Brent P. Krueger

Energy transfer in the peridinin chlorophyll-a protein of Amphidinium carterae studied by polarized transient absorption and target analysis.

The peridinin chlorophyll-a protein (PCP) of dinoflagellates differs from the well-studied light-harvesting complexes of purple bacteria and green plants in its large (4:1) carotenoid to chlorophyll ratio and the unusual properties of its primary pigment, the carotenoid peridinin. We utilized ultrafast polarized transient absorption spectroscopy to examine the flow of energy in PCP after initial excitation into the strongly allowed peridinin S2 state. Global and target analysis of the isotropic and anisotropic decays reveals that significant excitation (25-50%) is transferred to chlorophyll-a directly from the peridinin S2 state. Because of overlapping positive and negative features, this pathway was unseen in earlier single-wavelength experiments. In addition, the anisotropy remains constant and high in the peridinin population, indicating that energy transfer from peridinin to peridinin represents a minor or negligible pathway. The carotenoids are also coupled directly to chlorophyll-a via a low-lying singlet state S1 or the recently identified SCT. We model this energy transfer time scale as 2.3 +/- 0.2 ps, driven by a coupling of approximately 47 cm(-1). This coupling strength allows us to estimate that the peridinin S1/SCT donor state transition moment is approximately 3 D.

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.

Using Molecular Dynamics and Quantum Mechanics Calculations To Model Fluorescence Observables

We provide a critical examination of two different methods for generating a donor-acceptor electronic coupling trajectory from a molecular dynamics (MD) trajectory and three methods for sampling that coupling trajectory, allowing the modeling of experimental observables directly from the MD simulation. In the first coupling method we perform a single quantum-mechanical (QM) calculation to characterize the excited state behavior, specifically the transition dipole moment, of the fluorescent probe, which is then mapped onto the configuration space sampled by MD. We then utilize these transition dipoles within the ideal dipole approximation (IDA) to determine the electronic coupling between the probes that mediates the transfer of energy. In the second method we perform a QM calculation on each snapshot and use the complete transition densities to calculate the electronic coupling without need for the IDA. The resulting coupling trajectories are then sampled using three methods ranging from an independent sampling of each trajectory point (the independent snapshot method) to a Markov chain treatment that accounts for the dynamics of the coupling in determining effective rates. The results show that the IDA significantly overestimates the energy transfer rate (by a factor of 2.6) during the portions of the trajectory in which the probes are close to each other. Comparison of the sampling methods shows that the Markov chain approach yields more realistic observables at both high and low FRET efficiencies. Differences between the three sampling methods are discussed in terms of the different mechanisms for averaging over structural dynamics in the system. Convergence of the Markov chain method is carefully examined. Together, the methods for estimating coupling and for sampling the coupling provide a mechanism for directly connecting the structural dynamics modeled by MD with fluorescence observables determined through FRET experiments.

Observation of the S1 state of spheroidene in LH2 by two-photon fluorescence excitation

Abstract The two-photon fluorescence excitation spectrum of LH2, measured by directly exciting the carotenoid S 1 transition and monitoring fluorescence from the bacteriochlorophyll, provides the first direct experimental verification of carotenoid S 1 to bacteriochlorophyll energy transfer. It also provides an estimate of the in situ spheroidene S 1 transition energy of 13 900±150 cm −1 , slightly red-shifted from solution estimates. The relative abilities of the carotenoids spheroidene (10 conjugated double bonds) and rhodopin glucoside (11 conjugated double bonds) to transfer energy via their S 1 states are discussed in terms of spectral overlap factors and competing processes such as S 1 →S 0 internal conversion.

Hybrid molecular dynamics‐quantum mechanics simulations of solute spectral properties in the condensed phase: Evaluation of simulation parameters

We have explored the impact of a number of basic simulation parameters on the results of a recently developed hybrid molecular dynamics‐quantum mechanics (MD‐QM) method (Mercer et al., J Phys Chem B 1999, 103, 7720). The method utilizes MD simulations to explore the ground‐state configuration space of the system and QM evaluation of those structures to yield the time‐dependent electronic transition energy, which is transformed into the optical line‐broadening function using the second‐order cumulant expansion. Both linear and nonlinear optical spectra can then be generated for comparison to experiment. The dependence of the resulting spectra on the length of the MD trajectory, the QM sampling rate, and the QM model chemistry have all been examined. In particular, for the system of oxazine‐4 in methanol studied here, at least 20 ps of MD trajectory are needed for qualitative convergence of linear spectral properties, and > 100 ps is needed for quantitative convergence. Surprisingly, little difference is found between the 3‐21G and 6‐31G(d) basis sets, and the CIS and TD‐B3LYP methods yield remarkably similar spectra. The semiempirical INDO/s method yields the most accurate results, reproducing the experimental Stokes shift to within 5% and the FWHM to within 20%. Nonlinear 3‐pulse photon echo peak shift (3PEPS) decays have also been simulated. Decays are generally poorly reproduced, though the initial peak shift which depends on the overall coupling of motions to the solute transition energy is within 15% of experiment for all model chemistries other than those using the STO‐3G basis.

Carotenoid mediated B800–B850 coupling in LH2

Ab initio calculations of the excited state properties of a portion of the light harvesting 2 complex consisting of a supermolecule of two B850 bacteriochlorophyll (BChl), one B800 BChl, and one rhodopin glucoside carotenoid show significant mixing between the BChl and carotenoid states. Calculation of the B800–B850 coulombic coupling, through the transition density cube method, both with and without the carotenoid reveal that the carotenoid affects an increase in the B800–B850 coupling via an indirect (superexchange) mechanism. This carotenoid-mediated coupling explains a portion of the difference between the B800–B850 energy transfer rate determined experimentally and through traditional (Forster) calculations.

MD Simulations and FRET Studies of Dye-Labeled RNA

Fluorescence resonance energy transfer (FRET) is a powerful technique for understanding the structural transformations of RNA, DNA and proteins. With a few notable exceptions, the contribution of fluorophore and linker dynamics to these FRET measurements has not generally been investigated. Towards a better understanding of FRET on dye-labeled RNA, we present molecular dynamic (MD) simulations of 16mer double-stranded RNA with cyanine dyes attached at either the 3’ or 5’ ends with a 3 carbon linker. Water is included explicitly, and both dyes are in the ground state configuration. Differences in these two labeling strategies are discussed. We compare our simulations to data taken both on surface-attached and droplet-confined molecules. The effect of relative dye orientation and distance fluctuations due to the flexible linker are explicitly investigated.

Fluorescence Upconversion and ab initio Studies of the Light-Harvesting Function of Carotenoids in Bacterial Light-Harvesting Antenna

Fluorescence upconversion experiments and ab initio calculations show that S2-Qx electronic excitation transfer (to several Behl Qx transitions) is the primary pathway for carotenoid to Bchl energy transfer. Calculations on a carotenoid-B850-B850 aggregate suggest that the carotenoid may mediate B800–B850 EET by modifying the B850 transition density.