorption and fluorescence properties of this oligomeric membrane protein in the cubic phase, in comparison with its β-octyl glucoside solution. In these experiments, native LH2 and LH2 labeled by a fluorescent marker were used. The results indicate that the inclusion of LH2 into the cubic phase induced modifications in the carotenoid and B800 binding sites. Despite these significant perturbations, the protein seems to keep an oligomeric structure. The relevance of these observations for the possible crystallization of this protein in the cubic phase is discussed.

Two-dimensional structure of the native light-harvesting complex LH2 from Rubrivivax gelatinosus and of a truncated form

The light-harvesting complex LH2 of Rubrivivax gelatinosus has an oligomeric structure built from α-β heterodimers containing three bacteriochlorophylls and one carotenoid each. The α subunit (71 residues) presents a C-terminal hydrophobic extension (residues 51–71) which is prone to attack by an endogenous protease. This extension can also be cleaved by a mild thermolysin treatment, as demonstrated by electrophoresis and by matrix-assisted laser desorption-time of flight mass spectrometry. This cleavage does not affect the pigment binding sites as shown by absorption spectroscopy. Electron microscopy was used to investigate the structures of the native and thermolysin cleaved forms of the complexes. Two-dimensional crystals of the reconstituted complexes were examined after negative staining and cryomicroscopy. Projection maps at 10 Å resolution were calculated, demonstrating the nonameric ring-like organization of α-β subunits. The cleaved form presents the same structural features. We conclude that the LH2 complex is structurally homologous to the Rhodopseudomonas acidophila LH2. The hydrophobic C-terminal extension does not fold back in the membrane, but lays out on the periplasmic surface of the complex.

Effects of positional disorder on optical absorption spectra of light-harvesting antenna complexes in photosynthetic bacteria

We study theoretically the influence of both diagonal and off-diagonal disorder on the absorption spectra of the light-harvesting antenna complex LH2 consisting of two circular aggregates, B850 and B800, of bacteriochlorophyll pigments in photosynthetic purple bacteria. Off-diagonal disorder, i.e., randomness in excitonic couplings between molecules, is introduced by a model of disorder in the position of each pigment molecule embedded in proteins. We demonstrate that a large contribution of positional disorder provides a natural explanation for the experimental fact that the excitonic B850 absorption peak is broader than that of monomeric B800 in spite of motional narrowing.

The electronic structure of the light-harvesting LH2 complex in the light of single-molecule optical spectroscopy

The three-dimensional structure of LH2 is characterized by a high symmetry which imposes particular selection rules on the optical transitions involving the exciton states that are associated with the interacting bacteriochlorophyll a pigments. Therefore, this system offers a unique case for a detailed study of parameters that determine the electronic structure of light-harvesting complexes. Of particular interest are the actual interaction strengths between the pigments, the effect of intrinsic disorder, and the contribution of electron-phonon interactions. Many experiments have been designed to determine these parameters, but the supposed exciton structure of LH2 is masked by inhomogeneous broadening of the absorption lines due to heterogeneity of the sample. Here we report on the results obtained by single-molecule optical spectroscopy at low temperature, by which effects of inhomogenous broadening are largely eliminated. This allows us to obtain optical spectra with well-resolved structure, which offer a critical test for theoretical models of the LH2 complex. We will present the results that were obtained sofar, and discuss the implications for the electronic structure of LH2.

Heteronuclear 2D-correlations in a uniformly [13C, 15N] labeled membrane-protein complex at ultra-high magnetic fields.

One- and two-dimensional solid-state NMR experiments on a uniformly labeled intrinsic membrane-protein complex at ultra-high magnetic fields are presented. Two-dimensional backbone and side-chain correlations for a [U-13C, 15N] labeled version of the LH2 light-harvesting complex indicate significant resolution at low temperatures and under Magic Angle Spinning. Tentative assignments of some of the observed correlations are presented and attributed to the alpha-helical segments of the protein, mostly found in the membrane interior.

Rapid excitation-energy transfer to optically forbidden states in light-harvesting antennas of photosynthetic bacteria

The rate of excitation-energy transfer (EET) within the light-harvesting complex LH2 of photosynthetic purple bacteria is calculated, based on a new formula for EET between molecular aggregates with a small mutual distance, by taking into account both the exciton–phonon and exciton–disorder interactions. It is elucidated that EET from B800 to higher and optically forbidden exciton states in B850 is the dominant mechanism of the rapid EET.