Thijs J. Aartsma

Direct observation of tiers in the energy landscape of a chromoprotein: A single-molecule study

Single-molecule spectroscopic techniques were applied to individual pigments embedded in a chromoprotein. A sensitive tool to monitor structural fluctuations of the protein backbone in the local environment of the chromophore is provided by recording the changes of the spectral positions of the pigment absorptions as a function of time. The data provide information about the organization of the energy landscape of the protein in tiers that can be characterized by an average barrier height. Additionally, a correlation between the average barrier height within a distinct tier and the time scale of the structural fluctuations is observed.

Spectroscopy on the B850 Band of Individual Light-Harvesting 2 Complexes of Rhodopseudomonas acidophila I. Experiments and Monte Carlo Simulations

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.

Time-resolved spectroscopy at 10 K of the Photosystem II reaction center; deconvolution of the red absorption band

Abstract The red absorption band of isolated Photosystem II reaction centers at 10 K and its flash-induced changes due to formation of the primary radical pair, P+I−, and subsequent recombination to the triplet state, PT, were analysed. It is concluded that the primary electron donor P consists of two chlorophyll molecules, which show exciton interaction and are responsible both for the main long wavelength absorption band at 679.6 nm and a shoulder at 683.6 nm (these wavelengths vary somewhat between preparations). The angle between their Qy transitions is about 60° and the exciton splitting about 85 cm−1. Both bands are bleached in the states P+ and PT and replaced by a single band at 678 nm, attributed to one of the two chlorophylls, while the oxidized or triplet state is localized on the other, contributing little absorbance in the Qy region. The pheophytin which acts as the intermediary electron acceptor I has a 6.4 nm wide Qy band at 676.5 nm with about 2 3 of the amplitude of a chlorophyll Qy band. For the remaining pigments gaussian curve fitting of the absorption spectrum led to the following tentative assignments. The Qy band of the other pheophytin is spectrally indistinguishable from that of I. All accessory chlorophylls absorb in the 670 nm region; a good fit was obtained with two 4.7 nm wide bands peaking at 672.6 and 669.4 nm, respectively, and at least one Qy band at slightly shorter wavelength. However, neither an artifactual origin of the latter band nor the presence of two such bands can be excluded. The pigments of the PS II reaction center show little exciton interaction.

Spectroscopy on the B850 band of individual light-harvesting 2 complexes of Rhodopseudomonas acidophila. II. Exciton states of an elliptically deformed ring aggregate.

Spectroscopy of individual light-harvesting 2 complexes from purple photosynthetic bacteria revealed a deformation of the circular complex into C(2) symmetry. The present work relates the geometry of the deformed aggregate to its spectroscopic properties. Different models of elliptical deformation are discussed and compared with the experimental findings. It is shown that the model with smaller interpigment distances, where the curvature of the ellipse is small, provides the best agreement with fluorescence excitation spectra of individual complexes.

Energy transfer, charge separation and pigment arrangement in the reaction center of Photosystem II

Abstract Energy transfer and charge separation in the isolated Photosystem II reaction center at room temperature were studied with transient absorption difference spectroscopy upon selective excitation of the reaction center pigments. The measurements were performed with two dye lasers, which had a spectral bandwidth of less than 1 nm, and with an instrument response function of 5 or 18 ps depending on the type of experiment. Small changes with time constants of 0.6 ns and 120 ps are attributed to damaged reaction centers. Selective excitation of the long-wavelength pigments, presumably P680 and the pheophytins, led to charge separation in 3 ps. Selective excitation of the short-wavelength pigments, presumably accessory chlorophylls, led to charge separation in 30 ps with the same quantum efficiency. This excludes equilibration of the excited state between accessory chlorophyll and P680 in less than 30 ps. The overlap of the fluorescence spectrum of accessory chlorophyll with the absorption of P680 is very good and the slow energy transfer is attributed to an about 30 A center-to-center distance, which makes the histidines 118 in helix II of the D1 and D2 proteins likely binding sites of the chlorophylls nearest to the long-wavelength pigments, P680 and pheophytin. Reevaluation of the literature in the light of these data suggests that P680 is a dimer with nearly (anti) parallel Q Y -transition moments of the constituent monomers, making an angle with their connecting axis close to the magic angle, and that the geometry of P680 and the pheophytins is not C 2 -symmetrical around an axis perpendicular to the membrane.

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.

Spectroscopy of Individual Light-Harvesting 2 Complexes of Rhodopseudomonas acidophila: Diagonal Disorder, Intercomplex Heterogeneity, Spectral Diffusion, and Energy Transfer in the B800 Band

This paper reports a detailed spectroscopic study of the B800 absorption band of individual light-harvesting 2 (LH2) complexes of the photosynthetic purple bacterium Rhodopseudomonas acidophila at 1. 2 K. By applying single-molecule detection techniques to this system, details and properties can be revealed that remain obscured in conventional ensemble experiments. For instance, from fluorescence-excitation spectra of the individual complexes a more direct measure of the diagonal disorder could be obtained. Further spectral diffusion phenomena and homogeneous linewidths of individual bacteriochlorophyll a (BChl a) molecules are observed, revealing valuable information on excited-state dynamics. This work demonstrates that it is possible to obtain detailed spectral information on individual pigment-protein complexes, providing direct insight into their electronic structure and into the mechanisms underlying the highly efficient energy transfer processes in these systems.

Enhanced Photocurrent Generation by Photosynthetic Bacterial Reaction Centers through Molecular Relays, Light-Harvesting Complexes, and Direct Protein–Gold Interactions

The utilization of proteins as nanodevices for solar cells, bioelectronics, and sensors generally necessitates the transfer of electrons to or from a conducting material. Here we report on efforts to maximize photocurrent generation by bacterial photosynthetic reaction center pigment-protein complexes (RCs) interfaced with a metal electrode. The possibility of adhering RCs to a bare gold electrode was investigated with a view to minimizing the distance for electron tunneling between the protein-embedded electron-transfer cofactors and the metal surface. Substantial photocurrents were achieved despite the absence of coating layers on the electrode or engineered linkers to achieve the oriented deposition of RCs on the surface. Comparison with SAM-covered gold electrodes indicating enhanced photocurrent densities was achieved because of the absence of an insulating layer between the photoactive pigments and the metal. Utilizing RCs surrounded by light-harvesting 1 complex resulted in higher photocurrents, surprisingly not due to enhanced photoabsorption but likely due to better surface coverage of uniformly oriented RC-LH1 complexes and the presence of a tetraheme cytochrome that could act as a connecting wire. The introduction of cytochrome-c (cyt-c) as a molecular relay also produced increases in current, probably by intercalating between the adhered RCs or RC-LH1 complexes and the electrode to mediate electron transfer. Varying the order in which components were introduced to the electrode indicated that dynamic rearrangements of RCs and cyt-c occurred at the bare metal surface. An upper limit for current generation could not be detected within the range of the illumination power available, with the maximum current density achieved by RC-LH1 complexes being on the order of 25 μA/cm(2). High currents could be generated consecutively for several hours or days under ambient conditions.

Photon-echo relaxation in molecular mixed crystals

Abstract The results of a photon-echo relaxation study on the lowest 1 B 2u ← 1 A 1g transition of tetracene and pentacene in a p -terphenyl host crystal are reported. The photon-echo decay times were measured at 1.37 K and found to be 8.2 ± ns for tetracene and 13.8 ± 0.5 ns for pentacene. These relaxation times correspond to homogeneous linewidths of 19.4 and 11.5 MHz respectively and are over a factor of 10 3 smaller than the observed inhomogeneous linewidths in the optical absorption spectrum. Temperature dependent photon-echo intensity measurements further show that, below 3.5 K, optical relaxation is due to an orbach process with a presumably pseudolocalized phonon state. The resonant phonon frequency was measured to be 8.1 ± 1 cm −1 for tetracene and 21.2 ± 0.5 cm −1 for pentacene.

The enzyme mechanism of nitrite reductase studied at single-molecule level

A generic method is described for the fluorescence “readout” of the activity of single redox enzyme molecules based on Förster resonance energy transfer from a fluorescent label to the enzyme cofactor. The method is applied to the study of copper-containing nitrite reductase from Alcaligenes faecalis S-6 immobilized on a glass surface. The parameters extracted from the single-molecule fluorescence time traces can be connected to and agree with the macroscopic ensemble averaged kinetic constants. The rates of the electron transfer from the type 1 to the type 2 center and back during turnover exhibit a distribution related to disorder in the catalytic site. The described approach opens the door to single-molecule mechanistic studies of a wide range of redox enzymes and the precise investigation of their internal workings.