Characterization and deposition of various light-harvesting antenna complexes by electrospray atomization

Photosynthetic organisms have light-harvesting complexes that absorb and transfer energy efficiently to reaction centers. Light-harvesting complexes (LHCs) have received increased attention in order to understand the natural photosynthetic process and also to utilize their unique properties in fabricating efficient artificial and bio-hybrid devices to capture solar energy. In this work, LHCs with different architectures, sizes, and absorption spectra, such as chlorosomes, Fenna–Matthews–Olson (FMO) protein, LH2 complex, and phycobilisome have been characterized by an electrospray-scanning mobility particle-sizer system (ES-SMPS). The size measured by ES-SMPS for FMO, chlorosomes, LH2, and phycobilisome were 6.4, 23.3, 9.5, and 33.4?nm, respectively. These size measurements were compared with values measured by dynamic light scattering and those reported in the literature. These complexes were deposited onto a transparent substrate by electrospray deposition. Absorption and fluorescence spectra of the deposited LHCs were measured. It was observed that the LHCs have light absorption and fluorescence spectra similar to that in solution, demonstrating the viability of the process.     

Quantum chemical insights in energy dissipation and carotenoid radical cation formation in light harvesting complexes

Light harvesting complexes (LHCs) have been identified in all photosynthetic organisms. To understand their function in light harvesting and energy dissipation, detailed knowledge about possible excitation energy transfer (EET) and electron transfer (ET) processes in these pigment proteins is of prime importance. This again requires the study of electronically excited states of the involved pigment molecules, in LHCs of chlorophylls and carotenoids. This paper represents a critical review of recent quantum chemical calculations on EET and ET processes between pigment pairs relevant for the major LHCs of green plants (LHC-II) and of purple bacteria (LH2). The theoretical methodology for a meaningful investigation of such processes is described in detail, and benefits and limitations of standard methods are discussed. The current status of excited state calculations on chlorophylls and carotenoids is outlined. It is focused on the possibility of EET and ET in the context of chlorophyll fluorescence quenching in LHC-II and carotenoid radical cation formation in LH2. In the context of non-photochemical quenching of green plants, it is shown that replacement of the carotenoid violaxanthin by zeaxanthin in its binding pocket of LHC-II can not result in efficient quenching. In LH2, our computational results give strong evidence that the S(1) states of the carotenoids are involved in carotenoid cation formation. By comparison of theoretical findings with recent experimental data, a general mechanism for carotenoid radical cation formation is suggested.     

Two-photon study on the electronic interactions between the first excited singlet states in carotenoid-tetrapyrrole dyads

Electronic interactions between the first excited states (S(1)) of carotenoids (Car) of different conjugation lengths (8-11 double bonds) and phthalocyanines (Pc) in different Car-Pc dyad molecules were investigated by two-photon spectroscopy and compared with Car S(1)-chlorophyll (Chl) interactions in photosynthetic light harvesting complexes (LHCs). The observation of Chl/Pc fluorescence after selective two-photon excitation of the Car S(1) state allowed sensitive monitoring of the flow of energy between Car S(1) and Pc or Chl. It is found that two-photon excitation excites to about 80% to 100% exclusively the carotenoid state Car S(1) and that only a small fraction of direct tetrapyrrole two-photon excitation occurs. Amide-linked Car-Pc dyads in tetrahydrofuran demonstrate a molecular gear shift mechanism in that effective Car S(1) → Pc energy transfer is observed in a dyad with 9 double bonds in the carotenoid, whereas in similar dyads with 11 double bonds in the carotenoid, the Pc fluorescence is strongly quenched by Pc → Car S(1) energy transfer. In phenylamino-linked Car-Pc dyads in toluene extremely large electronic interactions between the Car S(1) state and Pc were observed, particularly in the case of a dyad in which the carotenoid contained 10 double bonds. This observation together with previous findings in the same system provides strong evidence for excitonic Car S(1)-Pc Q(y) interactions. Very similar results were observed with photosynthetic LHC II complexes in the past, supporting an important role of such interactions in photosynthetic down-regulation.     

Integrating DNA Photonic Wires into Light‐Harvesting Supramolecular Polymers

An approach combining DNA nanoscaffolds with supramolecular polymers for the efficient and directional propagation of light‐harvesting cascades has been developed. A series of photonic wires with different arrangements of fluorophores in DNA‐organized nanostructures were linked to light‐harvesting supramolecular phenanthrene polymers (SPs) in a self‐assembled fashion. Among them, a light‐harvesting complex (LHC) composed of SPs and a photonic wire of phenanthrene, Cy3, Cy5, and Cy5.5 chromophores reveals a remarkable energy transfer efficiency of 59 %. Stepwise transfer of the excitation energy collected by the light‐harvesting SPs via the intermediate Cy3 and Cy5 chromophores to the final Cy5.5 acceptor proceeds through a Förster resonance energy transfer mechanism. In addition, the light‐harvesting properties are documented by antenna effects ranging from 1.4 up to 23 for different LHCs.     

The Growth‐promoting Mechanism of Unusual Spectroscopic Form of LH2 (LH4) from Rhodopseudomonas palustris CGA009 in Low Light

The experimental evidence for the growth‐promoting mechanism and the efficiency of energy transfer (EET) of LH4 under low light are still not available. To elucidate the light adaption mechanism of LH4, we deleted the genes pucBAd involved in the synthesis of the α/β polypeptides of LH4 in Rhodopseudomonas palustris CGA009. Compared to wild strain, the growth rate of pucBAd mutant significantly decreased under low light, while there were no significant changes in the growth rate, the contents and compositions of photopigments, absorption spectra of cell lysates under high light. Moreover, the fluorescence quantum efficiency (FQE) was used to further compare the EET between LH2 and LH4. The FQE in LH4 increased up to 1.5‐fold than did in LH2. Collectively, this study showed that LH4 could provide more and high energetic state photons for promoting bacterial phototrophic growth in response to low‐light environment.     

Ultrafast time-resolved carotenoid to-bacteriochlorophyll energy transfer in LH2 complexes from photosynthetic bacteria

Steady-state and ultrafast time-resolved optical spectroscopic investigations have been carried out at 293 and 10 K on LH2 pigment-protein complexes isolated from three different strains of photosynthetic bacteria: Rhodobacter (Rb.) sphaeroides G1C, Rb. sphaeroides 2.4.1 (anaerobically and aerobically grown), and Rps. acidophila 10050. The LH2 complexes obtained from these strains contain the carotenoids, neurosporene, spheroidene, spheroidenone, and rhodopin glucoside, respectively. These molecules have a systematically increasing number of pi-electron conjugated carbon-carbon double bonds. Steady-state absorption and fluorescence excitation experiments have revealed that the total efficiency of energy transfer from the carotenoids to bacteriochlorophyll is independent of temperature and nearly constant at approximately 90% for the LH2 complexes containing neurosporene, spheroidene, spheroidenone, but drops to approximately 53% for the complex containing rhodopin glucoside. Ultrafast transient absorption spectra in the near-infrared (NIR) region of the purified carotenoids in solution have revealed the energies of the S1 (2(1)Ag-)-->S2 (1(1)Bu+) excited-state transitions which, when subtracted from the energies of the S0 (1(1)Ag-)-->S2 (1(1)Bu+) transitions determined by steady-state absorption measurements, give precise values for the positions of the S1 (2(1)Ag-) states of the carotenoids. Global fitting of the ultrafast spectral and temporal data sets have revealed the dynamics of the pathways of de-excitation of the carotenoid excited states. The pathways include energy transfer to bacteriochlorophyll, population of the so-called S* state of the carotenoids, and formation of carotenoid radical cations (Car*+). The investigation has found that excitation energy transfer to bacteriochlorophyll is partitioned through the S1 (1(1)Ag-), S2 (1(1)Bu+), and S* states of the different carotenoids to varying degrees. This is understood through a consideration of the energies of the states and the spectral profiles of the molecules. A significant finding is that, due to the low S1 (2(1)Ag-) energy of rhodopin glucoside, energy transfer from this state to the bacteriochlorophylls is significantly less probable compared to the other complexes. This work resolves a long-standing question regarding the cause of the precipitous drop in energy transfer efficiency when the extent of pi-electron conjugation of the carotenoid is extended from ten to eleven conjugated carbon-carbon double bonds in LH2 complexes from purple photosynthetic bacteria.     

Excitonic energy transfer in light-harvesting complexes in purple bacteria

Two distinct approaches, the Frenkel-Dirac time-dependent variation and the Haken-Strobl model, are adopted to study energy transfer dynamics in single-ring and double-ring light-harvesting (LH) systems in purple bacteria. It is found that the inclusion of long-range dipolar interactions in the two methods results in significant increase in intra- or inter-ring exciton transfer efficiency. The dependence of exciton transfer efficiency on trapping positions on single rings of LH2 (B850) and LH1 is similar to that in toy models with nearest-neighbor coupling only. However, owing to the symmetry breaking caused by the dimerization of BChls and dipolar couplings, such dependence has been largely suppressed. In the studies of coupled-ring systems, both methods reveal an interesting role of dipolar interactions in increasing energy transfer efficiency by introducing multiple intra/inter-ring transfer paths. Importantly, the time scale (4 ps) of inter-ring exciton transfer obtained from polaron dynamics is in good agreement with previous studies. In a double-ring LH2 system, non-nearest neighbor interactions can induce symmetry breaking, which leads to global and local minima of the average trapping time in the presence of a non-zero dephasing rate, suggesting that environment dephasing helps preserve quantum coherent energy transfer when the perfect circular symmetry in the hypothetic system is broken. This study reveals that dipolar coupling between chromophores may play an important role in the high energy transfer efficiency in the LH systems of purple bacteria and many other natural photosynthetic systems.     

THE MULTIPLE PIGMENT-PROTEINS OF THE PHOTOSYSTEM I ANTENNA*

A photosystem I (PS I) holocomplex was obtained from barley by ultracentrifugation of PS I-enriched stroma lamellae on sucrose gradients. Further solubilization with glycosidic surfactants followed by Deriphat-poly-acrylamide gel electrophoresis (PAGE) fractionated the holocomplex into its core complex (CC I) and individual light-harvesting I (LHC I) pigment-protein subcomplexes. The LHC I contains chlorophyll a, all of the chlorophyll A of PS I and xanthophylls but no carotenes. Sodium dodecylsulfate PAGE analysis of the subcomplexes shows that barley LHC I is composed of at least five apoproteins having sizes between 11 and 24 kDa. Isolation of a 17 kDa LHC Ic component by Deriphat-PAGE shows it to be a photosynthetic pigment-protein. Room-temperature absorption spectra indicate that LHC Ic is enriched in chlorophyll a in comparison to the LHC Ia and Ib components. The LHC Ic apoprotein is shown to be distinct from the subunit III and IV polypeptides of CC I. Analysis of PS I fractions obtained from sucrose gradients as well as from Deriphat-PAGE indicates that in higher plants an oligomeric structure of the PS I entity exists in vitro.     

Energy transfer between surface-immobilized light-harvesting chlorophyll a/b complex (LHCII) studied by surface plasmon field-enhanced fluorescence spectroscopy (SPFS)

The major light-harvesting chlorophyll a/b complex (LHCII) of the photosynthetic apparatus in green plants can be viewed as a protein scaffold binding and positioning a large number of pigment molecules that combines rapid and efficient excitation energy transfer with effective protection of its pigments from photobleaching. These properties make LHCII potentially interesting as a light harvester (or a model thereof) in photoelectronic applications. Most of such applications would require the LHCII to be immobilized on a solid surface. In a previous study we showed the immobilization of recombinant LHCII on functionalized gold surfaces via a 6-histidine tag (His tag) in the protein moiety. In this work the occurrence and efficiency of Fo?rster energy transfer between immobilized LHCII on a functionalized surface have been analyzed by surface plasmon field-enhanced fluorescence spectroscopy (SPFS). A near-infrared dye was attached to some but not all of the LHC complexes, serving as an energy acceptor to chlorophylls. Analysis of the energy transfer from chlorophylls to this acceptor dye yielded information about the extent of intercomplex energy transfer between immobilized LHCII.     

Excited state dynamics in photosynthetic reaction center and light harvesting complex 1

Key to efficient harvesting of sunlight in photosynthesis is the first energy conversion process in which electronic excitation establishes a trans-membrane charge gradient. This conversion is accomplished by the photosynthetic reaction center (RC) that is, in case of the purple photosynthetic bacterium Rhodobacter sphaeroides studied here, surrounded by light harvesting complex 1 (LH1). The RC employs six pigment molecules to initiate the conversion: four bacteriochlorophylls and two bacteriopheophytins. The excited states of these pigments interact very strongly and are simultaneously influenced by the surrounding thermal protein environment. Likewise, LH1 employs 32 bacteriochlorophylls influenced in their excited state dynamics by strong interaction between the pigments and by interaction with the protein environment. Modeling the excited state dynamics in the RC as well as in LH1 requires theoretical methods, which account for both pigment-pigment interaction and pigment-environment interaction. In the present study we describe the excitation dynamics within a RC and excitation transfer between light harvesting complex 1 (LH1) and RC, employing the hierarchical equation of motion method. For this purpose a set of model parameters that reproduce RC as well as LH1 spectra and observed oscillatory excitation dynamics in the RC is suggested. We find that the environment has a significant effect on LH1-RC excitation transfer and that excitation transfers incoherently between LH1 and RC.     

Toward assessment of density functionals for vibronic coupling in two‐photon absorption: A case study of 4‐nitroaniline

In this study, we predict vibronic two‐photon absorption (TPA) spectra for 4‐nitroaniline in vacuo. The simulations are performed using density functional theory and the approximate second‐order coupled‐cluster singles and doubles model CC2. Thereby we also demonstrate the possibility of simulations of vibronic TPA spectra with ab initio wavefunction methods that include electron correlation for medium‐sized systems. A special focus is put on the geometric derivatives of the second‐order transition moment and the dipole moment difference between the charge‐transfer excited state and the ground state. The results of CC2 calculations bring new insight into the vibronic coupling mechanism in TPA spectra of 4‐nitroniline and demonstrate that the mixed term is quite large and that it also exhibits a negative interference with the Franck‐Condon contribution. © 2015 Wiley Periodicals, Inc.     

Structure-Based Calculations of the Optical Spectra of the LH2 Bacteriochlorophyll-Protein Complex from Rhodopseudomonas acidophila

Abstract— The molecular structure of the light-harvesting complex 2 (LH2) bacteriochlorophyll-protein antenna complex from the purple non-sulfur photosynthetic bacterium Rhodopseudomonas acidophila , strain 10050 provides the positions and orientations of the 27 bacteriochlorophyll (BChl) molecules in the complex. Our structure-based model calculations of the distinctive optical properties (absorption, CD, polarization) of LH2 in the near-infrared region use a point-monopole approximation to represent the BChl Q_y transition moment. The results of the calculations support the assignment of the ring of 18 closely coupled BChl to B850 (BChl absorbing at 850 nm) and the larger diameter, parallel ring of 9 weakly coupled BChl to B800. All of the significantly allowed transitions in the near infrared are calculated to be perpendicular to the C9 symmetry axis, in agreement with polarization studies of this membrane-associated complex. To match the absorption maxima of the B800 and B850 components using a relative permittivity (dielectric constant) of 2.1, we assign different site energies (12 500 and 12260 cm⁻¹, respectively) for the Q_y transitions of the respective BChl in their protein binding sites. Excitonic coupling is particularly strong among the set of B850 chromophores, with pairwise interaction energies nearly 300 cm between nearest neighbors, comparable with the experimental absorption bandwidths at room temperature. These strong interactions, for the full set of 18 B850 chromophores, result in an excitonic manifold that is 1200 cm⁻¹ wide. Some of the upper excitonic states should result in weak absorption and perhaps stronger CD features. These predictions from the calculations await experimental verification.     

Separation of single‐walled carbon nanotubes with aromatic group functionalized polymethacrylates and building blocks contribution to the enrichment

We previously showed that in N,N‐dimethylformamide (DMF), poly(9‐anthracenylmethyl methacrylate) (PAMMA) and poly(2‐naphthylmethacrylate) selectively disperse semiconducting and metallic single‐walled carbon nanotubes (SWNTs), respectively. We have also proposed a new noncovalent polymer interaction based on photon induced dipole–dipole interaction to account for the metallicity‐based selectivity. In this article, we investigate two other polymethacrylates, that is, poly(benzyl methacrylate) (PBMA) and poly(methylmethacrylate)‐co‐(9‐anthracenylmethyl acrylate) (PMMA‐c‐PAMA) in the light of our previously proposed photon‐induced dipole–dipole interaction. We find that PBMA and PMM‐c‐PAMMA in DMF show no metallicity selectivity. The different selective behavior of the four polymers in DMF manifests the decisive influence of the side aromatic group in determining their metallicity selectivity. The nonpreferential energy transfer from PMMA‐c‐PAMA to SWNTs and the nonoverlap of PBMA fluorescence (in the ultraviolet range) with nanotube absorption account for their nonselectivity of specific nanotube species. Further, the parallel relationship between the diameters of extracted tube species and the affinity between polymers and solvents suggests the leading role of the polymeric conformation on the diameter selectivity. A sufficient (i.e., 2 weeks) standing time of the SWNTs solution after sonication, during which the polymers presumably optimize their conformation to the SWNTs, was found to be essential to the enrichment. © 2011 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys, 2011     

Reconstitution of bacterial photosynthetic unit in a lipid bilayer studied by single-molecule spectroscopy at 5 K

As a model of photosynthetic unit (PSU), self-assembled aggregates of pigment-protein complexes from photosynthetic bacteria were prepared in a lipid bilayer by reconstitution of the light-harvesting 2 (LH2) complex and light-harvesting 1-reaction center (LH1-RC) complex through detergent removal of their micelles in the presence of lipids. By performing polarization-controlled fluorescence and fluorescence-excitation spectroscopy on single aggregates at a temperature of 5 K, the composition of individual aggregates was determined and excitation energy transfer (EET) between constituent complexes was observed. LH2 and LH1-RC from a bacterium, Rhodobacter (Rb.) sphaeroides, were found to form a trimeric aggregate in which EET takes place from one LH2 to two LH1-RCs. In contrast, a heterodimer of LH2 and LH1-RC in which EET works was found to assemble from a combination of complexes of different bacterial species, that is, LH2 from Rb. sphaeroides and LH1-RC from Rhodopseudomonas (Rps.) palustris.     

Electronic coherence effects in photosynthetic light harvesting

Photosynthetic light harvesting is a paradigmatic example for quantum effects in biology. In this work, we review studies on quantum coherence effects in the LH2 antenna complex from purple bacteria to demonstrate how quantum mechanical rules play important roles in the speedup of excitation energy transfer, the stabilization of electronic excitations, and the robustness of light harvesting in photosynthesis. Subsequently, we present our recent theoretical studies on exciton dynamical localization and excitonic coherence generation in photosynthetic systems. We apply a variational-polaron approach to investigate decoherence of exciton states induced by dynamical fluctuations due to system-environment interactions. The results indicate that the dynamical localization of photoexcitations in photosynthetic complexes is significant and imperative for a complete understanding of coherence and excitation dynamics in photosynthesis. Moreover, we use a simple model to investigate quantum coherence effects in intercomplex excitation energy transfer in natural photosynthesis, with a focus on the likelihoods of generating excitonic coherences during the process. Our model simulations reveal that excitonic coherence between acceptor exciton states and transient nonlocal quantum correlation between distant pairs of chromophores can be generated through intercomplex energy transfer. Finally, we discuss the implications of these theoretical works and important open questions that remain to be answered.     

Explaining the visible and near-infrared circular dichroism spectra of light-harvesting 1 complexes from purple bacteria: a modeling study

In this work, we investigate the origin and characteristics of the circular dichroism (CD) spectrum of various light-harvesting 1 (LH1) complexes. The near-infrared (NIR) CD signal of these core antennae is strongly nonconservative, and the nature of this nonconservativity is under examination in this paper. So far, on the basis of the high-resolution structures of LH2, we have been able to model the absorption and CD spectra in the bacteriochlorophyll (BChl) Q(Y) and Q(X) regions of LH2 (Georgakopoulou et al., Biophys. J. 2002, 82, 2184-2197), as well as in the carotenoid region (Georgakopoulou et al., Biophys. J. 2004, 87, 3010-3022). We proceed by applying the same modeling method in order to reproduce the LH1 spectra. We assume a ring of dimers in a perfect circular arrangement with 16-fold symmetry, and account for all excitonic interactions within the ring. Because LH1 complexes exhibit Q(Y) and Q(X) CD signals of very low intensity, higher transitions can easily affect these regions. Therefore, we expand the model and take into account also the Soret and carotenoid transitions. We can now understand the shape of the absorption and CD spectra and contemplate the structure of the LH1 complex. The latter is similar to LH2 in that it is a very symmetric ring dominated by excitonic interactions. The larger number of symmetry and the bigger diameter of LH1, combined with small rotations of the BChl transition dipole moments, are responsible for the display of CD signals that are very low in intensity. The interaction of the Q(Y) with the carotenoid transitions results in complete loss of the conservativity. Interaction energies between all the pigments in the ring are calculated, and their values are in good accordance with what is reported in the literature.     

Selective assembly of photosynthetic antenna proteins into a domain-structured lipid bilayer for the construction of artificial photosynthetic antenna systems: structural analysis of the assembly using surface plasmon resonance and atomic force microscopy

Two types of photosynthetic membrane proteins, the peripheral antenna complex (LH2) and the core antenna/reaction center complex (LH1-RC), play an essential role in the primary process of purple bacterial photosynthesis, that is, capturing light energy, transferring it to the RC where it is used in subsequent charge separation. Establishment of experimental platforms is required to understand the function of the supramolecular assembly of LH2 and LH1-RC molecules into arrays. In this study, we assembled LH2 and LH1-RC arrays into domain-structured planar lipid bilayers placed on a coverglass using stepwise combinations of vesicle-to-planar membrane formation and vesicle fusion methods. First, it was shown that assembly of LH2 and LH1-RC in planar lipid bilayers, through vesicle-to-planar membrane formation, could be confirmed by absorption spectroscopy and high resolution atomic force microscopy (AFM). Second, formation of a planar membrane incorporating LH2 molecules made by the vesicle fusion method was corroborated by AFM together with quantitative analysis by surface plasmon resonance (SPR). By combining planar membrane formation and vesicle fusion, in a stepwise manner, LH2 and LH1-RC were successfully organized in the domain-structured planar lipid membrane. This methodology for construction of LH2/LH1-RC assemblies will be a useful experimental platform with which to investigate energy transfer from LH2 to LH1-RC where the relative arrangement of these two complexes can be controlled.     

Near-IR absorption and fluorescence spectra and AFM observation of the light-harvesting 1 complex on a mica substrate refolded from the subunit light-harvesting 1 complexes of photosynthetic bacteria Rhodospirillum rubrum

The subunit light-harvesting 1 (LH 1) complexes isolated from photosynthetic bacteria Rhodospirillum rubrum using n-octyl-beta-glucoside were reassociated and adsorbed on a mica substrate using spin-coat methods with the aim of using this LH complex in a nanodevice. The near-IR absorption and fluorescence spectra of the LH 1 complexes indicated that the LH 1 complex on the mica was stable, and efficient energy transfer from a carotenoid to a bacteriochlorophyll a was observed. Atomic force microscopy of the reassociated LH 1 complexes, under air, showed the expected ringlike structure. The outer and inner diameters of the ringlike structure of the LH 1 complex were approximately 30 and 8 nm, respectively, and the ringlike structure protruded by 0.2-0.6 nm.     

Molecular assembly of artificial photosynthetic antenna core complex on an amino-terminated ITO electrode

Bacterial photosynthetic membrane proteins, light-harvesting antenna complex (LH1), reaction center (RC), and their combined ‘core’ complex (LH1–RC) are functional elements in the primary photosynthetic events, i.e., capturing and transferring light energy and subsequent charge separation. These photosynthetic units (PSUs) isolated from Rhodospirillum rubrum (Rs. rubrum) were assembled onto an ITO electrode modified with 3-aminopropyltriethoxysilane (APS–ITO). The near IR absorption spectra of PSUs on the assembled electrodes were identical to those of solutions, indicating that the LH1 and LH1–RC core complexes were native on the electrode. Photocurrent response of PSUs on the electrode was examined upon illumination of the LH1 complex at 880 nm. The LH1–RC and a mixed assembly of LH1 and RC exhibited photocurrent response, but not LH1 only, consistent with the function of these PSUs, capturing light energy and transferring electron. This result provides useful methodology for building an artificial fabrication of PSUs on the electrode.