Herbert van Amerongen

Förster Excitation Energy Transfer in Peridinin-Chlorophyll-a-Protein

Time-resolved fluorescence anisotropy spectroscopy has been used to study the chlorophyll a (Chl a) to Chl a excitation energy transfer in the water-soluble peridinin-chlorophyll a-protein (PCP) of the dinoflagellate Amphidinium carterae. Monomeric PCP binds eight peridinins and two Chl a. The trimeric structure of PCP, resolved at 2 A (, Science. 272:1788-1791), allows accurate calculations of energy transfer times by use of the Förster equation. The anisotropy decay time constants of 6.8 +/- 0.8 ps (tau(1)) and 350 +/- 15 ps (tau(2)) are respectively assigned to intra- and intermonomeric excitation equilibration times. Using the ratio tau(1)/tau(2) and the amplitude of the anisotropy, the best fit of the experimental data is achieved when the Q(y) transition dipole moment is rotated by 2-7 degrees with respect to the y axis in the plane of the Chl a molecule. In contrast to the conclusion of, Biochemistry. 23:1564-1571) that the refractive index (n) in the Förster equation should be equal to that of the solvent, n can be estimated to be 1.6 +/- 0.1, which is larger than that of the solvent (water). Based on our observations we predict that the relatively slow intermonomeric energy transfer in vivo is overruled by faster energy transfer from a PCP monomer to, e.g., the light-harvesting a/c complex.

The Flow of Excitation Energy in LHCII Monomers: Implications for the Structural Model of the Major Plant Antenna

Spectral and kinetic information on energy transfer within the light-harvesting complex II (LHCII) monomer was obtained from this subpicosecond transient absorption study, by using selective excitation (663, 669, 672, 678, and 682 nm) of various Chl a absorption bands and detecting the induced changes over the entire Qy region (650-700 nm). It is shown that transfer from the pigment(s) absorbing around 663 nm to the low energy ones occurs in 5 +/- 1 ps, whereas the 670-nm excitation is delivered to the same destination in two phases (0.30 +/- 0.05 ps, and 12 +/- 2 ps), and a fast equilibration (lifetime 0.45 +/- 0.05 ps) takes place within the main absorption band (675-680 nm). From comparison with results from similar time-resolved measurements on trimeric samples, it can be concluded that the intramonomeric energy transfer completely determines the spectral equilibration observed in native LHCII complexes. To correlate the measured lifetimes and their associated spectra with the pigment organization within the available structural model of LHCII (. Nature. 367:614-621), extensive but straightforward theoretical modeling was used. Thus it is demonstrated that the pigment assignment (Chl a or Chl b) given by Kuhlbrandt and co-workers cannot simultaneously describe the dichroic spectra and the transient absorption results for the rather homologous LHCII and CP29 proteins. A more recent assignment for CP29, in which a Chl b molecule (Chl b5) is identified as a Chl a (Dr. R. Bassi, personal communication), leads to a much better description of both CP29 and LHCII. Furthermore, the orientations of the transition dipole moments, which have not been obtained in the crystal structure, are now assigned for most of the Chls.

Pathways for Energy Transfer in the Core Light-Harvesting Complexes CP43 and CP47 of Photosystem II

The pigment-protein complexes CP43 and CP47 transfer excitation energy from the peripheral antenna of photosystem II toward the photochemical reaction center. We measured the excitation dynamics of the chlorophylls in isolated CP43 and CP47 complexes at 77 K by time-resolved absorbance-difference and fluorescence spectroscopy. The spectral relaxation appeared to occur with rates of 0.2-0.4 ps and 2-3 ps in both complexes, whereas an additional relaxation of 17 ps was observed only in CP47. Using the 3.8-A crystal structure of the photosystem II core complex from Synechococcus elongatus (A. Zouni, H.-T. Witt, J. Kern, P. Fromme, N. Krauss, W. Saenger, and P. Orth, 2001, Nature, 409:739-743), excitation energy transfer kinetics were calculated and a Monte Carlo simulation of the absorption spectra was performed. In both complexes, the rate of 0.2-0.4 ps can be ascribed to excitation energy transfer within a layer of chlorophylls near the stromal side of the membrane, and the slower 2-3-ps process to excitation energy transfer to the calculated lowest excitonic state. We conclude that excitation energy transfer within CP43 and CP47 is fast and does not contribute significantly to the well-known slow trapping of excitation energy in photosystem II.

The quantitative relationship between structure and polarized spectroscopy in the FMO complex of Prosthecochloris aestuarii: refining experiments and simulations

New absorption, linear dichroism (LD) and circular dichroism (CD) measurements at low temperatures on the Fenna—Matthews—Olson complex from Prosthecochloris aestuarii are presented. Furthermore, the anisotropy of fluorescence excitation spectra is measured and used to determine absolute LD spectra, i.e. corrected for the degree of orientation of the sample. In contrast to previous studies, this allows comparison of not only the shape but also the amplitude of the measured spectra with that calculated by means of an exciton model. In the exciton model, the point-dipole approximation is used and the calculations are based on the trimeric structure of the complex. An improved description of the absorption and LD spectra by means of the exciton model is obtained by simply using the same site energies and coupling strengths that were given by Louwe et al. (1997, J Phys Chem B 101: 11280–11287) and including three broadening mechanisms, which proved to be essential: Inhomogeneous broadening in a Monte Carlo approach, homogeneous broadening by using the homogeneous line shape determined by fluorescence line-narrowing measurements [Wendling et al. (2000) J Phys Chem B 104: 5825–5831] and lifetime broadening. An even better description is obtained when the parameters are optimized by a global fit of the absorption, LD and CD spectra. New site energies and coupling strengths are estimated. The amplitude of the LD spectrum is described quite well. The shape of the CD spectrum is modelled in a satisfactory way but its size can only be simulated by using a rather large value for the index of refraction of the medium surrounding the chromophores. It is shown that the estimated coupling strengths are compatible with the value of the dipole strength of bacteriochlorophyll a, when using the empty-cavity model for the local-field correction factor.

Fluorescence and Absorption Spectroscopy of the Weakly Fluorescent Chlorophyll a in Cytochrome b6f of Synechocystis PCC6803

A spectroscopic characterization of the chlorophyll a (Chl) molecule in the monomeric cytochrome b6f complex (Cytb6f) isolated from the cyanobacterium Synechocystis PCC6803 is presented. The fluorescence lifetime and quantum yield have been determined, and it is shown that Chl in Cytb6f has an excited-state lifetime that is 20 times smaller than that of Chl in methanol. This shortening of the Chl excited state lifetime is not caused by an increased rate of intersystem crossing. Most probably it is due to quenching by a nearby amino acid. It is suggested that this quenching is a mechanism for preventing the formation of Chl triplets, which can lead to the formation of harmful singlet oxygen. Using site-selected fluorescence spectroscopy, detailed information on vibrational frequencies in both the ground and Qy excited states has been obtained. The vibrational frequencies indicate that the Chl molecule has one axial ligand bound to its central magnesium and accepts a hydrogen bond to its 13(1)-keto carbonyl. The results show that the Chl binds to a well-defined pocket of the protein and experiences several close contacts with nearby amino acids. From the site-selected fluorescence spectra, it is further concluded that the electron-phonon coupling is moderately strong. Simulations of both the site-selected fluorescence spectra and the temperature dependence of absorption and fluorescence spectra are presented. These simulations indicate that the Huang-Rhys factor characterizing the electron-phonon coupling strength is between 0.6 and 0.9. The width of the Gaussian inhomogeneous distribution function is 210 +/- 10 cm-1.

Probing the structure and dynamics of a DNA hairpin by ultrafast quenching and fluorescence depolarization

DNA hairpins have been investigated in which individual adenines were replaced by their fluorescent analog 2-aminopurine (2AP). The temperature dependence of the time evolution of polarized emission spectra was monitored with picosecond time resolution. Four isotropic decay components for each oligonucleotide indicated the coexistence of at least four conformations. The fluorescence for three of these was significantly quenched, which is explained by hole transfer from 2AP to guanine(s). An approximately 8-ps component is ascribed to direct hole transfer, the approximately 50-ps and approximately 500-ps components are ascribed to structural reorganization, preceding hole transfer. At room temperature, a fraction remains unquenched on a 10-ns timescale, in contrast to higher temperatures, where the flexibility increases. Besides quenching due to base stacking, a second quenching process was needed to describe the data. Evidence for both intrastrand and interstrand hole transfer was found. The extracted probability for stacking between neighboring bases in double-stranded regions was estimated to be approximately 75% at room temperature and approximately 25% at 80 degrees C, demonstrating structural disorder of the DNA. Fluorescence depolarization revealed both local dynamics of the DNA and overall dynamics of the entire oligonucleotide. Upon raising the temperature, the C-N terminus of the hairpin appears to melt first; the rest of the hairpin denatures above the average melting temperature.

Energy Transfer in Light-Harvesting Complexes LHCII and CP29 of Spinach Studied with Three Pulse Echo Peak Shift and Transient Grating

Three pulse echo peak shift and transient grating (TG) measurements on the plant light-harvesting complexes LHCII and CP29 are reported. The LHCII complex is by far the most abundant light-harvesting complex in higher plants and fulfills several important physiological functions such as light-harvesting and photoprotection. Our study is focused on the light-harvesting function of LHCII and the very similar CP29 complex and reveals hitherto unresolved excitation energy transfer processes. All measurements were performed at room temperature using detergent isolated complexes from spinach leaves. Both complexes were excited in their Chl b band at 650 nm and in the blue shoulder of the Chl a band at 670 nm. Exponential fits to the TG and three pulse echo peak shift decay curves were used to estimate the timescales of the observed energy transfer processes. At 650 nm, the TG decay can be described with time constants of 130 fs and 2.2 ps for CP29, and 300 fs and 2.8 ps for LHCII. At 670 nm, the TG shows decay components of 230 fs and 6 ps for LHCII, and 300 fs and 5 ps for CP29. These time constants correspond to well-known energy transfer processes, from Chl b to Chl a for the 650 nm TG and from blue (670 nm) Chl a to red (680 nm) Chl a for the 670 nm TG. The peak shift decay times are entirely different. At 650 nm we find times of 150 fs and 0.5-1 ps for LHCII, and 360 fs and 3 ps for CP29, which we can associate mainly with Chl b <--> Chl b energy transfer. At 670 nm we find times of 140 fs and 3 ps for LHCII, and 3 ps for CP29, which we can associate with fast (only in LHCII) and slow transfer between relatively blue Chls a or Chl a states. From the occurrence of both fast Chl b <--> Chl b and fast Chl b --> Chl a transfer in CP29, we conclude that at least two mixed binding sites are present in this complex. A detailed comparison of our observed rates with exciton calculations on both CP29 and LHCII provides us with more insight in the location of these mixed sites. Most importantly, for CP29, we find that a Chl b pair must be present in some, but not all, complexes, on sites A(3) and B(3). For LHCII, the observed rates can best be understood if the same pair, A(3) and B(3), is involved in both fast Chl b <--> Chl b and fast Chl a <--> Chl a transfer. Hence, it is likely that mixed sites also occur in the native LHCII complex. Such flexibility in chlorophyll binding would agree with the general flexibility in aggregation form and xanthophyll binding of the LHCII complex and could be of use for optimizing the role of LHCII under specific circumstances, for example under high-light conditions. Our study is the first to provide spectroscopic evidence for mixed binding sites, as well as the first to show their existence in native complexes.

Fluorescence Lifetime Heterogeneity in Aggregates of LHCII Revealed by Time-Resolved Microscopy

Two-photon excitation, time-resolved fluorescence microscopy was used to investigate the fluorescence quenching mechanisms in aggregates of light-harvesting chlorophyll a/b pigment protein complexes of photosystem II from green plants (LHCII). Time-gated microscopy images show the presence of large heterogeneity in fluorescence lifetimes not only for different LHCII aggregates, but also within a single aggregate. Thus, the fluorescence decay traces obtained from macroscopic measurements reflect an average over a large distribution of local fluorescence kinetics. This opens the possibility to resolve spatially different structural/functional units in chloroplasts and other heterogeneous photosynthetic systems in vivo, and gives the opportunity to investigate individually the excited states dynamics of each unit. We show that the lifetime distribution is sensitive to the concentration of quenchers contained in the system. Triplets, which are generated at high pulse repetition rates of excitation (>1 MHz), preferentially quench domains with initially shorter fluorescence lifetimes. This proves our previous prediction from singlet-singlet annihilation investigations (Barzda, V., V. Gulbinas, R. Kananavicius, V. Cervinskas, H. van Amerongen, R. van Grondelle, and L. Valkunas. 2001. Biophys. J. 80:2409-2421) that shorter fluorescence lifetimes originate from larger domains in LHCII aggregates. We found that singlet-singlet annihilation has a strong effect in time-resolved fluorescence microscopy of connective systems and has to be taken into consideration. Despite that, clear differences in fluorescence decays can be detected that can also qualitatively be understood.

Ultrafast single excitation transfer from carotenoids to chlorophylls via different pathways in light-harvesting complex II of greenplants.

Sub-picosecond excitation transfer from carotenoids to chlorophyll in light-harvesting complex II was studied at 77 K using pump-probe experiments. We did not find evidence for energy transfer from the carotenoids to chlorophyll b. However, transfer from carotenoid to chlorophyll a was found to occur with a time constant of 220±25 fs. It is observed that spectrally different chlorophyll a pools are populated upon selective excitation of different carotenoids.

Ultrafast energy transfer in LHC-II trimers from the Chl a b light-harvesting antenna of Photosystem II

Time-resolved absorption difference profiles were obtained for LHC-II trimers, isolated from Photosystem II in spinach with n-dodecyl β-d-maltoside, using one-color and two-color pump-probe techniques. The one-color isotropic signals are predominantly excited state absorption at 640 nm, and a combination of photobleaching and stimulated emission at wavelengths ≥ 665nm. At intermediate wavelengths, dynamic red-shifting due to downhill energy transfer among the chlorophyll (Chl) spectral forms produces a bipolar signal, in which prompt photo-bleaching/stimulated emission is superseded at later times by excited state absorption. Triexponential analyses of these profiles yield the lifetime components 2–6 ps (associated with the spectral shifting), 14–36 ps (possibly due to energy transfer between LHC-II monomers), and several hundred picoseconds. The one-color anisotropy decays are resolvable at 665–675 nm, with lifetimes of 4.3 to 6.3 ps. They are unresolvably fast (i.e., exhibit subpicosecond lifetimes) at 640–650 nm. The two-color isotropic absorption difference signals show clear spectral evolution arising from equilibration among the LHC-II spectral components for excitation wavelengths shorter than 670 nm. However, most of this spectral evolution occurs within less than 2.5 ps. No resolvable anisotropy decay was observed in the two-color experiments. Taken together, the one-color and two-color experiments indicate that both picosecond and subpicosecond energy transfer steps occur in this antenna. The faster processes appear to dominate the spectral equilibration; slower processes occur in isoenergetic energy transfers among the longer-wavelength Chl a spectral forms that absorb between 665 and 675 nm. The values of the long-time anisotropic r(x), measured in the one-color and two-color experiments, are qualitatively consistent with static linear dichroism spectra of these preparations.