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Photophysics of Biological and Synthetic Multichromophoric Systems : Spectroscopic Investigations of Bacterial Light-Harvesting Complexes and of Carbonyl-Bridged Triarylamine Derivatives
Abstract
Multichromophoric systems play a major role in natural photosynthetic processes. They are the basic building blocks for the absorption of light and the subsequent energy transfer within photosynthetic organisms. Optimized by evolutionary selection processes, they constitute an ideal blueprint for artificial, nature-inspired multichromophoric light-harvesting systems. This thesis investigates the energy transfer and the photophysical properties of different multichromophoric systems:
-Reaction centre light-harvesting complex I (RC-LH1), a natural light-harvesting complex from purple bacteria,
-a hybrid system built from a spherical gold nanoparticle (AuNP) and a light-harvesting complex II (LH2), another light-harvesting complex from purple bacteria,
-two novel artificial light-harvesting systems, based on carbonyl-bridged triarylamines (CBTs)
Different methods of optical spectroscopy and optical microscopy have been used to investigate these systems.
The first part of this work discusses the results of time-resolved optical spectroscopy in a picosecond-range on isolated RC-LH1 from Rhodopseudomonas palustris. Their fluorescence decay was recorded dependent on the excitation fluence and the repetition rate of the excitation laser. Both parameters were varied over three orders of magnitude. We observed three components in the decays with characteristic decay times of 40, 200 and 600 ps, respectively, that occurred in different amplitude ratios, dependent on the excitation parameters. A first suggestion that the decay times are an indicator of the redox state of the so called “special pair” (P) within the RC could be underpinned by two reference experiments. In those experiments, the redox state of P was influenced by a reducing agent or an oxidising agent, respectively, by stepwise increasing its concentration while the fluence and the repetition rate stayed fixed. Thus the decay times of 40, 200 and 600 ps could be assigned to different species of RC-LH1 with their special pair in the neutral state P, with the special pair in the reduced state P+ and with the reaction centre lacking or dysfunctional, respectively. Using these results as well as values from the literature we were able to design a detailed kinetic model of the energy transfer pathways in RC-LH1. The fluorescence decays of RC-LH1 could then be simulated by a global master equation approach based on a microstate description. Due to an excellent agreement between experiment and simulation this model allows to predict the relative populations of the aforementioned species for given excitation parameters as well as to predict the relative population of carotenoid triplet states on the LH1 rings.
In the second part of this work, a fluorescence microscope with single molecule sensitivity was used to show the plasmonic fluorescence enhancement of LH2 from Rhodobacter sphaeroides by spherical AuNP. The fluorescence intensities of single LH2 were measured in presence and in absence of AuNP, with the excitation wavelength in resonance and off resonance of the AuNP’s plasmon. Using a home-built evaluation algorithm the intensities of more than 4000 single LH2 could be retrieved. From these intensities, extensive distributions that show the relative frequencies of the different intensity values for all the aforementioned excitation conditions could be gained. When excited in resonance with the plasmon of the AuNP, the intensity distribution of the LH2 in presence of AuNPs as a whole shifted to higher intensity values as compared to the case in absence of AuNPs. The mean value of the intensity distribution in presence of the AuNP was about a factor of 2 higher than that of the intensity distribution in absence of the AuNP. When excited off the plasmon resonance, the intensity distributions of LH2 were almost identical in presence as well as in absence of AuNPs. These observations show the plasmonic origin of the fluorescence enhancement of LH2 and point towards a small spatial distance between LH2 and AuNP, which might indicate an adsorption of LH2 to the spherical AuNP. In a reference experiment a spacer layer of a thickness of 20 nm was introduced between AuNP and LH2. In this configuration, no fluorescence enhancement could be detected even at excitation of LH2 within the plasmon resonance. The result from this reference experiment thus defined a maximum distance between AuNP and LH2 of 20 nm when no spacer layer is present. Model calculations underpinned our observations and gave a direct hint towards an adsorption of LH2 to the AuNPs.
The third part of this work is concerned with the extensive optical characterisation of two novel organic light-harvesting systems. Two Sprache: Englisch
Multichromophoric systems play a major role in natural photosynthetic processes. They are the basic building blocks for the absorption of light and the subsequent energy transfer within photosynthetic organisms. Optimized by evolutionary selection processes, they constitute an ideal blueprint for artificial, nature-inspired multichromophoric light-harvesting systems. This thesis investigates the energy transfer and the photophysical properties of different multichromophoric systems:
-Reaction centre light-harvesting complex I (RC-LH1), a natural light-harvesting complex from purple bacteria,
-a hybrid system built from a spherical gold nanoparticle (AuNP) and a light-harvesting complex II (LH2), another light-harvesting complex from purple bacteria,
-two novel artificial light-harvesting systems, based on carbonyl-bridged triarylamines (CBTs)
Different methods of optical spectroscopy and optical microscopy have been used to investigate these systems.
The first part of this work discusses the results of time-resolved optical spectroscopy in a picosecond-range on isolated RC-LH1 from Rhodopseudomonas palustris. Their fluorescence decay was recorded dependent on the excitation fluence and the repetition rate of the excitation laser. Both parameters were varied over three orders of magnitude. We observed three components in the decays with characteristic decay times of 40, 200 and 600 ps, respectively, that occurred in different amplitude ratios, dependent on the excitation parameters. A first suggestion that the decay times are an indicator of the redox state of the so called “special pair” (P) within the RC could be underpinned by two reference experiments. In those experiments, the redox state of P was influenced by a reducing agent or an oxidising agent, respectively, by stepwise increasing its concentration while the fluence and the repetition rate stayed fixed. Thus the decay times of 40, 200 and 600 ps could be assigned to different species of RC-LH1 with their special pair in the neutral state P, with the special pair in the reduced state P+ and with the reaction centre lacking or dysfunctional, respectively. Using these results as well as values from the literature we were able to design a detailed kinetic model of the energy transfer pathways in RC-LH1. The fluorescence decays of RC-LH1 could then be simulated by a global master equation approach based on a microstate description. Due to an excellent agreement between experiment and simulation this model allows to predict the relative populations of the aforementioned species for given excitation parameters as well as to predict the relative population of carotenoid triplet states on the LH1 rings.
In the second part of this work, a fluorescence microscope with single molecule sensitivity was used to show the plasmonic fluorescence enhancement of LH2 from Rhodobacter sphaeroides by spherical AuNP. The fluorescence intensities of single LH2 were measured in presence and in absence of AuNP, with the excitation wavelength in resonance and off resonance of the AuNP’s plasmon. Using a home-built evaluation algorithm the intensities of more than 4000 single LH2 could be retrieved. From these intensities, extensive distributions that show the relative frequencies of the different intensity values for all the aforementioned excitation conditions could be gained. When excited in resonance with the plasmon of the AuNP, the intensity distribution of the LH2 in presence of AuNPs as a whole shifted to higher intensity values as compared to the case in absence of AuNPs. The mean value of the intensity distribution in presence of the AuNP was about a factor of 2 higher than that of the intensity distribution in absence of the AuNP. When excited off the plasmon resonance, the intensity distributions of LH2 were almost identical in presence as well as in absence of AuNPs. These observations show the plasmonic origin of the fluorescence enhancement of LH2 and point towards a small spatial distance between LH2 and AuNP, which might indicate an adsorption of LH2 to the spherical AuNP. In a reference experiment a spacer layer of a thickness of 20 nm was introduced between AuNP and LH2. In this configuration, no fluorescence enhancement could be detected even at excitation of LH2 within the plasmon resonance. The result from this reference experiment thus defined a maximum distance between AuNP and LH2 of 20 nm when no spacer layer is present. Model calculations underpinned our observations and gave a direct hint towards an adsorption of LH2 to the AuNPs.
The third part of this work is concerned with the extensive optical characterisation of two novel organic light-harvesting systems. Two derivatives of carbonyl-bridged triarylamines (CBTs) were investigated with absorption spectroscopy, photoluminescence (PL) emission and PL-excitation spectroscopy (all steady-state) as well as time-resolved emission spectroscopy on picosecond timescales. Both compounds consist of a CBT core that is decorated with either three peripheral naphthalimide (NI) molecules or three peripheral 4-(5-hexyl-2,2’-bithiophene)-naphthalimide (NIBT) molecules. These compounds are abbreviated as CBT-NI and CBT-NIBT, respectively. Additionally isolated CBT, NI and NIBT were investigated as reference compounds as well as mixtures of CBT with NI or NIBT, respectively, that were not covalently bound. For all compounds, we recorded the absorption spectra from the near ultraviolet to the near infrared range, the PL emission spectra in dependence of the excitation wavelength – which at the same time gave access to the PL-excitation spectra – as well as the PL quantum yield and the PL lifetime. For the mixtures of the isolated compounds the same parameters were recorded except for the PL quantum yield and the PL lifetime. The data showed that CBT-NI works as an energy funnel. Photoluminescence always occurred from the CBT core, no matter whether the excitation was tuned to the spectral region of the CBT core or the peripheral NI. For CBT-NIBT, the reverse behaviour could be observed. No matter which absorption band was excited, only PL from the NIBT periphery could be observed. In contrast to the concentrator abilities of CBT-NI, CBT-NIBT represents an energy distributor.
In summary this thesis presented three multichromophoric systems that act as examples to understand natural light-harvesting systems, to manipulate them and to imitate them. The experiments on RC-LH1 from Rhodopseudomonas palustris allow a deeper understanding of the energy transfer pathways within this system. The model derived from these experiments enables us to make detailed predictions of the photophysical behaviour of RC-LH1 dependent on the excitation parameters. The study on AuNP-LH2 hybrids represents a statistically solid proof-of-principle that shows the plasmonic fluorescence enhancement of single bacterial light-harvesting complexes by well-defined nanostructures. It gives a good example on how natural light-harvesting systems can be manipulated. The imitation and advancement of natural light-harvesting concepts is realised in the study of the two derivatives of CBT. The extensive optical characterisation of these novel compounds shows their potential as building blocks for molecular electronics, organic photovoltaics and as a promising model system for molecular energy transfer.
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The aim of this paper is to review and discuss the results obtained by fluorescence and absorption spectroscopy of bacterial chromatophores excited with picosecond pulses of varying power and intensity. It was inferred that spectral and kinetic characteristics depend essentially on the intensity, the repetition rate of the picosecond excitation pulses as well as on the optical density of the samples used. Taking the different experimental conditions properly into account, most of the discrepancies between the fluorescence and absorption measurements can be solved. At high pulse repetition rate (>10(6) Hz), even at moderate excitation intensities (10(10)-10(11) photons/cm(2) per pulse), relatively long-lived triplet states start accumulating in the system. These are efficient (as compared to the reaction centers) quenchers of mobile singlet excitations due to singlet-triplet annihilation. The singlet-triplet annihilation rate constant in Rhodospirillum rubrum was determined to be equal to 10(-9) cm(3) s(-1). At fluences >10(12) photons/cm(2) per pulse singlet-singlet annihilation must be taken into account. Furthermore, in the case of high pulse repetition rates, triplet-triplet annihilation must be considered as well. From an analysis of experimental data it was inferred that the singlet-singlet annihilation process is probably migration-limited. If this is the case, one has to conclude that the rate of excitation decay in light-harvesting antenna at low pumping intensities is limited by the efficiency of excitation trapping by the reaction center. The influence of annihilation processes on spectral changes is also discussed as is the potential of a local heating caused by annihilation processes. The manifestation of spectral inhomogeneity of light-harvesting antenna in picosecond fluorescence and absorption kinetics is analyzed.
A theory for Frenkel exciton dynamics in molecular aggregates which incorporates coupling to vibrational motions (intramolecular, intermolecular and solvent) with multiple spectral densities of arbitrary nature and interpolates between the coherent and the incoherent limits is developed. A rigorous procedure for identifying the relevant collective nuclear coordinates necessary to represent a given set of spectral densities is obtained. Additional coordinates are required as the temperature is lowered. Exciton dynamics is calculated by following the evolution of wavepackets representing the electronic density matrix in the collective coordinates phase space. The signatures of excitonic and nuclear motions in ultrafast fluorescence spectroscopy are explored using a hierarchy of reduction schemes with varying numbers of collective coordinates.
We investigate the optical properties of spherical gold and silver clusters with diameters of 20 nm and larger. The light scattering spectra of individual clusters are measured using dark-field microscopy, thus avoiding inhomogeneous broadening effects. The dipolar plasmon resonances of the clusters are found to have nearly Lorentzian line shapes. With increasing size we observe polaritonic red-shifts of the plasmon line and increased radiation damping for both gold and silver clusters. Apart from some cluster-to-cluster variations of the plasmon lines, agreement with Mie theory is reasonably good for the gold clusters. However, it is less satisfactory for the silver clusters, possibly due to cluster faceting or chemical effects.
Observations of the annihilation constant and the energy transfer rate of singlet excitons in naphthalene and anthracene are analyzed in terms of a recent theoretical framework. Problems that arise in traditional interpretations of the temperature dependence are resolved. It is shown that in certain temperature ranges the observables contain little information about exciton motion. Lower bounds for motion rates are diffusion constants are obtained.
The crystal structure of the light-harvesting antenna complex (LH2) from Rhodopseudomonas acidophila strain 10050 shows that the active assembly consists of two concentric cylinders of helical protein subunits which enclose the pigment molecules. Eighteen bacteriochlorophyll a molecules sandwiched between the helices form a continuous overlapping ring, and a further nine are positioned between the outer helices with the bacteriochlorin rings perpendicular to the transmembrane helix axis. There is an elegant intertwining of the bacteriochlorophyll phytol chains with carotenoid, which spans the complex.
Origin and early evolution of lifeEarliest evidence for life and photosynthesisThe nature of the earliest photosynthetic systemsThe origin and evolution of metabolic pathways with special reference to chlorophyll biosynthesisEvolutionary relationships among reaction centers and other electron transport componentsDo all photosynthetic reaction centers derive from a common ancestor?The origin of linked photosystems and oxygen evolutionOrigin of the oxygen-evolving complex and the transition to oxygenic photosynthesisAntenna systems have multiple evolutionary originsEndosymbiosis and the origin of chloroplastsOne versus several endosymbiotic events for chloroplast originsMost types of algae are the result of secondary endosymbiosisFollowing endosymbiosis, many genes were transferred to the nucleus, and proteins were reimported to the chloroplastEvolution of carbon metabolism pathways
Supramolecular chemistry offers a great toolbox of non-covalent interactions to organize chromophoric and multichromophoric systems into functional aggregates of precise size and order via a bottom-up approach. These interactions can be utilized to improve the photophysical properties and processes (e.g. transport of excitation energy) of the supramolecular aggregates. This thesis deals with the synthesis, the self-assembly and the investigation of photophysical properties of novel multichromophoric systems.
The first part of this thesis covers the influences of steric effects and hydrogen-bonding on the self-assembly, the stacking behavior and the optical properties of pyrene derivatives. The photophysical properties of the pyrene compounds were investigated in (i) highly diluted solutions, (ii) at increased concentrations in an intermediate self-assembly regime, and (iii) in the dry crystalline state, utilizing steady-state and time-resolved optical spectroscopy. The π stacking of the pyrene moieties into weakly coupling H aggregates ─ i.e. cofacially arranged chromophores ─ is the governing driving force initiating self-assembly in solution. In this regime, the ground state H-aggregates of the pyrene moieties give rise to excimer formation ─ i.e. an excited dimer. Hydrogen-bonding and steric effects have almost no influence on the self-assembly process, but they do determine the excimer formation rate within the supramolecular aggregates. Upon transition to the crystalline state the influence of the different intermolecular interactions becomes more striking. The excimer fluorescence vanishes with increasing order in the crystalline state, which is promoted by the formation of hydrogen-bonds and is decreased with the introduction of bulky groups.
The second part of this thesis deals with the synthesis and the photophysical properties of two novel carbonyl-bridged triarylamine derivatives. The C3 symmetric chromophore was connected in 2, 6, and 10 position via amide linkers and short alkyl spacers with either naphthalimides or 4-(5-hexyl-2,2’-bithiophene)-naphthalimides. The employed universal synthetic route allows for a versatile functionalization of carbonyl-bridged triarylamines. Both multichromophoric compounds show efficient energy transfer, evidenced by steady-state and time-resolved spectroscopy. The compound bearing naphthalimides in the periphery funnels the energy from the periphery to the carbonyl-bridged triarylamine core. In the second multichromophoric compound energy transfer proceeds vice versa, from the central carbonyl-bridged triarylamine to the lateral 4-(5-hexyl-2,2’-bithiophene)-naphthalimide units. Furthermore, this compound forms transparent fluorescent gels at very low concentration while retaining the optical and energy transfer properties.
The last part of this thesis is concerned with the self-assembly of the carbonyl-bridged triarylamine with 4-(5-hexyl-2,2’-bithiophene)-naphthalimide substituents which forms supramolecular nanofibers. A striking feature of these aggregates is the outstanding excitation energy transport along the fibers at room temperature. Electron and atomic force microscopy techniques revealed micrometer-long supramolecular aggregates with molecular diameter. Spectroscopic investigations of these nanofibers demonstrate that hydrogen-bonding of the amide linkers together with strong π-stacking of the carbonyl-bridged triarylamine are responsible for the uniaxial self-assembly. The threefold-symmetric nature of the hydrogen-bonds enforces a cofacial H-aggregation of the carbonyl-bridged triarylamine cores leading to a significant nearest neighbor coupling. The high level of order in combination with the coupling of the carbonyl-bridged triarylamine cores result in long-range energy transport over at least 4 µm, which corresponds to more than 10.000 molecules. This was verified for individual nanofibers using a confocal fluorescence microscope. For this supramolecular system the energy transport is most likely explained by quantum coherent effects. The self-assembly of this compound is an excellent example to demonstrate the enormous potential of supramolecular chemistry towards highly ordered non-covalently bonded architectures with remarkable properties and functionality.
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We studied the time-resolved fluorescence of isolated RC-LH1 complexes from Rhodopseudomonas palustris as a function of the photon fluence and the repetition rate of the excitation laser. Both parameters were varied systematically over three orders of magnitude. Based on a microstate description we developed a quantitative model for RC-LH1 and obtained very good agreement between experiments and elaborate simulations based on a global master equation approach. The model allows us predict the relative population of RC-LH1 complexes with the special pair in the neutral state or in the oxidized state P(+), and those complexes that lack a reaction centre.
The synthesis and photophysical properties of two novel multichromophoric compounds is presented. Their molecular design comprises a carbonyl-bridged triarylamine core and either naphthalimides or 4-(5-hexyl-2,2′-bithiophene)naphthalimides as second chromophore in the periphery. The lateral chromophores are attached to the core via an amide linkage and a short alkyl spacer. The synthetic approach demonstrates a straightforward functionalization strategy for carbonyl-bridged triarylamines. Steady-state and time-resolved spectroscopic investigations of these compounds, in combination with three reference compounds, provide clear evidence for energy transfer in both multichromophoric compounds. The direction of the energy transfer depends on the lateral chromophore used. Furthermore, the compound bearing the lateral 4-(bithiophene)naphthaimides is capable of forming fluorescent gels at very low concentrations in the sub-millimolar regime whilst retaining its energy transfer properties.
Decay of the bacteriochlorophyll excited state was measured in membranes of the purple bacteria Rhodospirillum (R.) rubrum, Rhodobacter (Rb.) sphaeroides wild type and Rb. sphaeroides mutant M21 using low intensity picosecond absorption spectroscopy. The excitation and probing pulses were chosen in the far red wing of the long wavelength absorption band, such that predominantly the minor antenna species B896 was excited. The decay of B896 was studied between 77 and 177K under conditions that the traps were active. In all species the B896 excited state decay is almost temperature independent between 100 and 177K, and probably between 100 and 300 K. In this temperature range the decay rates for the various species are very similar and close to 40 ps. Below 100 K this rate remains temperature independent in Rb. sphaeroides w. t. and M21, while in R. rubrum a steep decrease sets in. An analysis of this data with the theory of nuclear tunneling indicates an activation energy for the final transfer step from B896 to the special pair of 70cm(-1) for R. rubrum and 30cm(-1) or less for Rb. sphaeroides.
IntroductionVarious Approaches of Fluorescence SensingFluorescent pH IndicatorsDesign Principles of Fluorescent Molecular Sensors Based on Ion or Molecule RecognitionFluorescent Molecular Sensors of Metal IonsFluorescent Molecular Sensors of AnionsFluorescent Molecular Sensors of Neutral MoleculesFluorescence Sensing of GasesSensing DevicesRemote Sensing by Fluorescence LIDARSpectrophotometric and Spectrofluorometric pH TitrationsSingle-Wavelength MeasurementsDual-Wavelength MeasurementsDetermination of the Stoichiometry and Stability Constant of Metal Complexes from Spectrophotometric or Spectrofluorometric TitrationsDefinition of the Equilibrium ConstantsPreliminary Remarks on Titrations by Spectrophotometry and SpectrofluorometryFormation of a 1 : 1 Complex (Single-Wavelength Measurements)Formation of a 1 : 1 Complex (Dual-Wavelength Measurements)Formation of Successive Complexes ML and M2LCooperativityDetermination of the Stoichiometry of a Complex by the Method of Continuous Variations (Job's Method)Bibliography
The native bacteriopheophytin a in reaction centers of Rb. sphaeroides R26 has been exchanged with modified bacteriopheophytins (bacteriochlorins), as well as with plant-type pheophytins (chlorins). Emphasis is on four pigments, which differ by their C-3 substituents (vinyl or acetyl) or their state of oxidation (chlorin or bacteriochlorin). The native BPhe a, which is a member of this group, can be replaced by the other three at both binding sites, HA and HB. However, exchange at HB proceeds more readily. Optical spectra (absorption, cd) show characteristic shifts, and the cd spectra indicate induced interactions between HA,B and BA,B and possibly also with P. Upon flash illumination, all modified reaction centers show reversible electron transfer to QB with recombination times comparable to native reaction centers. Forward rates and electron-transfer yields are also reported for some of the pigments.
Transient EPR spectra of protonated and deuterated Zn-substituted reaction centres of Rhodobacter sphaeroides R-26, measured at a microwave frequency of 95 GHz and an external magnetic field of 3.5 T, are presented. The high-field/high-frequency spin-polarized spectrum of the correlated coupled radical pair P865+.QA−. after laser flash excitation is a very sensitive monitor of the relative orientation of the two g-matrices and the dipolar tensor with respect to each other. Therefore, detailed structural information of the RC concerning the relative orientation of the primary donor P865+. and the acceptor QA−. with respect to the axis connecting the two molecules in the RC can be derived. Together with the information obtained by high-field cw-EPR on single crystals, the enhanced resolution of the polarized high-field spectra allows an unambiguous assignment of the g-matrix of the donor P865+. to the molecular axis system. The experimental results are compared with earlier X-band (9 GHz) and K-band (24 GHz) EPR experiments.
Luminescent and redox-active heterotriangulene-based dendrimers (G1 and G2) of first and second generation have been synthesized, and their photophysical properties, oxidation behaviors, and applications in organic light emitting diodes (OLEDs) have been investigated. The dendrimers show efficient aggregation-induced emissions originated from the carbazole dendrons, heterotriangulene core, intramolecular charge-transfer (ICT), and intermolecular aggregation absorptions. Highest-occupied molecular orbital and lowest-unoccupied molecular orbital (HOMO and LUMO) values were acquired using UV–vis spectroscopy and cyclic voltammetry. The OLEDs fabricated with G1 and G2 as non-doping emitters exhibit exclusive aggregation-induced luminescence peaking at 600 and 630nm, respectively, and demonstrate a good performance with maximum luminance of 1586cdm−2 at current efficiency of 5.3cdA−1 for G1 and 827cdm−2 at current efficiency of 6.9cdA−1 for G2.
Compound 1 was synthesized by a palladium-catalyzed Sonogashira cross-coupling reaction of heterotriangulene derivative 3 with three equiv. of 1-dodecyloxy-4-ethynylbenzene 2 in good yield. After the dichloromethane solution of 1 was mixed with methanol, two-level self-assembly from nanowires to microrods based on 1 was obtained and characterized with scanning electron microscopy (SEM), transmission electron microscopy (TEM) and X-ray diffraction (XRD). The driving force of the two-level self-assembly is attributed to the strong π–π stacking interactions of heterotriangulene cores and the hydrophobic interactions of alkyl chains with solvent molecules.
The primary reaction of photosynthesis is light-driven charge
separation, carried out in reaction centres, which are complexes of
integral membrane proteins and cofactors. The recent determination of
the crystal structures of the reaction centres of two photosynthetic
bacteria provides a basis for a quantitative understanding of the
primary electron transfer processes of photosynthesis.
A new approach that uses a combination of semiempirical configuration interaction method and exciton theory to calculate electronic energies, eigenstates, absorption spectrum and circular dichroism (CD) spectrum of the LH2 antenna of Rhodopseudomonas acidophila is introduced. A statistical simulation that uses experimental homogeneous line widths was used to account for the inhomogeneous line width of the observed spectrum. Including the effect of orbital overlap of the close-lying pigments of the B850 ring and the effect of the pigment protein interaction in the B800 ring allowed a successful simulation of the experimental absorption and CD spectra of the antenna at room temperature. Two experimental parameters, the transition energy and the magnitude of the transition dipole moment of monomeric bacteriochlorophyll a (Bchl a), were used in the calculation. The dielectric constant of the protein matrix was taken as 2.1 [ε0]. The questions of localization lengths of the excitonic states and the energy transfer mechanism between the B800 and the B850 rings are discussed in light of the results obtained.
The remarkable efficiencies of solar energy conversion attained by photosynthetic organisms derive partly from the designs of the light-harvesting apparatuses. The strategy employed by nature is to capture sunlight over a wide spectral and spatial cross section in chromophore arrays, then funnel the energy to a trap (reaction center). Nature's blueprint has inspired the conception of a diversity of artificial light-harvesting antenna systems for applications in solar energy conversion or photonics. Despite numerous, wide-ranging studies, truly quantitative predictions for such multichromophoric assemblies are scarce because Förster theory in its standard form often seems to fail. We report here a new framework within which energy transfer in molecular assemblies can be modeled quantitatively using a generalization of Förster's theory. Our results show that the principles involved in optimization of energy transfer in confined molecular assemblies are not revealed in a simple way by the absorption and emission spectra because such spectra are insensitive to length scales on the order of molecular dimensions.
M subunit Trp252 is the only amino acid residue which is located between the bacteriopheophytin HA and the quinone QA in the photosynthetic reaction centre of Rhodobacter sphaeroides. Oligodeoxynucleotide-directed mutagenesis was employed to elucidate the influence of this aromatic amino acid on the electron transfer between these two chromophores. For this, M subunit Trp252 was changed to tyrosine or phenylalanine, and Thr222, which presumably forms a hydrogen bridge to the indole ring of M subunit Trp252, to valine. In all three mutated reaction centres, the electron-accepting ubiquinone QA is less firmly bound to its binding site than in the wild-type protein. The electron transfer from the reduced bacteriopheophytin HA− to QA proceeds in the wild-type and in the mutant ThrM222Val within 220 ps. However, in the mutants TrpM252Tyr and TrpM252Phe the time constants are 600 ps and 900 ps, respectively. This indicates that M subunit Trp252 participates in the binding of QA and reduction of this quinone.
Low temperature spin polarized X- and K-band transient EPR spectra of the state P+.(Q
A−.Mn2+) in reaction centres of Rhodobacter sphaeroides Y are presented. A strong component with a spectral width of ∼0.4 mT appears near g = 2 and a weak component with a width of ∼570 mT is observed between g ≈ 1 – 6. The narrow spectrum is primarily due to P+. and the broad component is assigned to (Q
A−.Mn2+). Both signals are polarized as a result of the correlation of the unpaired spins on P
865+. and (Q
A−.Mn2+). At times < 200 ns after the laser the P+. contribution has an AEA (A = absorption, E = emission) polarization pattern at both X- and K-band. The K-band spectrum has a somewhat higher spectral resolution and three distinct time constants in the polarization decay. At later times, the initial AEA pattern relaxes to a purely absorptive spectrum. The decay of the spin polarized (Q
A−.Mn2+) signal is about two orders of magnitude slower than that of P
854+.. However, its rise time is roughly the same as the decay of the AEA pattern near g = 2. Both contributions to the spectra can be observed up to temperatures above 200 K with little or no change in form. However, the transition from the early to late signal near g = 2 is faster at higher temperatures as a result of faster spin relaxation. The dominant relaxation rate follows Arrhenius behaviour. However, the fast component observed at K-band is independent of temperature. The effect of the magnetic properties of the metal on the spin polarization and the expected differences between the spectra of P+.(Q
A−.Mn2+) and P+.(Q
A−.Fe2+) are discussed.
In order to find an upper theoretical limit for the efficiency of p‐n junction solar energy converters, a limiting efficiency, called the detailed balance limit of efficiency, has been calculated for an ideal case in which the only recombination mechanism of hole‐electron pairs is radiative as required by the principle of detailed balance. The efficiency is also calculated for the case in which radiative recombination is only a fixed fraction f c of the total recombination, the rest being nonradiative. Efficiencies at the matched loads have been calculated with band gap and f c as parameters, the sun and cell being assumed to be blackbodies with temperatures of 6000°K and 300°K, respectively. The maximum efficiency is found to be 30% for an energy gap of 1.1 ev and f c = 1. Actual junctions do not obey the predicted current‐voltage relationship, and reasons for the difference and its relevance to efficiency are discussed.
The formation and decay of excited states of light-harvesting bacteriochlorophyll (BChl) molecules and primary charge separation in reaction centres (RCs) of pigment-protein complex B890 of the purple bacterium Chromatium minutissimum were studied as a function of excitation wavelength by means of picosecond absorbance difference spectroscopy. Selective excitation in the RC absorption band (at 800 nm) induced rapid bleaching of the absorption band of the RC BChl dimer (τ ∼ 1 ps) without absorption changes due to excited light-harvesting BChl. It is concluded that excitation energy transfer from the BChl dimer of the RC to light-harvesting BChl molecules is considerably slower than RC charge separation. Therefore, energy transfer from the light-harvesting antenna to the RC in bacterial photosynthesis may be described as a ‘migration-limited model’.
The efficiency of energy back-transfer from the reaction center to the antenna in chromatophores of the photosynthetic purple bacterium Rhodopseudomonas viridis was measured at room temperature by means of picosecond time-resolved fluorescence spectroscopy. It was found that 20 ± 5% of the excitation energy selectively absorbed by the reaction center pigments in the wavelength region around 830 nm is transferred back to the antenna and gives rise to antenna fluorescence. The measured yield of energy detrapping enabled calculation of the energy detrapping (10–13 ps)−1 and trapping (40–50 ps)−1 rates. These results confirm that in Rps. viridis, like in bacteriochlorophyll a-containing purple bacteria, the energy transfer step from antenna pigments to the reaction center is a rate limiting step in the overall energy trapping by the reaction center. We suggest that this situation is termed ‘transfer-to-trap-limited’ dynamics, to distinguish it from the situation where the charge separation is the rate limiting step (trap limited) or the overall energy diffusion through the antenna is limiting (diffusion limited).
Comparative measurements of time- and spectrally resolved picosecond fluorescence of the photosynthetic purple bacterium Rhodospirillum rubrum in a wide temperature range from room temperature down to 4 K have been performed with different excitation wavelengths within the main infrared absorption band employed. Several kinetically different spectral components have been characterized in the fluorescence decay at low temperatures. This gives substantial support to the viewpoint that the B880 light-harvesting antenna band of R. rubrum (and probably the core antenna of other purple bacteria) consists of more than just two spectral forms, as has been suggested by several authors. The rates of the antenna singlet excitation quenching by reaction centres in different states have been determined. Although almost unchanged in the temperature range from 300 to 77 K, these rates decrease by 2 to 4 times on temperature lowering down to 4 K. It was found that at 4 K, the oxidized primary donor is a stronger fluorescence quencher than its triplet state.
The molecular structure of the photosynthetic reaction centre from Rhodopseudomonas viridis has been elucidated using X-ray crystallographic analysis. The central part of the complex consists of two subunits, L and M, each of which forms five membrane-spanning helices. We present the first description of the high-resolution structure of an integral membrane protein.
One striking feature of bacterial reaction centers is that while they show a high degree of structural symmetry, function is entirely asymmetric: excitation of the primary electron donor, P, a bacteriochlorophyll (BChl) dimer, results almost exclusively in electron transfer along one of the two symmetric electron transfer pathways. Here another functional asymmetry of the reaction center is explored; i.e., the two monomer BChl molecules (B(A) and B(B)) have distinct interactions with P in the oxidized state, P(+). Previous work has suggested that the excited states of both B(A) and B(B) were quenched via energy transfer to P(+) within a few hundred femtoseconds. Here, it is shown that various excitation wavelengths, corresponding to different initial B(A) and B(B) excited states, result in distinct reaction pathways, and which pathway dominates depends both on the initial excited state formed and on the electronic structure of P(+). In particular, it is possible to specifically excite the Q(X) transition of B(B) by using excitation at 495 nm directly into the carotenoid S(2) state which then undergoes energy transfer to B(B). This results in the formation of a new state on the picosecond time scale that is both much longer lived and spectrally different than what one would expect for a simple excited state. Combining results from additional measurements using nonselective 600 or 800 nm excitation of both B(A) and B(B) to the Q(X) or Q(Y) states, respectively, it is found that B(B)* and B(A)* are quenched by P(+) with different kinetics and mechanisms. B(A)* formed using either Q(X) or Q(Y) excitation appears to decay rapidly (∼200 fs) without a detectable intermediate. In contrast, B(B)* formed via Q(X) excitation predominantly generates the long-lived state referred to above via an electron transfer reaction from the Q(X) excited state of B(B) to P(+). This reaction is in competition with intramolecular relaxation of the Q(X) state to the lowest singlet excited state. The Q(Y) excited state of B(B) appears to undergo the electron transfer reaction seen upon Q(X) excitation only to a very limited extent and is largely quenched via energy transfer to P(+). Finally, the ability of P(+) to quench B(B)* depends on the electronic structure of P(+). The asymmetric charge distribution between the two halves of P in the native reaction center is effectively reversed in the mutant HF(L168)/LH(L131), and in this case, the rate of quenching decreases significantly.
A new type of organic sensitizers incorporating a planar amine unit have been synthesized and demonstrated to be a highly efficient sensitizers, showing evidence of lateral interactions on the TiO2 surface. Under standard global air mass 1.5 solar conditions, the JK-98 sensitized cell gave a short circuit photocurrent density (J(sc)) of 16.78 mA cm(-2), an open-circuit voltage (V,,c) of 0.745 (V-oc) and a fill factor (ft) of 0.70, corresponding to an overall conversion efficiency (eta) of 8.71%.
We studied the optical response from more than 13, 000 individual photosynthetic pigment-protein complexes interacting with spherical gold nanoparticles. The nanodots were arranged in a quasi-hexagonal array by diblock copolymer micellar nanolithography. Exciting the proteins within the spectral range of the nanoparticles' plasmon resonance yields a clear enhancement of the protein fluorescence intensity, whereas excitation far out of the plasmon resonance features no effect. This result indicates a strategy for the construction of efficient hybrid light-harvesting devices.
We performed time-resolved spectroscopy on homoarrays of LH2 complexes from the photosynthetic purple bacterium Rhodopseudomonas acidophila. Variations of the fluorescence transients were monitored as a function of the excitation fluence and the repetition rate of the excitation. These parameters are directly related to the excitation density within the array and to the number of LH2 complexes that still carry a triplet state prior to the next excitation. Comparison of the experimental observations with results from dynamic Monte Carlo simulations for a model cluster of LH2 complexes yields qualitative agreement without the need for any free parameter and reveals the mutual relationship between energy transfer and annihilation processes.
We have employed time-resolved spectroscopy on the picosecond time scale in combination with dynamic Monte Carlo simulations to investigate the photophysical properties of light-harvesting 2 (LH2) complexes from the purple photosynthetic bacterium Rhodopseudomonas acidophila. The variations of the fluorescence transients were studied as a function of the excitation fluence, the repetition rate of the excitation and the sample preparation conditions. Here we present the results obtained on detergent solubilized LH2 complexes, i.e., avoiding intercomplex interactions, and show that a simple four-state model is sufficient to grasp the experimental observations quantitatively without the need for any free parameters. This approach allows us to obtain a quantitative measure for the singlet-triplet annihilation rate in isolated, noninteracting LH2 complexes.
A flexible organic dyad consisting of a perylene bisimide antenna covalently linked to a [60]fullerene has been synthesized and studied by electrochemistry, steady-state spectroscopy, and time-resolved spectroscopy. We found that the energy absorbed by the perylene bisimide is transferred to the fullerene with an efficiency close to 100%. The fullerene in turn undergoes intersystem crossing followed by triplet energy transfer back to the perylene bisimide with an efficiency of at least 20%. Hence the perylene bisimide unit acts as an antenna for the fullerene, i.e., effectively extending the fullerene absorption far into the visible spectral range, while at the same time the fullerene acts as a triplet sensitizer for the perylene bisimide. This has severe consequences for the exploitation of the dye antenna-fullerene concept for light harvesting in solar cells.
Photosynthetic reaction centres (RCs) catalyze light-driven electron, transport across photosynthetic membranes. The photosynthetic bacterium Rhodobacter, sphaeroides is often used for studies of RCs, and three groups have determined the structure of its reaction centre. There are discrepancies between these structures, however, and to resolve these we have determined the structure to higher resolution than before, using a new crystal form.
The new structure provides a more detailed description of the Rb. sphaeroides RC, and allows us to compare it with the structure of the RC from Rhodopseudomonas viridis. We find no evidence to support most of the published differences in cofactor binding between the RCs from Rps. viridis and Rb. sphaeroides. Generally, the mode of cofactor binding is conserved, particularly along the electron transfer pathway. Substantial differences are only found at ring V of one bacteriochlorophyll of the 'special pair' and for the secondary quinone, QB. A water chain with a length of about 23 A including 14 water molecules extends from the QB to the cytoplasmic side of the RC.
The cofactor arrangement and the mode of binding to the protein seem to be very similar among the non-sulphur bacterial photosynthetic RCs. The functional role of the displaced QB molecule, which might be present as quinol, rather than quinone, is not yet clear. The newly discovered water chain to the QB binding site suggests a pathway for the protonation of the secondary quinone QB.
The theory of the singlet-singlet annihilation in quasi-homogeneous photosynthetic antenna systems is developed further. In the new model, the following important contributions are taken into account: 1) the finite excitation pulse duration, 2) the occupation of higher excited states during the annihilation, 3) excitation correlation effects, and 4) the effect of local heating. The main emphasis is concentrated on the analysis of pump-probe kinetic measurements demonstrating the first two above possible contributions. The difference with the results obtained from low-intensity fluorescence kinetic measurements is highlighted. The experimental data with picosecond time resolution obtained for the photosynthetic bacterium Rhodospirillum rubrum at room temperature are discussed on the basis of this theory.
The crystal structure of a bacterial light-harvesting membrane protein complex shows a simple modular design and explains how solar energy is collected and passed on to the reaction centre.
Background:
The light-harvesting complexes II (LH-2s) are integral membrane proteins that form ring-like structures, oligomers of alpha beta-heterodimers, in the photosynthetic membranes of purple bacteria. They contain a large number of chromophores organized optimally for light absorption and rapid light energy migration. Recently, the structure of the nonameric LH-2 of Rhodopseudomonas acidophila has been determined; we report here the crystal structure of the octameric LH-2 from Rhodospirillum molischianum. The unveiling of similarities and differences in the architecture of these proteins may provide valuable insight into the efficient energy transfer mechanisms of bacterial photosynthesis.
Results:
The crystal structure of LH-2 from Rs. molischianum has been determined by molecular replacement at 2.4 A resolution using X-ray diffraction. The crystal structure displays two concentric cylinders of sixteen membrane-spanning helical subunits, containing two rings of bacteriochlorophyll-a (BChl-a) molecules. One ring comprises sixteen B850 BChl-as perpendicular to the membrane plane and the other eight B800 BChl-as that are nearly parallel to the membrane plane; eight membrane-spanning lycopenes (the major carotenoid in this complex) stretch out between the B800 and B850 BChl-as. The B800 BChl-as exhibit a different ligation from that of Rps. acidophila (aspartate is the Mg ligand as opposed to formyl-methionine in Rps. acidophila).
Conclusions:
The light-harvesting complexes from different bacteria assume various ring sizes. In LH-2 of Rs. molischianum, the Qy transition dipole moments of neighbouring B850 and B800 BChl-as are nearly parallel to each other, that is, they are optimally aligned for Föster exciton transfer. Dexter energy transfer between these chlorophylls is also possible through interactions mediated by lycopenes and B850 BChl-a phytyl tails; the B800 BChl-a and one of the two B850 BChl-as associated with each heterodimeric unit are in van der Waals distance to a lycopene, such that singlet and triplet energy transfer between lycopene and the BChl-as can occur by the Dexter mechanism. The ring structure of the B850 BChl-as is optimal for light energy transfer in that it samples all spatial absorption and emission characteristics and places all oscillator strength into energetically low lying, thermally accessible exciton states.
Carotenoids are usually considered to perform two major functions in
photosynthesis. They serve as accessory light harvesting pigments,
extending the range of wavelengths over which light can drive
photosynthesis, and they act to protect the chlorophyllous pigments from
the harmful photodestructive reaction which occurs in the presence of
oxygen. Drawing upon recent work with photosynthetic bacteria, evidence
is presented as to how the carotenoids are organized within both
portions of the photosynthetic unit (the light harvesting antenna and
the reaction centre) and how they discharge both their functions. The
accessory pigment role is a singlet-singlet energy transfer from the
carotenoid to the bacteriochlorophyll, while the protective role is a
triplet-triplet energy transfer from the bacteriochlorophyll to the
carotenoid.
The intensity dependence of picosecond kinetics in the light-harvesting antenna of the photosynthetic bacterium Rhodospirillum rubrum is studied at 77 K. By changing either the average excitation intensity or the pulse intensity we have been able to discriminate singlet-singlet and singlet-triplet annihilation. It is shown that the kinetics of both annihilation types are well characterized by the concept of percolative excitation dynamics leading to the time-dependent annihilation rates. The time dependence of these two types of annihilation rates is qualitatively different, whereas the dependencies can be related through the same adjustable parameter-a spectral dimension of fractal-like structures. The theoretical dependencies give a good fit to the experimental kinetics if the spectral dimension is equal to 1.5 and the overall singlet-singlet annihilation rate is close to the value obtained at room temperature. The percolative transfer is a consequence of spectral inhomogeneous broadening. The effect is more pronounced at lower temperatures because of the narrowing of homogeneous spectra.
The effect of chemical oxidation on the absorption and fluorescence emission spectra of the LH1 complex from Rhodobium marinum was investigated. Mild chemical oxidation of the LH1 complex, by addition of 10 mM potassium ferricyanide, caused a 2-3% bleaching of the 880-nm Qy absorption band. In contrast, at the same ferricyanide concentration, fluorescence emission intensity of the LH1 complex was quenched by about 50%. This result demonstrates that oxidation of very few bacteriochlorophyll (BChl) molecules in the LH1 ring is enough to completely quench its fluorescence.
Three photosynthetic complexes, light-harvesting complex 2 (LH2), light-harvesting complex 1 (LH1), and the reaction centre-light-harvesting complex 1 photounit (RC-LH1), were purified from a single species of a purple bacterium, Rhodobacter sphaeroides, and reconstituted into two-dimensional (2-D) crystals. Vesicular 2-D crystals of LH1 and RC-LH1 were imaged in negative stain and projection maps at 25 A resolution were produced. The rings formed by LH1 have approximately the same mean diameter as the LH1 rings from Rhodospirillum rubrum ( approximately 90 A) and therefore are likely to be composed of 15 to 17 alphabeta subunits. In the projection map calculated from the RC-LH1 2-D crystals, the reaction centre is represented by an additional density in the centre of the ring formed by the LH1 subunits. The marked improvement of shape and fine structure after a rotational pre-alignment of the RC-LH1 unit cells before averaging strongly suggests that the RC is not in a unique orientation within the LH1 rings. Tubular crystals of LH2 showed a high degree of order and allowed calculation of a projection map at 6 A resolution from glucose-embedded specimens. The projection structure shows a ring of nine alphabeta subunits. Variation of the alpha-helical projection densities suggests that the 9-fold symmetry axis is tilted with respect to the membrane normal.
The electronic structure of the circular aggregate of 18 bacteriochlorophyll a (BChl a) molecules responsible for the B850 absorption band of the light-harvesting 2 (LH2) complex of the photosynthetic purple bacterium Rhodopseudomonas acidophila has been studied by measuring fluorescence-excitation spectra of individual complexes at 1.2 K. The spectra reveal several well-resolved bands that are obscured in the single, broad B850 band observed in conventional absorption measurements on bulk samples. They are interpreted consistently in terms of the exciton model for the circular aggregate of BChl a molecules. From the energy separation between the different exciton transitions a reliable value of the intermolecular interaction is obtained. The spectra of the individual complexes allow for a distinction between the intra- and the intercomplex disorder. In addition to the random disorder, a regular modulation of the interaction has to be assumed to account for all the features of the observed spectra. This modulation has a C(2) symmetry, which strongly suggests a structural deformation of the ring into an ellipse.
Singlet-singlet annihilation is used to study exciton delocalization in the light harvesting antenna complex LH2 (B800-B850) from the photosynthetic purple bacterium Rhodobacter sphaeroides. The characteristic femtosecond decay constants of the high intensity isotropic and the low intensity anisotropy kinetics of the B850 ring are related to the hopping time tau(h) and the coherence length N(coh) of the exciton. Our analysis yields N(coh) = 2.8+/-0.4 and tau(h) = 0.27+/-0.05 ps. This approach can be seen as an extension to the concept of the spectroscopic ruler.
The new characteristics of multichromophoric Förster resonance energy transfer (MC-FRET) theory was discussed. It was clarified that the far-field linear spectroscopic information was insufficient to determine reaction rate and that distance dependence of the rate can vary with the disorder and temperature. The theory was applied to an important energy transfer process in light harvesting complex LH2. Application of the theory to the LH2 provided new evidence for very efficient and dispersive energy transfer dynamics caused by the MC effects.
The structure at 100K of integral membrane light-harvesting complex II (LH2) from Rhodopseudomonas acidophila strain 10050 has been refined to 2.0A resolution. The electron density has been significantly improved, compared to the 2.5A resolution map, by high resolution data, cryo-cooling and translation, libration, screw (TLS) refinement. The electron density reveals a second carotenoid molecule, the last five C-terminal residues of the alpha-chain and a carboxy modified alpha-Met1 which forms the ligand of the B800 bacteriochlorophyll. TLS refinement has enabled the characterisation of displacements between molecules in the complex. B850 bacteriochlorophyll molecules are arranged in a ring of 18 pigments composed of nine approximate dimers. These pigments are strongly coupled and at their equilibrium positions the excited state dipole interaction energies, within and between dimers, are approximately 370cm(-1) and 280cm(-1), respectively. This difference in coupling energy is similar in magnitude to changes in interaction energies arising from the pigment displacements described by TLS tensors. The displacements appear to be non-random in nature and appear to be designed to optimise the modulation of pigment energy interactions. This is the first time that LH2 pigment displacements have been quantified experimentally. The calculated energy changes indicate that there may be significant contributions to inter-pigment energy interactions from molecular displacements and these may be of importance to photosynthetic energy transfer.