Spectral trends in the fluorescence of single bacterial light-harvesting complexes: experiments and modified redfield simulations

In this work we present and discuss the single-molecule fluorescence spectra of a variety of species of light-harvesting complexes: LH2 of Rhodopseudomonas acidophila, Rhodobacter sphaeroides, and Rhodospirillum molischianum and LH1 of Rhodobacter sphaeroides. The emission spectrum of these complexes varies as a function of time as was described in earlier work. For each type of complex, we observe a pronounced and well-reproducible characteristic relationship between the fluorescence spectral parameters of the peak wavelength, width, and asymmetry. This dependence for the LH2 complexes can be quantitatively explained on the basis of a disordered exciton model by varying the static disorder and phonon coupling parameters. In addition, a correlation of the pigment site energies has to be assumed to interpret the behavior of the LH1 complex.     

Dynamics of the emission spectrum of a single LH2 complex: interplay of slow and fast nuclear motions

We have studied the relationship between the realizations of static disorder and the emission spectra observed for a single LH2 complex. We show that the experimentally observed spectral fluctuations reflect realizations of the disorder in the B850 ring associated with different degrees of exciton delocalization and different effective coupling of the excitons to phonon modes. The main spectral features cannot be explained using models with correlated disorder associated with elliptical deformations of the ring. A quantitative explanation of the measured single-molecule spectra is obtained using the modified Redfield theory and a model of the B850 ring with uncorrelated disorder of the site energies. The positions and spectral shapes of the main exciton components in this model are determined by the disorder-induced shift of exciton eigenvalues in combination with phonon-induced effects (i.e., reorganization shift and broadening, that increase in proportion to the inverse delocalization length of the exciton state). Being dependent on the realization of the disorder, these factors produce different forms of the emission profile. In addition, the different degree of delocalization and effective couplings to phonons determines a different type of excitation dynamics for each of these realizations. We demonstrate that experimentally observed quasistable conformational states are characterized by excitation energy transfer regimes varying from a coherent wavelike motion of a delocalized exciton (with a 100-fs pass over half of the ring) to a hopping-type motion of the wavepacket (with a 350-fs jump between separated groups of 3-4 molecules) and self-trapped excitations that do not move from their localization site.     

Excitation dynamics in Photosystem I trapped in TiO2 mesopores

Photosynthesis Research - Excitation decay in closed Photosystem I (PSI) isolated from cyanobacterium Synechocystis sp. PCC 6803 and dissolved in a buffer solution occurs predominantly with...     

Initial steps of signal generation in photoactive yellow protein revealed with femtosecond mid-infrared spectroscopy

Photoactive yellow protein (PYP) is a bacterial blue light sensor that induces Halorhodospira halophila to swim away from intense blue light. Light absorption by PYP's intrinsic chromophore, p-coumaric acid, leads to the initiation of a photocycle that comprises several distinct intermediates. Here we describe the initial structural changes of the chromophore and its nearby amino acids, using visible pump/mid-infrared probe spectroscopy. Upon photoexcitation, the trans bands of the chromophore are bleached, and shifts of the phenol ring bands occur. The latter are ascribed to charge translocation, which probably plays an essential role in driving the trans to cis isomerization process. We conclude that breaking of the hydrogen bond of the chromophore's C=O group with amino acid Cys69 and formation of a stable cis ground state occur in approximately 2 ps. Dynamic changes also include rearrangements of the hydrogen-bonding network of the amino acids around the chromophore. Relaxation of the coumaryl tail of the chromophore occurs in 0.9-1 ns, which event we identify with the I(0) to I(1) transition observed in visible spectroscopy.     

Circular dichroism of carotenoids in bacterial light-harvesting complexes: experiments and modeling

In this work we investigate the origin and characteristics of the circular dichroism (CD) spectrum of rhodopin glucoside and lycopene in the light-harvesting 2 complex of Rhodopseudomonas acidophila and Rhodospirillum molischianum, respectively. We successfully model their absorption and CD spectra based on the high-resolution structures. We assume that these spectra originate from seven interacting transition dipole moments: the first corresponds to the 0-0 transition of the carotenoid, whereas the remaining six represent higher vibronic components of the S2 state. From the absorption spectra we get an estimate of the Franck-Condon factors of these transitions. Furthermore, we investigate the broadening mechanisms that lead to the final shape of the spectra and get an insight into the interaction energy between carotenoids. Finally, we examine the consequences of rotations of the carotenoid transition dipole moment and of deformations in the light-harvesting 2 complex rings. Comparison of the modeled carotenoid spectra with modeled spectra of the bacteriochlorophyll QY region leads to a refinement of the modeling procedure and an improvement of all calculated results. We therefore propose that the combined carotenoid and bacteriochlorophyll CD can be used as an accurate reflection of the overall structure of the light-harvesting complexes.     

Contrasting the excited-state dynamics of the photoactive yellow protein chromophore: protein versus solvent environments

Wavelength- and time-resolved fluorescence experiments have been performed on the photoactive yellow protein, the E46Q mutant, the hybrids of these proteins containing a nonisomerizing “locked” chromophore, and the native and locked chromophores in aqueous solution. The ultrafast dynamics of these six systems is compared and spectral signatures of isomerization and solvation are discussed. We find that the ultrafast red-shifting of fluorescence is associated mostly with solvation dynamics, whereas isomerization manifests itself as quenching of fluorescence. The observed multiexponential quenching of the protein samples differs from the single-exponential lifetimes of the chromophores in solution. The locked chromophore in the protein environment decays faster than in solution. This is due to additional channels of excited-state energy dissipation via the covalent and hydrogen bonds with the protein environment. The observed large dispersion of quenching timescales observed in the protein samples that contain the native pigment favors both an inhomogeneous model and an excited-state barrier for isomerization.     

Stark spectroscopy on photoactive yellow protein,E46Q,and a nonisomerizing derivative,probes photo-induced charge motion

The change in the electrostatic properties on excitation of the cofactor of wild-type photoactive yellow protein (WT-PYP) have been directly determined using Stark-effect spectroscopy. We find that, instantaneously on photon absorption, there is a large change in the permanent dipole moment, /Delta(-->)mu/, (26 Debye) and in the polarizability, (-)Deltaalpha, (1000 A(3)). We expect such a large degree of charge motion to have a significant impact on the photocycle that is associated with the important blue-light negative phototactic response of Halorhodospira halophila. Furthermore, changing E46 to Q in WT-PYP does not significantly alter its electrostatic properties, whereas, altering the chromophore to prevent it from undergoing trans-cis isomerization results in a significant diminution of /Delta(-->)mu/ and (-)Deltaalpha. We propose that the enormous charge motion that occurs on excitation of 4-hydroxycinnamyl thioester, the chromophore in WT-PYP, plays a crucial role in initiating the photocycle by translocation of the negative charge, localized on the phenolate oxygen in the ground state, across the chromophore. We hypothesize that this charge motion would consequently increase the flexibility of the thioester tail thereby decreasing the activation barrier for the rotation of this moiety in the excited state.     

Electric field effects on the chlorophylls,pheophytins, and beta-carotenes in the reaction center of photosystem II

We present an electric field modulated absorption spectroscopy (Stark effect) study of isolated photosystem II reaction center complexes, including a preparation in which the inactive pheophytin H(B) was exchanged for 13(1)-deoxo-13(1)-hydroxy-pheophytin. The results reveal that the Stark spectrum of the Q(x) and Q(y) transitions of the pheophytins has a second-derivative line shape, indicating that the Stark effect is dominated by differences in the dipole moment between the ground and the electronically excited states of these transitions (Delta mu). The Delta mu values for the Q(x) and Q(y) transitions of H(B) are small (Delta mu = 0.6-1.0 D f(-1)), whereas that of the Q(x) transition of the active pheophytin H(A) is remarkably large (Delta mu = 3 D f(-1)). The Stark spectrum of the red-most absorbing pigments also shows a second-derivative line shape, but this spectrum is considerably red-shifted as compared to the second derivative of the absorption spectrum. This situation is unusual but has been observed before in heterodimer special pair mutants of purple bacterial reaction centers [Moore, L. J., Zhou, H., and Boxer, S. G. (1999) Biochemistry 38, 11949-11960]. The red-shifted Stark spectra can be explained by a mixing of exciton states with a charge-transfer state of about equal energy. We conclude that the charge transfer state involves H(A) and its immediate chlorophyll neighbor (B(A)), and we suggest that this (B(A)(delta+)H(A)(delta-)) charge transfer state plays a crucial role in the primary charge separation reaction in photosystem II. In contrast to most other carotenes, the two beta-carotene molecules of the photosystem II reaction center display a very small Delta mu, which can most easily be explained by excitonic coupling of both molecules. These results favor a model that locates both beta-carotene molecules at the same side of the complex.     

Linear-dichroism spectroscopy for the study of structural properties of proteins

This review gives an experiment directed survey of the application of linear-dichroism (LD) spectroscopy to the study of proteins. LD spectroscopy is a relatively simple technique that provides information on the orientation of chromophores in molecules, on molecular characteristies such as shape, size and electronic properties, and on binding parameters in molecular complexes. Since LD is only observed when the molecules are non-randomly oriented in the sample, particular attention is paid to various orientation techniques, viz. in electric and flow fields, in polymer films and gels, and by light induction (photoselection). Examples are given on bacteriorhodopsin and retinals, chlorosomes, lens crystallins, aspartate aminotransferase, and the interaction of gene32-and recA-protein with DNA.Abbreviations AAT aspartate aminotransferase - BR bacteriorhodopsin - CD circular dichroism - LD linear dichroism - PLP pyridoxal-5-phosphate - ss single stranded - TDM transition dipole moment