Effect of the in situ electrochemical oxidation on the pigment-protein arrangement and energy transfer in light-harvesting complex from Rhodobacter sphaeroides 601

The oxidation of bacteriochlorophylls (BChls) in peripheral light-harvesting complexes (LH2) from Rhodobacter sphaeroides was investigated by spectroelectrochemistry of absorption, fluorescence emission, and femtosecond (fs) pump-probe, with the aim obtaining information about the effect of in situ electrochemical oxidation on the pigment-protein arrangement and energy transfer within LH2. The experimental results revealed that: (a) the generation of the BChl radical cation in both B800 and B850 rings dramatically induced bleaching of the characteristic absorption in the NIR region and quenching of the fluorescence emission from the B850 ring for the electrochemical oxidized LH2; (b) the BChl-B850 radical cation might act as an additional channel to compete with the unoxidized BChl-B850 molecules for rapidly releasing the excitation energy, however the B800-B850 energy transfer rate remained almost unchanged during the oxidation process.     

Pathways of energy flow through the light-harvesting antenna of the photosynthetic purple bacterium rhodobacter sphaeroides

Using low intensity picosecond absorption spectroscopy with independently tunable excitation and probing infrared pulses, we have studied the pathways of energy transport through the light-harvesting antenna pigments of the photosynthetic purple bacterium Rhodobacter sphaeroides. From the observed excited-state rise time of the red-most pigment B896 as a function of excitation wavelength it is concluded that the B850 pigment of LH2 is spectrally heterogeneous. For excitations originating in the B850 pigment this results in a fast channel (9 ps) that is mainly excited in the peak of the B850 absorption band, and a slow channel (35 ps) that is predominantly excited at ~840 nm. Upon excitation of B800, more than 90% of the excitations follow the fast path. From the observed kinetics it is concluded that the majority of the LH2 → LH1 energy transfer takes place within at most a few picoseconds. The rate-limiting step in the whole energy transfer sequence appears to be the B896 → reaction center transfer. The origin of the B850 heterogeneity and the slow 35-ps component is at the moment unclear. Possibly it represents a highly extended form of LH2 in which transfer to LH1 takes a relatively long time, due to a large number of transfer steps.     

Band Structure and Local Dynamics of Excitons in Bacterial Light-harvesting Complexes Revealed by Spectrally Selective Spectroscopy

Hole-burned absorption and line-narrowed fluorescence spectra are studied at 5 K in wild type and mutant LH1 and LH2 antenna preparations from the photosynthetic purple bacterium Rhodobacter sphaeroides. Evidence was found in all samples, even in intact membranes, of the presence of a broad distribution of bacteriochlorophyll species that are unable to communicate energy between each other and to the exciton states of functional antenna complexes. The distribution maximum of these localized species determined by zero phonon hole action spectroscopy is at 783.5 nm in purified LH1 complexes and at 786.8 nm in B850-only mutant LH2 complexes. A well-resolved peak at 807 nm in LH1 complexes is assigned to the exciton band structure of functional core antenna complexes. Similar structure in LH2 complexes overlaps with the distribution of localized species. Off-diagonal (structural) disorder may be responsible for this exciton band structure. Our data also imply that pair-wise inter-chlorophyll couplings determine the resonance fluorescence lineshape of excitonic polarons.     

Direct visualization of exciton reequilibration in the LH1 and LH2 complexes of Rhodobacter sphaeroides by multipulse spectroscopy

The dynamics of the excited states of the light-harvesting complexes LH1 and LH2 of Rhodobacter sphaeroides are governed, mainly, by the excitonic nature of these ring-systems. In a pump-dump-probe experiment, the first pulse promotes LH1 or LH2 to its excited state and the second pulse dumps a portion of the excited state. By selective dumping, we can disentangle the dynamics normally hidden in the excited-state manifold. We find that by using this multiple-excitation technique we can visualize a 400-fs reequilibration reflecting relaxation between the two lowest exciton states that cannot be directly explored by conventional pump-probe. An oscillatory feature is observed within the exciton reequilibration, which is attributed to a coherent motion of a vibrational wavepacket with a period of ∼150 fs. Our disordered exciton model allows a quantitative interpretation of the observed reequilibration processes occurring in these antennas.     

Sequential photoisomerisation dynamics of the push-pull azobenzene Disperse Red 1

The ultrafast dynamics of the push-pull azobenzene Disperse Red 1 following photoexcitation at λ(pump) = 475 nm in solution in 2-fluorotoluene have been probed by broadband transient absorption spectroscopy and fluorescence up-conversion spectroscopy. The measured two-dimensional spectro-temporal absorption map features a remarkable "fast" excited-state absorption (ESA) band at λ ≈ 570 nm appearing directly with the excitation laser pulse and showing a sub-100 fs lifetime with a rapid spectral blue-shift. Moreover, its ultrafast decay is paralleled by rising distinctive ESA at other wavelengths. Global fits to the absorption-time profiles using a consecutive kinetic model yielded three time constants, τ(1) = 0.08 ± 0.03 ps, τ(2) = 0.99 ± 0.02 ps, and τ(3) = 6.0 ± 0.1 ps. Fluorescence-time profiles were biexponential with time constants τ(1)' = 0.12 ± 0.06 ps and τ(2)' = 0.70 ± 0.10 ps, close to the absorption results. Based on the temporal evolution of the transient spectra, especially the "fast" excited-state absorption band at λ ≈ 570 nm, and on the global kinetic analysis of the time profiles, τ(1) is assigned to an ultrafast transformation of the optically excited ππ* state to an intermediate state, which may be the nπ* state, τ(2) to the subsequent isomerisation and radiationless deactivation time to the S(0) electronic ground state, and τ(3) to the eventual vibrational cooling of the internally "hot" S(0) molecules.     

Observation of the energy-level structure of the low-light adapted B800 LH4 complex by single-molecule spectroscopy

Low-light adapted B800 light-harvesting complex 4 (LH4) from Rhodopseudomonas palustris is a complex in which the arrangement of the bacteriochloropyll a pigments is very different from the well-known B800-850 LH2 complex. For bulk samples, the main spectroscopic feature in the near-infrared is the occurrence of a single absorption band at 802 nm. Single-molecule spectroscopy can resolve the narrow bands that are associated with the exciton states of the individual complexes. The low temperature (1.2 K) fluorescence excitation spectra of individual LH4 complexes are very heterogeneous and display unique features. It is shown that an exciton model can adequately reproduce the polarization behavior of the complex, the experimental distributions of the number of observed peaks per complex, and the widths of the absorption bands. The results indicate that the excited states are mainly localized on one or a few subunits of the complex and provide further evidence supporting the recently proposed structure model.     

Excited-state structure and isomerization dynamics of the retinal chromophore in rhodopsin from resonance Raman intensities.

Resonance Raman excitation profiles have been measured for the bovine visual pigment rhodopsin using excitation wavelengths ranging from 457.9 to 647.1 nm. A complete Franck-Condon analysis of the absorption spectrum and resonance Raman excitation profiles has been performed using an excited-state, time-dependent wavepacket propagation technique. This has enabled us to determine the change in geometry upon electronic excitation of rhodopsin's 11-cis-retinal protonated Schiff base chromophore along 25 normal coordinates. Intense low-frequency Raman lines are observed at 98, 135, 249, 336, and 461 cm-1 whose intensities provide quantitative, mode-specific information about the excited-state torsional deformations that lead to isomerization. The dominant contribution to the width of the absorption band in rhodopsin results from Franck-Condon progressions in the 1,549 cm-1 ethylenic normal mode. The lack of vibronic structure in the absorption spectrum is shown to be caused by extensive progressions in low-frequency torsional modes and a large homogeneous linewidth (170 cm-1 half-width) together with thermal population of low-frequency modes and inhomogeneous site distribution effects. The resonance Raman cross-sections of rhodopsin are unusually weak because the excited-state wavepacket moves rapidly (approximately 35 fs) and permanently away from the Franck-Condon geometry along skeletal stretching and torsional coordinates.     

The long-wavelength chlorophyll states of plant LHCI at room temperature: a comparison with PSI-LHCI

The red antenna states of the external antenna complexes of higher plant photosystem I, known as LHCI, have been analyzed by measurement of their preequilibrium fluorescence upon direct excitation at 280 K. In addition to the previously detected F735 state, a hitherto undetected low-energy state with emission maximum around 713 nm was observed. The 280 K bandwidths (FWHM) are 55 nm for the F735 state and approximately 27 nm for the F713-nm state, much greater than for non-red-shifted antenna chlorophylls. The origin absorption band for the F735-nm state was directly detected by determination of its excitation (action) spectrum and lies at 709-710 nm. The absorption spectrum for F735, calculated using the Stepanov expression, closely overlaps the excitation spectrum, indicating that the very large Stokes shift (25 nm) is due to vibrational relaxation within the excited-state manifold and solvent effects can be excluded. Fluorescence anisotropy measurements, with direct excitation of F735, indicate that the transition dipoles of the two red states are parallel. Similar experiments performed in the long-wavelength absorbing tail of PSI-LHCI indicate the presence of emission state(s) that are red-shifted with respect to F735 of isolated LHCI. It is suggested that these are brought about by interactions between the complexes in PSI-LHCI, which occur in some yet undefined way, and which are broken upon solubilization of the component parts.     

The observation of ultrafast excited-state dynamical evolution in B800- partially or completely released LH2 of Rhodobacter sphaeroides 601 at room temperature

Photodynamics of two kinds of peripheral antenna complexes (LH2 of Rhodobacter sphaeroides, native LH2 (RS601) and B800-released LH2 where B800-BChls were partially or completely removed with different pH treatments), were studied using femtosecond pump-probe technique at different laser wavelengths. The obtained results for these samples with different B800/B850 ratios demonstrated that under the excitation around B800 nm, the photoabsorption and photobleaching dynamics were caused by the direct excitation of upper excitonic levels of B850 and excited state of B800 pigments, respectively. Furthermore, the removal of B800 pigments had little effect on the energy transfer processes of B850 interband/intraband transfer.     

Two-photon excitation spectrum of fluorescence of the light-harvesting complex B800–850 from Allochromatium minutissimum within 1200–1500 (600–750) nm spectral range is not carotenoid mediated

Two types of peripheral light-harvesting complexes LH2 (B800–850) from photosynthetic purple bacterium Allochromatium minutissimum were studied. First type containing carotenoids was prepared from wild type cells. The other one was obtained from carotenoid depleted cells grown with diphenylamine. We have shown that under laser femtosecond excitation within absorption 1200–1500 nm wavelength range the two-photon excitation of LH2 complexes takes place. This can be observed as fluorescence of bacteriochlorophyll (BChl) spectral form B850 (BChl molecules of circular aggregate with strong exciton interaction in 850 nm spectral domain). LH2 fluorescence excitation spectra under two-photon excitation are the same for carotenoid-containing and carotenoidless preparations. In both cases the broad band with peak near 1350 (675) nm (FWHM ～ 240 (120) nm) was found. It is concluded that the broad band with peak near 1350 (675) nm in two-photon excitation spectra of LH2 complexes from Allochromatium minutissimum cannot be interpreted as two-photon excitation band of the optically forbidden S₀ → S₁ transition of carotenoids (rhodopin). Possible nature of this band is discussed.     

Excitation dynamics in strongly bounded associates of B800–850 and B800–830 complexes from photosynthetic purple bacterium Thiorhodospira sibirica

Strongly bounded associates of B800–850 (LH2) and B800–830 (LH3) complexes from photosynthetic purple bacterium Thiorhodospira sibirica were investigated. It was shown that associates contain 8–10 complexes (LH2:LH3 ≈ 1:1). Absorption spectra of the monomer LH2 and the monomer LH3 complexes were calculated. Excitation of B800 absorption band of associates results in: (i) intracomplex excitation energy transfer from B800 to B830 or B850 with time constant of about 500 fs; (ii) intercomplex excitation energy transfer from B820 band of LH3 complex to B850 band of LH2 complex with time constant of about 2.5 ps; (iii) excitation deactivation in B850 band of LH2 complex with time constant of about 800 ps. Signal polarization at long-wavelength side of associates absorption spectrum near 900 nm was negative (?0.1). The interaction of LH3 and LH2 complexes in associates is, to some extent, analogous to the interaction of LH2 and LH1 complexes in chromatophores. Time constant of excitation energy transfer between LH3 and LH2 complexes in associates may be regarded as a minimal time constant for energy transfer between the peripheral and core antenna complexes.     

Identifying the lowest electronic states of the chlorophylls in the CP47 core antenna protein of photosystem II

CP47 is a pigment-protein complex in the core of photosystem II that tranfers excitation energy to the reaction center. Here we report on a spectroscopic investigation of the isolated CP47 complex. By deconvoluting the 77 K absorption and linear dichroism, red-most states at 683 and 690 nm have been identified with oscillator strengths corresponding to approximately 3 and approximately 1 chlorophyll, respectively. Both states contribute to the 4 K emission, and the Stark spectrum shows that they have a large value for the difference polarizability between their ground and excited states. From site-selective polarized triplet-minus-singlet spectra, an excitonic origin for the 683 nm state was found. The red shift of the 690 nm state is most probably due to strong hydrogen bonding to a protein ligand, as follows from the position of the stretch frequency of the chlorophyll 13(1) keto group (1633 cm(-)(1)) in the fluorescence line narrowing spectrum at 4 K upon red-most excitation. We discuss how the 683 and 690 nm states may be linked to specific chlorophylls in the crystal structure [Zouni, A., Witt, H.-T., Kern, J., Fromme, P., Krauss, N., Saenger, W., and Orth, P. (2001) Nature 409, 739-743].     

Two-photon excitation fluorescence spectrum of the light-harvesting complex LH2 from Chromatium minutissimum within 650-745 nm range is determined by two-photon absorption of bacteriochlorophyll rather than of carotenoids

Two-photon fluorescence excitation spectra of the peripheral light-harvesting complex LH2 from the purple photosynthetic bacterium Chromatium minutissimum were examined within the expected spectral range of the optically forbidden S1 singlet state of carotenoids. LH2 preparations isolated from wild-type and carotenoid-depleted cells were used. 100-fs laser pulses in the range of 1300-1490 nm with an energy of 7-9 mW (corresponding to one-photon absorption between 650 and 745 nm) were used for two-photon fluorescence excitation. It was shown that two-photon fluorescence excitation spectra of LH2 complex from wild and carotenoid-depleted cells are very similar to each other and to the two-photon fluorescence excitation spectrum of bacteriochlorophyll a in acetone. It was concluded that direct two-photon excitation of bacteriochlorophyll a determines the fluorescence of both samples within the 650-745 nm spectral range.     

On the influence of pigment-protein interactions on the energy transfer processes in photosynthetic membrane structures. 2. LH2 complex of Rhodobacter sphaeroides

Low-temperature heterogeneous absorption and circular dichroism spectra of the Rb. sphaeroides LH2 complexes are calculated within the framework of the mini-exciton theory and diagonal static random disorder for the pure electronic transitions of the monomeric Bchl molecules. The coupling of Bchl molecules with the surrounding amino acid residues has been shown to change both the exciton distribution between the pigment molecules in each of the exciton states. The value of the delocalization index depends on the excitation wavelength and varies between 2-6 Bchl molecules. The optical transitions occurring at 780-790 and 820 nm have been found to be strongly mixed so that all Bchl molecules of the LH2 complex predetermine absorption in these spectral regions. On the other hand, absorption at 800 and 850 nm is mainly determined by the cycles of 9 and 18 Bchl molecules, respectively. Thus, the light energy absorbed by the B800 molecules at 800 nm is transferred to the B850 molecules by the interlevel exciton relaxation processes due to the population of the heavily mixed 820-nm exciton levels. The width of the heterogeneous absorption band for the cyclic monomeric aggregate has been shown to decrease as compared with the monomeric absorption band by square root(Ndel) time, where Ndel is the mean number of pigments over which the exciton is delocalized within the excited absorption band.     

The Observation of Ultrafast Excited-state Dynamical Evolution In B800- Partially or Completely Released LH2 of Rhodobacter sphaeroides 601 at Room Temperature

Abstract

Photodynamics of two kinds of peripheral antenna complexes (LU2 of Rhodobacter sphaeroides, native LH2 (RS601) and B800-released LH2 where B800-BChls were partially or completely removed with different pH treatments), were studied using femtosecond pump-probe technique at different laser wavelengths. The obtained results for these samples with different B800/B850 ratios demonstrated that under the excitation around B800 nm, the photoabsorption and photobleaching dynamics were caused by the direct excitation of upper excitonic levels of B850 and excited state of B800 pigments, respectively. Furthermore, the removal of B800 pigments had little effect on the energy transfer processes of B850 interband/intraband transfer.     

Spectral inhomogeneity of photosystem I and its influence on excitation equilibration and trapping in the cyanobacterium Synechocystis sp. PCC6803 at 77 K.

Ultrafast transient absorption spectroscopy was used to probe excitation energy transfer and trapping at 77 K in the photosystem I (PSI) core antenna from the cyanobacterium Synechocystis sp. PCC 6803. Excitation of the bulk antenna at 670 and 680 nm induces a subpicosecond energy transfer process that populates the Chl a spectral form at 685--687 nm within few transfer steps (300--400 fs). On a picosecond time scale equilibration with the longest-wavelength absorbing pigments occurs within 4-6 ps, slightly slower than at room temperature. At low temperatures in the absence of uphill energy transfer the energy equilibration processes involve low-energy shifted chlorophyll spectral forms of the bulk antenna participating in a 30--50-ps process of photochemical trapping of the excitation by P(700). These spectral forms might originate from clustered pigments in the core antenna and coupled chlorophylls of the reaction center. Part of the excitation is trapped on a pool of the longest-wavelength absorbing pigments serving as deep traps at 77 K. Transient hole burning of the ground-state absorption of the PSI with excitation at 710 and 720 nm indicates heterogeneity of the red pigment absorption band with two broad homogeneous transitions at 708 nm and 714 nm (full-width at half-maximum (fwhm) approximately 200--300 cm(-1)). The origin of these two bands is attributed to the presence of two chlorophyll dimers, while the appearance of the early time bleaching bands at 683 nm and 678 nm under excitation into the red side of the absorption spectrum (>690 nm) can be explained by borrowing of the dipole strength by the ground-state absorption of the chlorophyll a monomers from the excited-state absorption of the dimeric red pigments.     

Fluorescence spectral fluctuations of single LH2 complexes from Rhodopseudomonas acidophila strain 10050

We have investigated the energy landscape of the bacterial photosynthetic peripheral light-harvesting complex LH2 of purple bacterium Rhodopseudomonas acidophila by monitoring sequences of fluorescence spectra of single LH2 assemblies, at room temperature, with different excitation intensities as well as at elevated temperatures, utilizing a confocal microscope. The fluorescence peak wavelength of individual LH2 complexes was found to abruptly move between quasi-stable levels differing by up to 30 nm. These spectral shifts either to the blue or to the red were accompanied by a broadening and decrease of the intensity of the fluorescence spectrum. The frequency and size of these fluorescence peak movements were found to increase linearly with excitation intensity. Using the modified Redfield theory, changes in the realization of the static disorder accounted for the observed changes in spectral shape and intensity. Long lifetimes of the quasi-stable states suggest large free energy barriers between the different realizations.     

Fluorescence spectroscopy of conformational changes of single LH2 complexes

We have investigated the energy landscape of the bacterial photosynthetic peripheral light-harvesting complex LH2 of purple bacterium Rhodopseudomonas acidophila by monitoring sequences of fluorescence spectra of single LH2 assemblies, at room temperature, with different excitation intensities as well as at elevated temperatures, utilizing a confocal microscope. The fluorescence peak wavelength of individual LH2 complexes was found to abruptly move between long-lived quasi-stable levels differing by up to 30 nm. The frequency and size of these fluorescence peak movements were found to increase linearly with the excitation intensity. These spectral shifts either to the blue or to the red were accompanied by a broadening and decrease of the intensity of the fluorescence spectrum. The probability for a particle to undergo significant spectral shift in either direction was found to be roughly the same. Using the modified Redfield theory, the observed changes in spectral shape and intensity were accounted for by changes in the realization of the static disorder. Long lifetimes of the quasi-stable states suggest large energetic barriers between the states characterized by different emission spectra.     

Circular symmetry of the light-harvesting 1 complex from Rhodospirillum rubrum is not perturbed by interaction with the reaction center

The effect of the interaction of the reaction center (RC) upon the geometrical arrangement of the bacteriochlorophyll a (BChla) pigments in the light-harvesting 1 complex (LH1) from Rhodospirillum rubrum has been examined using single molecule spectroscopy. Fluorescence excitation spectra at 1.8 K obtained from single detergent-solubilized as well as single membrane-reconstituted LH1-RC complexes showed predominantly (>70%) a single broad absorption maximum at 880-900 nm corresponding to the Q(y) transition of the LH1 complex. This absorption band was independent of the polarization direction of the excitation light. The remaining complexes showed two mutually orthogonal absorption bands in the same wavelength region with moderate splittings in the range of DeltaE = 30-85 cm(-1). Our observations are in agreement with simulated spectra of an array of 32 strongly coupled BChla dipoles arranged in perfect circular symmetry possessing only a diagonal disorder of 相似文献   

Spectroscopic studies of two spectral variants of light-harvesting complex 2 (LH2) from the photosynthetic purple sulfur bacterium Allochromatium vinosum

Two spectral forms of the peripheral light-harvesting complex (LH2) from the purple sulfur photosynthetic bacterium Allochromatium vinosum were purified and their photophysical properties characterized. The complexes contain bacteriochlorophyll a (BChl a) and multiple species of carotenoids. The composition of carotenoids depends on the light conditions applied during growth of the cultures. In addition, LH2 grown under high light has a noticeable split of the B800 absorption band. The influence of the change of carotenoid distribution as well as the spectral change of the excitonic absorption of the bacteriochlorophylls on the light-harvesting ability was studied using steady-state absorption, fluorescence and femtosecond time-resolved absorption at 77K. The results demonstrate that the change of the distribution of the carotenoids when cells were grown at low light adapts the absorptive properties of the complex to the light conditions and maintains maximum photon-capture performance. In addition, an explanation for the origin of the enigmatic split of the B800 absorption band is provided. This spectral splitting is also observed in LH2 complexes from other photosynthetic sulfur purple bacterial species. According to results obtained from transient absorption spectroscopy, the B800 band split originates from two spectral forms of the associated BChl a monomeric molecules bound within the same complex.