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.     

Computational prediction of absorbance maxima for a structurally diverse series of engineered green fluorescent protein chromophores

By virtue of its self-sufficiency to form a visible wavelength chromophore within the confines of its tertiary structure, the Aequorea victoria green fluorescent protein (GFP) is single-handedly responsible for the ever-growing popularity of fluorescence imaging of recombinant fusion proteins in biological research. Engineered variants of GFP with altered excitation or emission wavelength maxima have helped to expand the range of applications of GFP. The engineering of the GFP variants is usually done empirically by genetic modifications of the chromophore structure and/or its environment in order to find variants with new photophysical properties. The process of identifying improved variants could be greatly facilitated if augmented or guided by computational studies of the chromophore ground and excited-state properties and dynamics. In pursuit of this goal, we now report a thorough investigation of computational methods for prediction of the absorbance maxima for an experimentally validated series of engineered GFP chromophore analogues. The experimental dataset is composed of absorption maxima for 10 chemically distinct GFP chromophore analogues, including a previously unreported Y66D variant, measured under identical denaturing conditions. For each chromophore analogue, excitation energies and oscillator strengths were calculated using configuration interaction with single excitations (CIS), CIS with perturbative correction for double substitutions [CIS(D)], and time-dependent density functional theory (TD DFT) using several density functionals with solvent effects included using a polarizable continuum model. Comparison of the experimental and computational results show generally poor quantitative agreement with all methods attempted. However, good linear correlations between the calculated and experimental excitation energies (R2>0.9) could be obtained. Oscillator strengths obtained with TD DFT using pure density functionals also correlate well with the experimental values. Interestingly, most of the computational methods used in this work fail in the case of nonaromatic Y66S and Y66L protein chromophores, which may be related to a significant contribution of double excitations to their excited-state wavefunctions. These results provide an important benchmark of the reliability of the computational methods as applied to GFP chromophore analogues and lays a foundation for the computational design of GFP variants with improved properties for use in biological imaging.     

Quantum chemical simulation of excited states of chlorophylls, bacteriochlorophylls and their complexes

The present review describes the use of quantum chemical methods in estimation of structures and electronic transition energies of photosynthetic pigments in vacuum, in solution and imbedded in proteins. Monomeric Mg-porphyrins, chlorophylls and bacteriochlorophylls and their solvent 1:1 and 1:2 complexes were studied. Calculations were performed for Mg-porphyrin, Mg-chlorin, Mg-bacteriochlorin, mesochlorophyll a, chlorophylls a, b, c(1), c(2), c(3), d and bacteriochlorophylls a, b, c, d, e, f, g, h, plus several homologues. Geometries were optimised with PM3, PM3/CISD, PM5, ab initio HF (6-31G*/6-311G**) and density functional B3LYP (6-31G*/6-311G**) methods. Spectroscopic transition energies were calculated with ZINDO/S CIS, PM3 CIS, PM3 CISD, ab initio CIS, time-dependent HF and time-dependent B3LYP methods. Estimates for experimental transition energies were obtained from linear correlations of the calculated transition energies of 1:1 solvent complexes against experimentally recorded solution energies (scaling). According to the calculations in five-coordinated solvent complexes the magnesium atom lies out of the porphyrin plane, while in six-coordinated complexes the porphyrin is nearly planar. Charge densities on magnesium and nitrogen atoms were strongly dependent on the computational method deployed. Several dark states of low oscillator strength below the main Soret band were predicted for solvent complexes and chlorophylls and bacteriochlorophylls in protein environment. Such states, though not yet identified experimentally, might serve as intermediate states for excitation energy transfer in photosynthetic complexes. Q(y), Q(x) and Soret transition energies were found to depend on the orientation of the acetyl group and external pressure. A method to estimate site energies and dimeric interaction energies and to simulate absorption and CD spectra of photosynthetic complexes is described. Simulations for the light harvesting complexes Rhodospirillum molischianum, chlorosomes of Chlorobium tepidum and Chloroflexus aurantiacus, and LHC-II of Spinacia oleracea are presented as examples.     

Photosynthetic Light-Harvesting (Antenna) Complexes—Structures and Functions

Chlorophylls and bacteriochlorophylls, together with carotenoids, serve, noncovalently bound to specific apoproteins, as principal light-harvesting and energy-transforming pigments in photosynthetic organisms. In recent years, enormous progress has been achieved in the elucidation of structures and functions of light-harvesting (antenna) complexes, photosynthetic reaction centers and even entire photosystems. It is becoming increasingly clear that light-harvesting complexes not only serve to enlarge the absorption cross sections of the respective reaction centers but are vitally important in short- and long-term adaptation of the photosynthetic apparatus and regulation of the energy-transforming processes in response to external and internal conditions. Thus, the wide variety of structural diversity in photosynthetic antenna “designs” becomes conceivable. It is, however, common for LHCs to form trimeric (or multiples thereof) structures. We propose a simple, tentative explanation of the trimer issue, based on the 2D world created by photosynthetic membrane systems.     

Mimicking the role of the antenna in photosynthetic photoprotection

One mechanism used by plants to protect against damage from excess sunlight is called nonphotochemical quenching (NPQ). Triggered by low pH in the thylakoid lumen, NPQ leads to conversion of excess excitation energy in the antenna system to heat before it can initiate production of harmful chemical species by photosynthetic reaction centers. Here we report a synthetic hexad molecule that functionally mimics the role of the antenna in NPQ. When the hexad is dissolved in an organic solvent, five zinc porphyrin antenna moieties absorb light, exchange excitation energy, and ultimately decay by normal photophysical processes. Their excited-state lifetimes are long enough to permit harvesting of the excitation energy for photoinduced charge separation or other work. However, when acid is added, a pH-sensitive dye moiety is converted to a form that rapidly quenches the first excited singlet states of all five porphyrins, converting the excitation energy to heat and rendering the porphyrins kinetically incompetent to readily perform useful photochemistry.     

Unique Properties of Heme Binding of the Porphyromonas gingivalis HmuY Hemophore-like Protein Result from the Evolutionary Adaptation of the Protein Structure

To acquire heme, Porphyromonas gingivalis uses a hemophore-like protein (HmuY). HmuY sequesters heme from host hemoproteins or heme-binding proteins produced by cohabiting bacteria, and delivers it to the TonB-dependent outer-membrane receptor (HmuR). Although three-dimensional protein structures of members of the novel HmuY family are overall similar, significant differences exist in their heme-binding pockets. Histidines (H134 and H166) coordinating the heme iron in P. gingivalis HmuY are unique and poorly conserved in the majority of its homologs, which utilize methionines. To examine whether changes observed in the evolution of these proteins in the Bacteroidetes phylum might result in improved heme binding ability of HmuY over its homologs, we substituted histidine residues with methionine residues. Compared to the native HmuY, site-directed mutagenesis variants bound Fe(III)heme with lower ability in a similar manner to Bacteroides vulgatus Bvu and Tannerella forsythia Tfo. However, a mixed histidine-methionine couple in the HmuY was sufficient to bind Fe(II)heme, similarly to T. forsythia Tfo, Prevotella intermedia PinO and PinA. Double substitution resulted in abolished heme binding. The structure of HmuY heme-binding pocket may have been subjected to evolution, allowing for P. gingivalis to gain an advantage in heme acquisition regardless of environmental redox conditions.     

Changes in the energy distribution between chlorophyll-protein complexes of thylakoid membranes from pea mutants with modified pigment content. I. Changes due to the modified pigment content

The low-temperature (77 K) emission and excitation chlorophyll fluorescence spectra in thylakoid membranes isolated from pea mutants were investigated. The mutants have modified pigment content, structural organization, different surface electric properties and functions [Dobrikova et al., Photosynth. Res. 65 (2000) 165]. The emission spectra of thylakoid membranes were decomposed into bands belonging to the main pigment protein complexes. By an integration of the areas under them, the changes in the energy distribution between the two photosystems as well as within each one of them were estimated. It was shown that the excitation energy flow to the light harvesting, core antenna and RC complexes of photosystem II increases with the total amount of pigments in the mutants, relative to the that to photosystem I complexes. A reduction of the fluorescence ratio between aggregated trimers of LHC II and its trimeric and monomeric forms with the increase of the pigment content (chlorophyll a, chlorophyll b, and lutein) was observed. This implies that the closer packing in the complexes with a higher extent of aggregation regulates the energy distribution to the PS II core antenna and reaction centers complexes. Based on the reduced energy flow to PS II, i.e., the relative increased energy flow to PS I, we hypothesize that aggregation of LHC II switches the energy flow toward LHC I. These results suggest an additive regulatory mechanism, which redistributes the excitation energy between the two photosystems and operates at non-excess light intensities but at reduced pigment content.     

Proton-Coupled Electron Transfer between 4-Cyanophenol and Photoexcited Rhenium(I) Complexes with Different Protonatable Sites

Two rhenium(I) tricarbonyl diimine complexes, one of them with a 2,2'-bipyrazine (bpz) and a pyridine (py) ligand in addition to the carbonyls ([Re(bpz)(CO)(3)(py)](+)), and one tricarbonyl complex with a 2,2'-bipyridine (bpy) and a 1,4-pyrazine (pz) ligand ([Re(bpy)(CO)(3)(pz)](+)) were synthesized, and their photochemistry with 4-cyanophenol in acetonitrile solution was explored. Metal-to-ligand charge transfer (MLCT) excitation occurs toward the protonatable bpz ligand in the [Re(bpz)(CO)(3)(py)](+) complex while in the [Re(bpy)(CO)(3)(pz)](+) complex the same type of excitation promotes an electron away from the protonatable pz ligand. This study aimed to explore how this difference in electronic excited-state structure affects the rates and the reaction mechanism for photoinduced proton-coupled electron transfer (PCET) between 4-cyanophenol and the two rhenium(I) complexes. Transient absorption spectroscopy provides clear evidence for PCET reaction products, and significant H/D kinetic isotope effects are observed in some of the luminescence quenching experiments. Concerted proton-electron transfer is likely to play an important role in both cases, but a reaction sequence of proton transfer and electron transfer steps cannot be fully excluded for the 4-cyanophenol/[Re(bpz)(CO)(3)(py)](+) reaction couple. Interestingly, the rate constants for bimolecular excited-state quenching are on the same order of magnitude for both rhenium(I) complexes.     

Global analysis of fluorometric titration curves in the presence of excited-state association and quenching

The fluorometric determination of the ground-state dissociation constant K(d) of a complex between ligand and titrant with 1:1 stoichiometry in the presence of excited-state association and quenching is discussed. This report extends the results of a previous study (Novikov, E.; Stobiecka, A.; Boens, N. J. Phys. Chem. A 2000, 104, 5388), where the direct parametric fit of the fluorometric titration was used to recover reliable estimates for K(d). Here, we show that in the presence of excited-state association and quenching the unique value of K(d) can be obtained from global analysis of four fluorometric titration curves measured at two emission wavelengths and two excitation wavelengths. The same identifiability criterion is applicable for systems where quenching can be neglected. The linked parameters in the global analysis are rational functions of the rate constants, independent of the excitation and emission wavelengths. The developed algorithms for the global parametric fit of fluorometric titration curves are explored using simulations.     

Identification of an additional low-lying excited state of carotenoid radical cations

Carotenoids are the crucial pigments involved in photoprotection and in scavenging harmful free radicals in all living organisms. The underlying chemical processes are charge transfer and free radical reactions, both of them leading to carotenoid radical cation (Car*+) formation. Accurate knowledge of the molecular properties of Car*+ is thus a prerequisite for understanding of their function as photoprotective and antioxidant agents. Despite their fundamental importance in nonphotochemical quenching in green plants, only little is known about the Car*+ excited states and their dynamics. Our combined experimental and theoretical investigation employing femtosecond time-resolved pump-probe spectroscopy and quantum chemical calculations proves the existence of a second low-lying pipi* excited-state energetically below the well-known strongly allowed excited-state responsible for the intense absorption of Car*+ in the near-IR region. Hence, we suggest denoting the latter state as D3 state in the future. Our findings have also implications for nonphotochemical quenching in green plants, since direct quenching of chlorophyll excited states by Forster energy transfer to Car*+ is possible and efficient.     

Hydrogen bonding and reactivity of water to azines in their S1 (n,π*) electronic excited states in the gas phase and in solution

A unified picture is presented of water interacting with pyridine, pyridazine, pyrimidine, and pyrazine on the S(1) manifold in both gas-phase dimers and in aqueous solution. As (n,π*) excitation to the S(1) state removes electrons from the ground-state hydrogen bond, this analysis provides fundamental understanding of excited-state hydrogen bonding. Traditional interpretations view the excitation as simply breaking hydrogen bonds to form dissociated molecular products, but reactive processes such as photohydrolysis and excited-state proton coupled electron transfer (PCET) are also possible. Here we review studies performed using equations-of-motion coupled-cluster theory (EOM-CCSD), multireference perturbation theory (CASPT2), time-dependent density-functional theory (TD-DFT), and excited-state Monte Carlo liquid simulations, adding new results from symmetry-adapted-cluster configuration interaction (SAC-CI) and TD-DFT calculations. Invariably, gas-phase molecular dimers are identified as stable local minima on the S(1) surface with energies less than those for dissociated molecular products. Lower-energy biradical PCET minima are also identified that could lead to ground-state recombination and hence molecular dissociation, dissociation into radicals or ions, or hydration reactions leading to ring cleavage. For pyridine.water, the calculated barriers to PCET are low, suggesting that this mechanism is responsible for fluorescence quenching of pyridine.water at low energies rather than accepted higher-energy Dewar-benzene based "channel three" process. Owing to (n,π*) excitation localization, much higher reaction barriers are predicted for the diazines, facilitating fluorescence in aqueous solution and predicting that the as yet unobserved fluorescence from pyridazine.water and pyrimidine.water should be observable. Liquid simulations based on the assumption that the solvent equilibrates on the fluorescence timescale quantitatively reproduce the observed spectral properties, with the degree of (n,π*) delocalization providing a critical controlling factor.     

Modeling of photosynthetic light-harvesting: From structure to function

In order to bridge the gap between the crystal structure of photosynthetic pigment-protein complexes and the data gathered in optical experiments, two essential problems need to be solved. On one hand, theories of optical spectra and excitation energy transfer have to be developed that take into account the pigment-pigment (excitonic) and the pigment-protein (exciton-vibrational) coupling on an equal footing. On the other hand, the parameters entering these theories need to be calculated from the structural data. Good agreement between simulations and experimental data then allows to draw conclusions on structure-function relationships of these complexes and to make predictions. In the development of theory, a delicate question is how to describe the interplay between the quantum dynamics of excitons and the dephasing of coherences by the coupling of excitons to protein vibrations. Quantum mechanic coherences are utilized for efficient light harvesting. In the reaction centers of purple bacteria an energy sink is created by a coherent coupling of exciton states to intermolecular charge transfer states. The dephasing of coherences can be monitored, e.g., by the temperature dependent shift of optical lines. In the Fenna-Matthews-Olson protein, which acts as an excitation energy wire between the outer chlorosome antenna and the reaction center complex, an energy funnel for efficient light-harvesting is formed by the pigment-protein coupling. The protein shifts the local transition energies of the pigments, the so-called site energies in a specific way, such that pigments facing the reaction center are redshifted with respect to those on the chlorosome side. In the light-harvesting complex of higher plants an excitation energy funnel is created by the use of two different types of chlorophyll (Chl) pigments, Chla and Chlb and by the pigment-protein coupling that creates an energy sink at Chla 610 located in the stromal layer at the periphery of the complex. The close contact between Chla and Chlb gives rise to ultrafast subpicosecond exciton transfer, whereas dynamic localization effects are inferred to lead to long ps relaxation times between the majority of Chla pigments.     

Excited-state proton transfer in complexes of poly(methacrylic acid) with dodecyltrimethylammonium chloride

Proton-transfer reactions in aqueous solutions of poly(methacrylic acid) (PMA) were studied using a fluorescent probe and Fourier transform infrared (FTIR) spectroscopy. Protolytic photodissociation of 1-hydroxypyrene (HP) in water was found to be very slow. The PMA polyanion appeared to be very inefficient as a proton acceptor in the excited-state reaction with HP. However, a drastic increase in the deprotonation efficiency was observed in PMA solutions with the same pH values close to neutral when dodecyltrimethylammonium chloride (DTAC) was added. The protonated form of HP, as well as its anion, was shown to be solubilized in polyion-covered micelles. Time-resolved fluorescence data suggested at least two localization sites with different reactivities toward PMA. FTIR spectroscopy was used to quantify the degree of ionization of PMA in PMA-DTAC mixtures. The IR data indicated that protolytic dissociation of PMA could be well described by the Henderson-Hasselbach equation with an apparent pK of 6.6. In contrast, the fluorescent data revealed cooperative protonation of the PMA groups interacting with HP localized within surfactant assemblies. This selective protonation at a pH close to neutral may be associated with a conformational transition in the polymer-surfactant complex.     

Photophysics and multifunctionality of hypericin-like pigments in heterotrich ciliates: a phylogenetic perspective

In this paper, we review the literature and present some new data to examine the occurrence and photophysics of the diverse hypericin-like chromophores in heterotrichs, the photoresponses of the cells, the various roles of the pigments and the taxa that might be studied to advance our understanding of these pigments. Hypericin-like chromophores are known chemically and spectrally so far only from the stentorids and Fabrea, the latter now seen to be sister to stentorids in the phylogenetic tree. For three hypericin-like pigments, the structures are known but these probably do not account for all the colors seen in stentorids. At least eight physiological groups of Stentor exist depending on pigment color and presence/absence of zoochlorellae, and some species can be bleached, leading to many opportunities for comparison of pigment chemistry and cell behavior. Several different responses to light are exhibited among heterotrichs, sometimes by the same cell; in particular, cells with algal symbionts are photophilic in contrast to the well-studied sciaphilous (shade-loving) species. Hypericin-like pigments are involved in some well-known photophobic reactions but other pigments (rhodopsin and flavins) are also involved in photoresponses in heterotrichs and other protists. The best characterized role of hypericin-like pigments in heterotrichs is in photoresponses and they have at least twice evolved a role as photoreceptors. However, hypericin and hypericin-like pigments in diverse organisms more commonly serve as predator defense and the pigments are multifunctional in heterotrichs. A direct role for the pigments in UV protection is possible but evidence is equivocal. New observations are presented on a folliculinid from deep water, including physical characterization of its hypericin-like pigment and its phylogenetic position based on SSU rRNA sequences. The photophysics of hypericin and hypericin-like pigments is reviewed. Particular attention is given to how their excited-state properties are modified by the environment. Dramatic changes in excited-state behavior are observed as hypericin is moved from the homogeneous environment of organic solvents to the much more structured surroundings provided by the complexes it forms with proteins. Among these complexes, it is useful to consider the differences between environments where hypericin is not found naturally and those where it is, notably, for example, in heterotrichs. It is clear that interaction with a protein modifies the photophysics of hypericin and understanding the molecular basis of this interaction is one of the outstanding problems in elucidating the function of hypericin and hypericin-like chromophores.     

Resonance Raman and surface-enhanced resonance Raman spectra of LH2 antenna complex from Rhodobacter sphaeroides and Ectothiorhodospira sp. excited in the Qx and Qy transitions

Well-resolved vibrational spectra of LH2 complex isolated from two photosynthetic bacteria, Rhodobacter sphaeroides and Ectothiorhodospira sp., were obtained using surface-enhanced resonance Raman scattering (SERRS) exciting into the Qx and the Qy transitions of bacteriochlorophyll a. High-quality SERRS spectra in the Qy region were accessible because the strong fluorescence background was quenched near the roughened Ag surface. A comparison of the spectra obtained with 590 nm and 752 nm excitation in the mid- and low-frequency regions revealed spectral differences between the two LH2 complexes as well as between the LH2 complexes and isolated bacteriochlorophyll a. Because peripheral modes of pigments contribute mainly to the low-frequency spectral region, frequencies and intensities of many vibrational bands in this region are affected by interactions with the protein. The results demonstrate that the microenvironment surrounding the pigments within the two LH2 complexes is somewhat different, despite the fact that the complexes exhibit similar electronic absorption spectra. These differences are most probably due to specific pigment-pigment and pigment-protein interactions within the LH2 complexes, and the approach might be useful for addressing subtle static and dynamic structural variances between pigment-protein complexes from different sources or in complexes altered chemically or genetically.     

De novo design and characterization of copper centers in synthetic four-helix-bundle proteins.

The design and chemical synthesis of de novo metalloproteins on cellulose membranes with the structure of an antiparallel four-helix bundle is described. All possible combinations of three different sets of amphiphilic helices were assembled on cyclic peptide templates which were bound by a cleavable linker to the cellulose. In the hydrophobic interior, the four-helix bundle proteins carry a cysteine and several histidines at various positions for copper ligation. This approach was used successfully to synthesize, for the first time, copper proteins based on a four-helix bundle. UV-vis spectra monitored on the solid support showed ligation of copper(II) by about one-third out of the 96 synthesized proteins and tetrahedral complexes of cobalt(II) by most of these proteins. Three of the most stable copper-binding proteins were synthesized in solution and their structural properties analyzed by spectroscopic methods. Circular dichroism, one-dimensional NMR, and size-exclusion chromatography indicate a folding into a compact state containing a high degree of secondary structure with a reasonably ordered hydrophobic core. They displayed UV-vis absorption, resonance Raman, and EPR spectra intermediate between those of type 1 and type 2 copper centers. The present approach provides a sound basis for further optimizing the copper binding and its functional properties by using combinatorial protein chemistry guided by rational principles.     

Photochemical hole-burning in photosynthetic reaction centers

The change in absorbance (hole spectrum) of the primary electron donor (P870) in bacterial photosynthetic reaction centers has been studied at 1.5–2.1 K following narrow-band excitation at several wavelengths within the P870 absorption band. The hole width is very large, suggesting a homogeneous linewidth on the order of 200–300 cm⁻¹. Possible interpretations of this highly unusual result, including ultra-fast excited-state decay, are discussed.     

Structural aspects, IR study, and molecular photophysics of the cumulene-bridged complexes with rhenium(I), ruthenium(II), and osmium(II) centers

The synthesis of a series of ReI, RuII, and OsII complexes that contain rigid polyphosphine/cumulene spacers is reported here. These cumulenic ligands, namely, 1,1',3,3'-tetrakis(diphenylphosphino)allene (C3P4) and 1,1',4,4'-tetrakis(diphenylphosphino)cumulene (C4P4), utilize diphenylphosphino linkage components to coordinate to the metal-polypyridyl or metal-carbonyl units. Characterization of all mono-, homo-, and heterobimetallic complexes is achieved using 31P(1H) NMR, IR, and fast atom bombardment mass spectroscopy (FAB/MS) and elemental analysis. The two ReI homobimetallic complexes were also characterized by single-crystal X-ray structure determination, which provided the structural evidence of a 90 degrees rotation between the C3 and C4 adducts causing a change in the electrochemical behavior. The ground-state electronic absorption and redox interactions, along with the excited-state photophysical characteristics, are also explored. Electrochemical studies showed that an increase in the carbon chain length resulted in a greater amount of sigma-donation from the ligand to the metal centers, as well as a greater amount of electronic communication between the metal termini of the bimetallic species. The electronic absorption and emission spectra of the new complexes were also determined and characterized. The lifetimes of the excited-state luminescence of the ReI mono- and homobimetallic complexes were found to be an order of magnitude shorter than the lifetimes of the heterobimetallic complexes containing the RuII and OsII moieties. Excited-state energy transfer was observed from the higher MLCT excited state of the ReI centers to the lower energy MLCT excited state of the RuII and OsII centers on the following basis: no ReI-based emission was detected in the steady-state emission measurements, the time-resolved decay traces were fitted to only single-exponential decays, and the quantum yields were identical for each compound at two different excitation wavelengths where different percentages of the metal-based chromophores were excited.     

Single-molecule four-color FRET visualizes energy-transfer paths on DNA origami

Fluorescence resonance energy transfer (FRET) represents a mechanism to transport light energy at the nanoscale, as exemplified by nature's light-harvesting complexes. Here we used DNA origami to arrange fluorophores that transport excited-state energy from an input dye to an output dye. We demonstrate that energy-transfer paths can be controlled on the single-molecule level by the presence of a "jumper" dye that directs the excited-state energy either to a red or to an IR output dye. We used single-molecule four-color FRET with alternating laser excitation to sort subpopulations and to visualize the control of energy transfer.