Arvi Freiberg

Picosecond dynamics of directed excitation transfer in spectrally heterogeneous light-harvesting antenna of purple bacteria

Picosecond spectrally resolved fluorescence kinetics measurements, together with model simulations of the obtained data, based on coupled rate equations, have been employed to determine the rates and the pathways of heterogeneous excitation transfer in Rhodobacter sphaeroides and Chromatium minutissimum . The presence of a directed excitation flow from the short-wavelength bacteriochlorophyll forms to the long-wavelength one and from the latter to reaction centres has been revealed. As a result, the overall excitation trapping time in the bacteria investigated has been found to be about 60 ps both at 77 K and at room temperature, i.e. the same as in Rhodospirillum rubrum , although the number of antenna bacteriochlorophyll molecules per reaction centre is several times larger. A comparison of the experimental and theoretical kinetic data shows that, besides obvious spectral heterogeneity of the bacteriochlorophyll antenna represented by well-resolved B800, B850 and B875 spectra forms, an intrinsic spectral inhomogeneiry of these forms is likely to play an essential role in the excitation transfer. The obtained picture of the mutual arrangement of different complexes in membranes is similar to the one suggested earlier, except that the presence of at least two typesof B800-850 complexes, the ones closely associated with B875 and the more remote ones,has been discovered. The excitation transfer to B875 has been shown to take about 10 and 50 ps for the first and the second type of B850 molecules, respectively. The intracomplex B800 → B850 transfer time is an order of magnitude smaller, about 1 ps. These three time constants seem to be practically independent of the reaction centre state and the temperature in the 300 K-77 K interval. At high excitation intensities (more than 1 · 1010 photons per pulse) a shortening of the long-wavelength fluorescence decay time and a short-wavelength shiftof the corresponding band maximum have been observed. Both effects are due to the annihilation of singlet and triplet excitations.

Detrapping of excitation energy from the reaction centre in the photosynthetic purple bacterium Rhodospirillum rubrum

Abstract Using picosecond absorption and fluorescence spectroscopy we have measured the yield of energy detrapping from the reaction centre of the photosynthetic purple bacterium Rhodospirillum rubrum at room temperature. By selective excitation of the reaction centre pigments at approx. 800 nm and probing of the antenna bleaching or fluorescence, 25 ± 5% and 40 ± 5% of the excitation energy was found to be transferred back to the antenna for photochemically fully active (PBIQ) and prereduced (PBIQ − ) reaction centres, respectively. Based on these data and the solution of a simple kinetic model, conclusions regarding the overall trapping time, antenna aggregation state and reaction centre coordination were obtained. Thus, the overall trapping time was concluded to be approx. 35 ps, in very good agreement with previous measurements, and the light-harvesting antenna is suggested to be organized in ( αβ ) 2 clusters which surround the reaction centre in a six-fold coordination.

Directed picosecond excitation transport in purple photosynthetic bacteria

Abstract Picosecond spectrally resolved fluorescent measurements together with the coupled kinetic rate equation model simulations of the data have been employed to determine the rates and pathways of heterogeneous excitation transport in the membranes of photosynthetic bacteria Rhodobacter sphaeroides and Chromatium minutissimum . Excitation transport from bacteriochlorophyll molecules B800 to B850, belonging to the same pigment-pigment-protein complex B800–850, proceeds in 1–2 ps at room temperature and at 77 K. The intercomplex excitation transport from B850 to B875 takes about 10 ps for most excitations. In the minor part of B800–850 complexes, the excitation transport to B875 complex takes much longer, about 50 ps. The macroscopic rate constant of excitation trapping by open reaction centres is shown to be the same for all the bacteria studied, although the number of antenna molecules per reaction centre differs significantly. This seems to be an indication of the intrinsic homogeneity of the long-wavelength bacteriochlorophyll band, which facilitates an additional localization of excitations in the vicinity of the reaction centre and, due to the shortening of the trapping time, increases the overall quantum yield of photosynthesis.

Picosecond kinetics of light excitations in photosynthetic purple bacteria in the temperature range of 300-4 K

Abstract Comparative measurements of time- and spectrally resolved picosecond fluorescence of the photosynthetic purple bacterium Rhodospirillum rubrum in a wide temperature range from room temperature down to 4 K have been performed with different excitation wavelengths within the main infrared absorption band employed. Several kinetically different spectral components have been characterized in the fluorescence decay at low temperatures. This gives substantial support to the viewpoint that the B880 light-harvesting antenna band of R. rubrum (and probably the core antenna of other purple bacteria) consists of more than just two spectral forms, as has been suggested by several authors. The rates of the antenna singlet excitation quenching by reaction centres in different states have been determined. Although almost unchanged in the temperature range from 300 to 77 K, these rates decrease by 2 to 4 times on temperature lowering down to 4 K. It was found that at 4 K, the oxidized primary donor is a stronger fluorescence quencher than its triplet state.

Kinetic model of primary energy transfer and trapping in photosynthetic membranes

The picosecond time-domain incoherent singlet excitation transfer and trapping kinetics in core antenna of photosynthetic bacteria are studied in case of low excitation intensities by numerical integration of the appropriate master equation in a wide temperature range of 4-300 K. The essential features of our two-dimensional-lattice model are as follows: Förster excitation transfer theory, spectral heterogeneity of both the light-harvesting antenna and the reaction center, treatment of temperature effects through temperature dependence of spectral bands, inclusion of inner structure of the trap, and transition dipole moment orientation. The fluorescence kinetics is analyzed in terms of distributions of various kinetic components, and the influence of different inhomogeneities (orientational, spectral) is studied.A reasonably good agreement between theoretical and experimental fluorescence decay kinetics for purple photosynthetic bacterium Rhodospirillum rubrum is achieved at high temperatures by assuming relatively large antenna spectral inhomogeneity: 20 nm at the whole bandwidth of 40 nm. The mean residence time in the antenna lattice site (it is assumed to be the aggregate of four bacteriochlorophyll a molecules bound to proteins) is estimated to be approximately 12 ps. At 4 K only qualitative agreement between model and experiment is gained. The failure of quantitative fitting is perhaps due to the lack of knowledge about the real structure of antenna or local heating and cooling effects not taken into account.

Pressure effects on spectra of photosynthetic light-harvesting pigment-protein complexes

Abstract The influence of high (up to 9 kbar) hydrostatic pressure on the absorption and fluorescence emission spectra of photosynthetic light-harvesting pigment-protein complexes isolated from purple bacteria Rhodospirillum rubrum has been studied at room temperature and at 77 K. Under pressure at room temperature all spectral bands exhibit a red-shift at a rate of between 30 and 120 cm −1 /kbar for different bands. From these pressure shifts the compressibility of the protein matrix can be estimated. The compressibility is remarkably different for the protein surrounding bacteriochlorophyll a molecules (κ≥25±5 Mbar −1 ) than for the one surrounding spirilloxanthin molecules (κ=10±2 Mbar −1 ). This indicates that the elastic properties of a protein are locally specific.

Energy trapping and detrapping in the photosynthetic bacterium Rhodopseudomonas viridis: transfer-to-trap-limited dynamics

Abstract The efficiency of energy back-transfer from the reaction center to the antenna in chromatophores of the photosynthetic purple bacterium Rhodopseudomonas viridis was measured at room temperature by means of picosecond time-resolved fluorescence spectroscopy. It was found that 20 ± 5% of the excitation energy selectively absorbed by the reaction center pigments in the wavelength region around 830 nm is transferred back to the antenna and gives rise to antenna fluorescence. The measured yield of energy detrapping enabled calculation of the energy detrapping (10–13 ps)−1 and trapping (40–50 ps)−1 rates. These results confirm that in Rps. viridis, like in bacteriochlorophyll a-containing purple bacteria, the energy transfer step from antenna pigments to the reaction center is a rate limiting step in the overall energy trapping by the reaction center. We suggest that this situation is termed ‘transfer-to-trap-limited’ dynamics, to distinguish it from the situation where the charge separation is the rate limiting step (trap limited) or the overall energy diffusion through the antenna is limiting (diffusion limited).

Picosecond fluorescence of simple photosynthetic membranes: Evidence of spectral inhomogeneity and directed energy transfer

Abstract The picosecond time-domain singlet excitation transfer and trapping kinetics in photosynthetic membranes in case of low excitation intensities is studied by numerical integration of the appropriate master equation. The essential features of our two-dimensional-lattice random walk model are spectral heterogeneity of the light-harvesting antenna, inclusion of temperature effects, nonabsolute excitation trap, correlation between spectral and spatial parameters. A reasonably good agreement between theoretical and experimental fluorescence decay kinetics for purple photosynthetic bacterium Rhodospirillum rubrum is achieved only by assuming relatively large spectral inhomogeneity. From this comparison the average excitation lifetime on the lattice site is estimated to be 5–8 ps at the effective nearest neighbour lattice distance of 32 A. If the model is correct, the relatively slow hopping rate determines that excitation transfer and trapping in R. rubrum at active photosynthesis conditions is a diffusion-limited process. The invariably present spectral disorder of photosynthetic systems promoting directed energy transfer serves for higher light-utilizing efficiency.

Picosecond processes in chromatophores at various excitation intensities

The aim of this paper is to review and discuss the results obtained by fluorescence and absorption spectroscopy of bacterial chromatophores excited with picosecond pulses of varying power and intensity. It was inferred that spectral and kinetic characteristics depend essentially on the intensity, the repetition rate of the picosecond excitation pulses as well as on the optical density of the samples used. Taking the different experimental conditions properly into account, most of the discrepancies between the fluorescence and absorption measurements can be solved. At high pulse repetition rate (>106 Hz), even at moderate excitation intensities (1010–1011 photons/cm2 per pulse), relatively long-lived triplet states start accumulating in the system. These are efficient (as compared to the reaction centers) quenchers of mobile singlet excitations due to singlet-triplet annihilation. The singlet-triplet annihilation rate constant in Rhodospirillum rubrum was determined to be equal to 10-9 cm3 s-1. At fluences >1012 photons/cm2 per pulse singlet-singlet annihilation must be taken into account. Furthermore, in the case of high pulse repetition rates, triplet-triplet annihilation must be considered as well. From an analysis of experimental data it was inferred that the singlet-singlet annihilation process is probably migration-limited. If this is the case, one has to conclude that the rate of excitation decay in light-harvesting antenna at low pumping intensities is limited by the efficiency of excitation trapping by the reaction center. The influence of annihilation processes on spectral changes is also discussed as is the potential of a local heating caused by annihilation processes. The manifestation of spectral inhomogeneity of light-harvesting antenna in picosecond fluorescence and absorption kinetics is analyzed.

Pico- and nanosecond fluorescence kinetics of Photosystem II reaction centre and its complex with CP47 antenna

Abstract Spectrally resolved pico- and nanosecond fluorescence kinetics of two types of Photosystem II core complex: D1/D2/cyt b559 reaction centres (RCs) and RCs together with CP47 proximal antenna have been studied at room temperature and at 77 K. The kinetics at room temperature were measured with the RCs being in different functional states. In the photoactive RCs at room temperature a picosecond decay with the lifetime components 13 ± 3 ps and 110 ± 30 ps is followed by the complex nanosecond kinetics. In the case of RC + CP47 complexes picosecond decays are slower: 25 ± 10 ps and 190 ± 30 ps, but nanosecond decay has similar behaviour. The data are analyzed by a simple three-state kinetic model postulating the formation of the primary radical pair in an unrelaxed form and allowing a back-recombination from that state. If this is correct, it is the proof that in the charge-separated state nuclear coordinate relaxation takes place on the picosecond time-scale. The following conclusions considering the nature and temporal characteristics of light excitations have been made: (i) At room temperature excitations are in equilibrium between P680 and the accessory chlorophylls including CP47 antenna and, therefore, only the average trapping time could be observed. This time is equal to 13 ± 3 ps in RCs and 25 ± 10 ps in RC + CP47 complexes. (ii) All other decays at room temperature (except probably part of the 5 ± 0.5 ns component) are of a recombination origin and reflect complex relaxation of the metastable radical pair state. (iii) At low temperatures an energetically directed excitation transfer, qualitatively very similar to the one observed in the core antenna of some purple bacteria takes place. This energy transfer is relatively slow with an apparent transfer time 10–20 ps at 77 K. (iv) Not only P680, but also P+ 680 and Pheo− are very efficient quenchers of excitations.