R. van Grondelle

Measurement and global analysis of the absorbance changes in the photocycle of the photoactive yellow protein from Ectothiorhodospira halophila.

The photocycle of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila was examined by time-resolved difference absorption spectroscopy in the wavelength range of 300-600 nm. Both time-gated spectra and single wavelength traces were measured. Global analysis of the data established that in the time domain between 5 ns and 2 s only two intermediates are involved in the room temperature photocycle of PYP, as has been proposed before (Meyer T.E., E. Yakali, M. A. Cusanovich, and G. Tollin. 1987. Biochemistry. 26:418-423; Meyer, T. E., G. Tollin, T. P. Causgrove, P. Cheng, and R. E. Blankenship. 1991. Biophys. J. 59:988-991). The first, red-shifted intermediate decays biexponentially (60% with tau = 0.25 ms and 40% with tau = 1.2 ms) to a blue-shifted intermediate. The last step of the photocycle is the biexponential (93% with tau = 0.15 s and 7% with tau = 2.0 s) recovery to the ground state of the protein. Reconstruction of the absolute spectra of these photointermediates yielded absorbance maxima of about 465 and 355 nm for the red- and blue-shifted intermediate with an epsilon max at about 50% and 40% relative to the epsilon max of the ground state. The quantitative analysis of the photocycle in PYP described here paves the way to a detailed biophysical analysis of the processes occurring in this photoreceptor molecule.

Temperature dependence of energy transfer from the long wavelength antenna BChl-896 to the reaction center in Rhodospirillum rubrum, Rhodobacter sphaeroides (w.t. and M21 mutant) from 77 to 177K, studied by picosecond absorption spectroscopy.

Decay of the bacteriochlorophyll excited state was measured in membranes of the purple bacteria Rhodospirillum (R.) rubrum, Rhodobacter (Rb.) sphaeroides wild type and Rb. sphaeroides mutant M21 using low intensity picosecond absorption spectroscopy. The excitation and probing pulses were chosen in the far red wing of the long wavelength absorption band, such that predominantly the minor antenna species B896 was excited. The decay of B896 was studied between 77 and 177K under conditions that the traps were active. In all species the B896 excited state decay is almost temperature independent between 100 and 177K, and probably between 100 and 300 K. In this temperature range the decay rates for the various species are very similar and close to 40 ps. Below 100 K this rate remains temperature independent in Rb. sphaeroides w. t. and M21, while in R. rubrum a steep decrease sets in. An analysis of this data with the theory of nuclear tunneling indicates an activation energy for the final transfer step from B896 to the special pair of 70cm-1 for R. rubrum and 30cm-1 or less for Rb. sphaeroides.

Energy transfer within the bacteriochlorophyll antenna of purple bacteria at 77 K, studied by picosecond absorption recovery

Abstract The dynamics of energy transfer within the bacteriochlorophyll antenna of Rhodobacter sphaeroides and Rhodospirillum rubrum, with closed reaction centers, has been studied at 77 K using low-intensity infrared picosecond absorption recovery. Measurements of isotropic decay as well as decay of induced anisotropy yielded a detailed picture of the energy transfer pathways in the two antenna systems. The BChl antenna of Rb. sphaeroides seems to consist of at least four different BChl a species: BChl 800, BChl 850, BChl 875, and BChl 896. Upon excitation of the highest-energy pigment, a transfer sequence towards lower energy is initiated. The transfer steps between the different pigment pools are characterized by the following time constants: BChl 800 → BChl 850, т = 2 ± 1 ps ; BChl 850 → BChl 875, т = 40 ± 5 ps ; BChl 875 → BChl 896, т = 20 ± 5 ps . Once the excitations are localized on B896 a slower quenching, т = 190 ± 10 ps , by closed reaction centers (P+) occurs. From measurements of decay of induced anisotropy it is further concluded that efficient transfer between BChl 800 molecules takes place on a time-scale comparable to the BChl 800 → BChl 850 transfer. A marked increase in anisotropy in the red wing of the absorption spectrum offers a clear evidence of the presence of the long-wavelength antenna component B896. The BChl antenna of R. rubrum is composed of two BChl a species, BChl 880, and BChl 896, and the energy transfer kinetics are observed to be very similar to the corresponding part of Rb. sphaeroides. Some evidence of further spectral inhomogeneity (apart from B896) or spectral shifts induced by excitation of the B880 antenna pigment was also obtained. Several possible models are discussed for the origin and organization of the B896 pigment.

Spectroscopic properties of antenna complexes of Rhodobacter sphaeroides in vivo

Abstract Intact membranes of antenna mutants of Rhodobacter sphaeroides obtained by chemical mutagenesis containing only the B800–850 or the B875 reaction center complex were used to study the spectral properties of these antenna complexes separately in vivo. Wild-type spectral characteristics were restored to each mutant, following complementation by the relevant gene. It is shown that the absorption spectra of recombinant strains and of wild-type Rhodobacter sphaeroides can be analyzed in terms of those of the separate complexes as observed in the mutants. Distinct differences occur between the spectra of the antenna complexes isolated by means of detergent solubilization of the membrane and those of the mutants. Measurements of absorption and flash-induced absorption difference spectra and of linear dichroism and fluorescence polarization spectra at low temperature indicate that in the intact membrane the previously characterized bacteriochlorophyll Q y absorption bands near 800, 850 and 875 nm display an optical inhomogeneity and that they all contain a relatively weak transition at longer wavelength, the orientation of which is more parallel to the membrane plane than the orientation of the main transitions. Rapid and efficient energy transfer to the long-wave component (BChl 870 ) in the B800–850 complex could be demonstrated. Some of the long-wave transitions are also observable at room temperature. They may reflect the mode of aggregation of the complexes in their lipid environment and, by increased overlap between donor emission and acceptor absorbance, serve to facilitate energy transfer within the antenna system.

Energy transfer dynamics of isolated B800–850 and B800–820 pigment-protein complexes of Rhodobacter sphaeroides and Rhodopseudomonas acidophila

We have compared the energy-transfer dynamics of different B800–850 and B800–820 isolated pigment-protein complexes. Picosecond absorption spectroscopy under annihilation-free conditions has been used to measure the energy-transfer rate from BChl 800 to BChl 850/820 at 296 and 77 K. It is found that at room temperature the BChl 800 → BChl 850/820 transfer is very fast (< 1 ps in all studied complexes). At 77 K in the type 1 B800–850 complex of Rhodobacter sphaeroides and Rhodopseudomonas acidophila the BChl 800 to BChl 850 transfer is about 1–2 ps, whereas in the other complexes the decay of the BChl 800 excited state occurs within less than 1 ps, also at 77 K. The observed subtle differences between the different complexes give insight into the fine details of energy transfer. The excited state of BChl 850/820 has a long lifetime (≈ 1 ns), but measurements of induced anisotropy reveal very fast energy transfer between more or less identical BChl 850 molecules.

Spectral broadening of interacting pigments: Polarized absorption by photosynthetic proteins.

Excitonic interaction between pigment molecules is largely responsible for the static and dynamic spectroscopic properties of photosynthetic pigment-proteins. This paper provides a new description of its effect on polarized absorption spectroscopy, in particular on circular dichroism (CD). We investigate excitonic spectra of finite width and use spectral moments to compare 1) inhomogeneously broadened excitonic spectra, 2) spectra that are (homogeneously broadened by vibrations or electron-phonon interaction, and 3) spectra that are simulated by applying convolution after the interaction has been evaluated. Two cases are distinguished. If the excitonic splitting is smaller than the width of the interacting absorption bands, the broadening of the excitonic spectrum can be approximated by a convolution approach, although a correction is necessary for CD spectra. If the excitonic splitting exceeds the bandwidth, the well-known exchange narrowing occurs. We demonstrate that this is accompanied by redistribution of dipole strength and spectral shifts. The magnitude of a CD spectrum is conveniently expressed by its first spectral moment. As will be shown, this is independent of spectral broadening as well as dispersive shifts induced by pigment-protein interactions. Consequently, it provides a simple tool to relate the experimental CD spectrum of a pigment complex to the excitonic interactions from which it originates. To illustrate the potential of the presented framework, the spectroscopy of the LH2 pigment-protein complex from purple bacteria is analyzed and compared for dimer-like and ring-like structures. Furthermore, it is demonstrated that the variability of the CD of chlorosomes from green bacteria can be explained by small changes in the structure of their cylindrical bacteriochlorophyll c subunits.

Spectroscopic characterisation of the reaction centre of photosystem II using polarised light: Evidence for β-carotene excitons in PS II reaction centres

The absorption and linear dichroism spectra of the isolated D1/D2/cytochrome b -559 Photosystem II reaction centre from pea ( Pisum sativum ) measured at 77 K are shown to depend on the concentration of the detergent Triton X-100 (TX-100) in the supporting buffer. This implies that the PS II reaction centre adopts different conformations in different concentrations of this detergent. In the case of the absorption due to β-carotene, additional transitions are observed when the reaction centre is suspended in a medium containing low levels (0.02% TX-100) when compared with higher concentrations (0.20%) of TX-100. Differences are also seen in the lowest energy Q y (0−0) band. Suspension of the PS II reaction centre in higher concentrations of TX-100 results in a slight decrease (7%) in the dipole strength of one or more of long wavelength transitions relative to that measured in 0.01% TX-100, resulting in a large shift of the absorption maximum from 677.5 nm to 672 nm. The linear dichroism spectrum of the PS II reaction centre complex measured in a buffer containing 0.20% (w/v) TX-100 indicates the presence of one spectral form of β-carotene. When measured in a buffer containing 0.02% (w/v) TX-100, the linear dichroism spectrum has more transitions in the β-carotene absorption region. We discuss the possibility that the differences are due to excitonic interactions between molecules of β-carotene which lie close together at lower detergent concentrations. The effect of the detergent TX-100 on the conformation of the PS II reaction centre is discussed in relation to the number of β-carotene molecules in the preparation.

The construction and properties of a mutant of rhodobacter sphaeroides with the lh1 antenna as the sole pigment protein

Recent work on the spectroscopic properties of the B875 (LH1) antenna of Rb. sphaeroides has been performed on the complex purified from membranes of the wild-type solubilised in lithium dodecyl sulphate. In order to facilitate an examination of the properties of the membrane-bound antenna free from other complexes, mutant M2192 was constructed from mutant M21, which contains reaction centres and LH1 but lacks the B800–850 (LH2)complex. This was accomplished by insertion of transposon Tn5 into the puf L gene which encodes the L polypeptide of the reaction centre. This manipulation leaves B875 as the sole pigment protein, which has been confirmed by Southern and Northern hybridisation, gel electrophoresis and fluorescence and absorbance spectroscopy. Evidence from Gaussian deconvolution of the Qy absorbance region, and from fluorescence polarisation, suggests that the long-wavelength species BChl 896 is present, and may be an inherent property of the LH1 antenna.

Singlet-singlet excitation annihilation measurements on the antenna of Rhodospirillum rubrum between 300 and 4 K

Bymeans of fluorescence measurements singlet-singlet excitation annihilation in the antenna of Rhodospirillum rubrum chromatophores was studied at temperatures between 300 and 4 K. Two fluorescence bands had to be assumed to explain the data at 4 K. These bands (F911 and F918) are attributed to emission from the main antenna pigment BChl 880 and from a minor spectral component BChl 896, respectively. The latter pigment is supposed to be associated with the reaction center (Van Grondelle, R., Bergstrom, H., Sundstrom, V., Van Dorssen, R.J., Vos, M. and Hunter, C.N. (1988) in Photosynthetic Light-Harvesting Systems (Scheer, H. and Schneider, S., eds.), pp. 519–530, Walter de Gruyter, Berlin). At 4 K 90% of the fluorescence originates from this pigment. Analysis of the data at 4 K indicated that BChl 8% is arranged in clusters of 21 ± 9 BChls. Since there are presumably only 5 or 6 BChls 896 per reaction center, this indicates that several reaction centers are clustered. Annihilation measurements on the isolated B880 antenna complex indicate that, at 4 K, the spectral heterogeneity still exists and that the presence of BChl 896 is therefore not caused by interactions between the reaction center and the antenna. The domain size of BChl 880 was estimated to be between 35 and 75 BChls in chromatophores at 4 K. A wavelength dependence of the annihilation, due to the presence of the two types of BChl, was observed between about100 and 4 K. Measurements of fluorescence polarization suggest that this wavelength dependence may be caused by a reduction of the rate of energy transfer from BChl 896 to BChl 880 upon cooling. An increased annihilation between 100 and 300 K can, at least partly, be ascribed to an enhanced rate of energy transfer between individual BChls. This rate increases from an average valueof (4 ± 1) · 10 11 s−1 at 100 K to (1.5 ± 0.5) 1012 s−1 at 300 K.

A perturbed two-level model for exciton trapping in small photosynthetic systems.

The study of exciton trapping in photosynthetic systems provides significant information about migration kinetics within the light harvesting antenna (LHA) and the reaction center (RC). We discuss two random walk models for systems with weakly coupled pigments, with a focus on the application to small systems (10-40 pigments/RC). Details of the exciton transfer to and from the RC are taken into consideration, as well as migration within the LHA and quenching in the RC. The first model is obtained by adapting earlier local trap models for application to small systems. The exciton lifetime is approximated by the sum of three contributions related to migration in the LHA, trapping by the RC, and quenching within the RC. The second model is more suitable for small systems and regards the finite rate of migration within the LHA as a perturbation of the simplified model, where the LHA and the RC are each represented by a single pigment level. In this approximation, the exciton lifetime is the sum of a migration component and a single nonlinear expression for the trapping and quenching of the excitons. Numerical simulations demonstrate that both models provide accurate estimates of the exciton lifetime in the intermediate range of 20-50 sites/RC. In combination, they cover the entire range of very small to very large photosynthetic systems. Although initially intended for regular LHA lattices, the models can also be applied to less regular systems. This becomes essential as more details of the structure of these systems become available. Analysis with these models indicates that the excited state decay in LH1 is limited by the average rate at which excitons transfer to the RC from neighboring sites in the LHA. By comparing this to the average rate of transfer within the LHA, various structural models that have been proposed for the LH1 core antenna are discussed.