Van Grondelle Research Articles

Fluorescence Lifetime Heterogeneity in Aggregates of LHCII Revealed by Time-Resolved Microscopy

Two-photon excitation, time-resolved fluorescence microscopy was used to investigate the fluorescence quenching mechanisms in aggregates of light-harvesting chlorophyll a/ b pigment protein complexes of photosystem II from green plants (LHCII). Time-gated microscopy images show the presence of large heterogeneity in fluorescence lifetimes not only for different LHCII aggregates, but also within a single aggregate. Thus, the fluorescence decay traces obtained from macroscopic measurements reflect an average over a large distribution of local fluorescence kinetics. This opens the possibility to resolve spatially different structural/functional units in chloroplasts and other heterogeneous photosynthetic systems in vivo, and gives the opportunity to investigate individually the excited states dynamics of each unit. We show that the lifetime distribution is sensitive to the concentration of quenchers contained in the system. Triplets, which are generated at high pulse repetition rates of excitation (>1 MHz), preferentially quench domains with initially shorter fluorescence lifetimes. This proves our previous prediction from singlet-singlet annihilation investigations (Barzda, V., V. Gulbinas, R. Kananavicius, V. Cervinskas, H. van Amerongen, R. van Grondelle, and L. Valkunas. 2001. Biophys. J. 80:2409–2421) that shorter fluorescence lifetimes originate from larger domains in LHCII aggregates. We found that singlet-singlet annihilation has a strong effect in time-resolved fluorescence microscopy of connective systems and has to be taken into consideration. Despite that, clear differences in fluorescence decays can be detected that can also qualitatively be understood.

Electronic and Vibrational Coherence in the Core Light-Harvesting Antenna of Rhodopseudomonas viridis

In this paper we explain the wavelength-dependent oscillatory features in the pump−probe kinetics of the core LH1 antenna of Rhodopseudomonas viridis (Monshouwer, R.; Baltuška, A.; van Mourik, F.; van Grondelle, R. J. Phys. Chem. A 1998, 102, 4360). A quantitative fit of the data was obtained using the doorway−window representation of the nonlinear optical response in the vibrational eigenstate basis. In contrast to LH1/LH2 complexes from the BChl a-containing species, the LH1 antenna of Rps. viridis is characterized by a strong coupling of the excitonic states with two underdamped low-frequency modes (58 and 110 cm-1 at 77 K). Following a short femtosecond excitation pulse, this gives rise to the intense oscillations observed in the pump−probe traces, including their time and excitation/detection wavelength dependence. Furthermore, it leads to a pronounced and specific heterogeneity of the major absorption band due to the combined effects of the exciton splitting, disorder, and the presence of vibrational sidebands. The sharp maxima in the second derivative of the low-temperature absorption spectrum (Monshouwer, R.; Visschers, R. W.; van Mourik, F.; Freiberg, A.; van Grondelle, R. Biochim. Biophys. Acta 1995, 1229, 373) were assigned to the lowest exciton−vibrational transitions. The wavelength dependence of the experimentally observed oscillatory pattern suggests a different vibrational coherence decay for the ground- and excited-state wave packets. This can be explained by assuming the (incoherent) migration of the delocalized exciton (polaron) around the ringlike antenna with a characteristic time constant of 0.9−1.5 ps at 77K.

Ubiquinone binding, ubiquinone exclusion, and detailed cofactor conformation in a mutant bacterial reaction center.

The X-ray crystal structure of a Rhodobacter sphaeroides reaction center with the mutation Ala M260 to Trp (AM260W) has been determined. Diffraction data were collected that were 97.6% complete between 30.0 and 2.1 A resolution. The electron density maps confirm the conclusions of a previous spectroscopic study, that the Q(A) ubiquinone is absent from the AM260W reaction center (Ridge, J. P., van Brederode, M. E., Goodwin, M. G., van Grondelle, R., and Jones, M. R. (1999) Photosynthesis Res. 59, 9-26). Exclusion of the Q(A) ubiquinone caused by the AM260W mutation is accompanied by a change in the packing of amino acids in the vicinity of the Q(A) site that form part of a loop that connects the DE and E helices of the M subunit. This repacking minimizes the volume of the cavity that results from the exclusion of the Q(A) ubiquinone, and further space is taken up by a feature in the electron density maps that has been modeled as a chloride ion. An unexpected finding is that the occupancy of the Q(B) site by ubiquinone appears to be high in the AM260W crystals, and as a result the position of the Q(B) ubiquinone is well-defined. The high quality of the electron density maps also reveals more precise information on the detailed conformation of the reaction center carotenoid, and we discuss the possibility of a bonding interaction between the methoxy group of the carotenoid and residue Trp M75. The conformation of the 2-acetyl carbonyl group in each of the reaction center bacteriochlorins is also discussed.

Excited States of the 5-Chlorophyll Photosystem II Reaction Center

Results of 4.2 K hole burning, chemical reduction (sodium dithionite, in dark and with illumination), and oxidation (ferricyanide) experiments are reported for the isolated PS II reaction center containing five chlorophyll (Chl) molecules (RC-5). Qy states at 679.6 and 668.3 nm are identified as being highly localized on pheophytin a of the D1 branch (Pheo1) and pheophytin a of the D2 branch (Pheo2), respectively. The Pheo1−Qx and Pheo2−Qx transitions were found to lie on the low and high energy sides of the single Pheo−Qx absorption band, at 544.4 and 541.2 nm, respectively. The Qy band of the 684 nm absorbing Chl, which is more apparent in absorption in RC-5 than in RC-6 samples, is assigned to the peripheral Chl on the D1 side. The results are consistent with that peripheral Chl being Chlz (Stewart, D. H.; Cua, A.; Chisholm, D. A.; Diner, B. A.; Bocian, D. F.; Brudvig, G. W. Biochemistry 1998, 37, 10040). The results indicate that P680, the primary electron donor, is the main acceptor for energy transfer from the Pheo1−Qy state and that excitation energy transfer from the Pheo1−Qy state and P680* to the 684 nm Chl is inefficient. It is concluded that the procedure used to prepare RC-5 has only a small effect on the energies of the Qy states associated with the core cofactors of the 6-Chl RC as well as the 684 nm Chl. Implications of the results for the multimer model (Durrant, J. R.; Klug, D. R; Kwa, S. L. S.; van Grondelle, R.; Porter, G.; Dekker, J. P. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 4798) are considered. In that model the Qy-states of the core are significantly delocalized over several cofactors. The results presented provide no support for this model.

The exciton transfer in the antenna systems of rhodopseudomonas acidophila

The photosynthetic apparatus of the purple bacterium Rhodobacter sphaeroides is organised so that light energy absorbed by the peripheral antenna (LH2) complexes migrates towards the core (LH1) complex, before being trapped by the reaction centre (RC). This migration and trapping process has been studied in mutants where the energy levels of the LH2 BChls have been raised by mutagenesis of the C-terminal aromatic residues (Fowler, G.J.S., Visschers, R.W., Grief, G.G., Van Grondelle, R. and Hunter, C.N. (1992) Nature 355, 848–850), and in a mutant which lacks the core complex. In the former case, the alterations to the LH2 complexes did not prevent efficient energy transfer to the LHI-RC complex, but fluorescence emission spectra indicated that the equilibrium of energy within the system was affected so that back transfer from the LH1-RC core is minimised. This mimics the situation found in some other bacteria such as Rhodopseudomonas acidophila and Rps. cryptolactis. In the mutant lacking LH1, energy is transferred from LH2 directly to the RC, despite the absence of the core antenna. Energy transfer efficiencies for carotenoids and LH2 to LH1 were measured for the blue-shifted LH2 mutants, and were found to be high (70%) in each case. These data, together with measurements of excitation annihilation as a function of incident excitation energy, were used to estimate the domain sizes for energy transfer in these mutants. In the LH2 mutants, domains of about 50 to 170 core BChls were found, depending on the type of mutation. One effect of the removal of LH1 appears to be the reorganisation of the peripheral LH2 antenna to form domains of at least 250 BChls.

Supramolecular organization of the photosynthetic apparatus of Rhodobacter sphaeroides.

Native tubular membranes were purified from the purple non-sulfur bacterium Rhodobacter sphaeroides. These tubular structures contain all the membrane components of the photosynthetic apparatus, in the relative ratio of one cytochrome bc1 complex to two reaction centers, and approximately 24 bacteriochlorophyll molecules per reaction center. Electron micrographs of negative-stained membranes diffract up to 25 A and allow the calculation of a projection map at 20 A. The unit cell (a = 198 A, b = 120 A and gamma = 103 degrees) contains an elongated S-shaped supercomplex presenting a pseudo-2-fold symmetry. Comparison with density maps of isolated reaction center and light-harvesting complexes allowed interpretation of the projection map. Each supercomplex is composed of light-harvesting 1 complexes that take the form of two C-shaped structures of approximately 112 A in external diameter, facing each other on the open side and enclosing the two reaction centers. The remaining positive density is tentatively attributed to one cytochrome bc1 complex. These features shed new light on the association of the reaction center and the light-harvesting complexes. In particular, the organization of the light-harvesting complexes in C-shaped structures ensures an efficient exchange of ubihydroquinone/ubiquinone between the reaction center and the cytochrome bc1 complex.

Excitations and excitons in photosystem I

The elementary requirements for energy transfer in a photosynthetic antenna are discussed. Energy migration between chlorophyll molecules in the PSI antenna reaction centre complex was modelled using a geometry based on the recent X–ray structure. The migration is trap–limited and an intrinsic electron transfer rate constant of 1.3 ps−1 was obtained by modifying the model to account for experimental results obtained from antennae of different sizes. Exciton coupling between pairs of Chlorophyll molecules was calculated and it is found that to prevent self–quenching their interplanar separation must be greater than or equal to 5 A. In the PSI reaction centre, exciton coupling between adjacent chlorophylls was found to be around 300 cm−1 with no particularly large value between the special pair being necessary to describe the experimentally observed transient spectra of P +700− P 700 and A −− A . Electrochromic interaction is particularly important in reproducing the A −− A spectrum.

The interaction of solutes with their environments

The origins of electronic spectral broadening in solutions, glasses and proteins are discussed via three pulse stimulated echo peak–shift measurements. Spectral densities for dye molecules in polar solutions are presented and discussed in terms of molecular models for the dynamics. The breakdown of the harmonic bath picture at low frequencies is presented. Finally, echo measurements on light harvesting complexes of purple bacteria are presented and it is shown that the energy transfer timescale can be obtained from peak–shift measurements.

The P700 triplet state in an intact environment detected by ODMR: A well resolved triplet minus singlet spectrum

Optically detected magnetic resonance (ODMR) of P700 triplet state has been performed in large photosystem I (PSI) particles. The `intactness' of the P700 environment in the PSI particles has been proved by the comparison of the spectra with those obtained in thylakoids and leaves. Effect of degradation of the samples on the spectra has also been tested. When detected in `native' PSI centers the microwaves induced triplet-minus-singlet absorption spectra (T−S) show very sharp features which allow to estimate the minimum number of bands involved in the change of the electronic state of the primary donor (singlet to triplet), and can be interpreted in terms of interactions among pigments in the reaction center. The analogy with the reaction centers of purple bacteria is discussed. The previously published spectra (H.J. den Blanken, A.J. Hoff, Biochim. Biophys. Acta 724 (1983) 52–61; J. Vrieze, P. Gast, A.J. Hoff, J. Phys. Chem. 100 (1996) 9960–9967) obtained in particles having smaller antenna size, resemble the ones obtained in our degraded samples: it is suggested that the isolation procedure to small particles may produce an increase in the heterogeneity of the environment of the primary donor and/or a change of the mutual orientations and interactions among pigments. The fluorescence detected magnetic resonance (FDMR) in large PSI particles supports an excitation energy transfer model derived from time resolved optical studies (R. Van Grondelle, J.P. Dekker, T. Gillbro, V. Sundstrom, Biochim. Biophys. Acta 1187 (1994) 1–65) and adapted for helium temperature, characterized by the presence of antenna pools emitting at 690, 720 and 735 nm, respectively. The FDMR signal of P700 changes sign (from positive to negative) when detected at emission wavelength shorter than 700 nm. Comparison of FDMR of P700 triplet state in large PSI-200 particles, in thylakoids and, for the first time, in leaves has been done.

Electronic Energy Transfer within the Hexamer Cofactor System of Bacterial Reaction Centers

Flow of excitation energy within the bacteriochlorin cofactor system of bacterial reaction centers has been studied by multicolor transient absorption spectroscopy using differently shaped excitation pulses of 30 fs, both at room temperature and at 15 K. This approach, which includes the analysis of free induction decay signals, helps to disentangle the processes of electronic dephasing, energy transfer, internal conversion, and nuclear motion, all taking place on the time scale of ∼100 fs. Part of the excitations (estimated at 80−90% at room temperature) flow via the scheme H* → B* → P+* → P-*, in which H* and B* represent excited bacteriopheophytin and bacteriochlorophyll monomers and P+* and P-* the upper and lower excited states of the bacteriochlorophyll dimer P, respectively. H* → B* takes less than 100 fs. B* → P+* (∼200 fs) energy transfer is a slower process than the internal conversion process within P* (50−100 fs) and therefore is rate limiting for P-* formation. At low temperature, electronic dephasing associated with the B* and P+* states takes place on a similar time scale as P* internal conversion. Upon excitation with pulses centered at 820 nm, estimated to spectrally overlap the close-lying B and P+ bands to equal extent, more than 90% of the P- band bleaches instantaneously and B* → P* transfer occurs for less than 10%. This might be indicative of an unexpectedly strong contribution of P+ to the 800 nm band. Alternatively, we propose that under these conditions the strongly coupled B* and P+* states are coherently excited. This possibility is consistent with B* → P+* electronic energy transfer occurring in the strong coupling regime under conditions where B* is more selective populated. The observed time scales are temperature-insensitive and furthermore similar in Rhodobacter sphaeroides R26 and Rhodopseudomonas viridis, which eliminates the possibility of direct B* → P-* energy transfer. At room temperature, part of the excitation energy flow deviates from the above scheme (Lin, S.; Taguchi, A. K. W; Woodbury, N. W. J. Phys. Chem. 1996, 100, 17067−17078), resulting in excitation-wavelength-dependent distributions of excited and/or radical pair states on the picosecond time scale. In general agreement with previous low-temperature work on a mutant reaction center (van Brederode, M. E.; Jones, M. R.; van Mourik, F.; van Stokkum I. H. M.; van Grondelle, R. Biochemistry 1997, 36, 6855−6861), we found that this deviation is particularly strong (up to ∼50%) at low temperature. Upon excitation in H and B, a high quantum yield is observed of radical pairs involving HL, with characteristics in the HL QY region, differing from those of P+HL- and suggesting that they can be identified as BL+HL-.

The role of betaArg-10 in the B800 bacteriochlorophyll and carotenoid pigment environment within the light-harvesting LH2 complex of Rhodobacter sphaeroides.

Previous work has suggested that the betaArg-10 residue forms part of the binding site for the B800 bacteriochlorophyll in the LH2 complex of Rhodobactersphaeroides [Crielaard, W., Visschers, R. W., Fowler, G. J. S., van Grondelle, R., Hellingwerf, K. J., Hunter, C. N. (1994) Biochim. Biophys. Acta1183, 473-482], and this is consistent with the X-ray crystallographic data that have been subsequently obtained for the related LH2 complex from Rhodopseudomonas acidophila [McDermott, G., Prince, S. M., Freer, A. A., Hawthornthwaite-Lawless, A. M., Papiz, M. Z., Cogdell, R. J., Isaacs, N. W. (1995) Nature 374, 517-521]. Therefore, in order obtain more information about the B800 binding site and its effect on the B800 absorption band, betaArg-10 was replaced by residues Met, His, Asn, Leu, and Lys (in addition to the Glu mutant described in our previous work); these residues were thought to represent a suitable range of amino acid shape, charge, and hydrogen-bonding ability. This new series of betaArg-10 mutants, in the form of LH2 complexes in the native membrane, has been characterized using a variety of biochemical and spectroscopic techniques in order to determine the ways in which the mutants differ from wild-type (WT) LH2. For example, most of the mutant LH2 complexes were found to have blue-shifted B800 absorption bands ranging from 794 to 783 nm at 77 K; the exception to this trend is the betaArg-10 to Met mutant, which absorbs maximally at 798 nm. These blue shifts decrease the spectral overlap between the "B800" and B850 pigments, which allowed us to examine the nature of the B800 to B850 transfer step for the betaArg-10 mutant LH2 complexes by carrying out a series of room temperature subpicosecond energy transfer measurements. The results of these measurements demonstrated that the reduced overlap leads to a slower B800 to B850 transfer, although the alterations at betaArg-10 were found to have little effect on the efficiency of internal energy transfer within LH2. Similarly, carotenoid to bacteriochlorophyll energy transfer was largely unaffected, although shifts in the excitation spectra in the carotenoid region were noted. These betaArg-10 mutant complexes provide an opportunity to investigate the structural requirements for the binding of monomeric bacteriochlorophyll and to examine the basis of the red shift seen for bacteriochlorophyll in photosynthetic complexes, in addition to providing new information about the environment of the carotenoid pigments in this complex.

P-E2-18 A spectroscopic characterization of LHCII reconstituted with different pigment ratios: Kleima FJ, 1 Calkoen F, 1 Urbanus ML, 1 Borst HC, 1 Peterman EJG, 1 van Grondelle R, 1 van Amerongen H, 1 Hobe S, 2 Paulsen H. 2 1 Dept. of Physics and Astronomy and Inst. for Molecular Biological Sciences, Vrije Univ. Amsterdam (NL), 2 Botanisches Inst. III

P-E2-18 A spectroscopic characterization of LHCII reconstituted with different pigment ratios: Kleima FJ, 1 Calkoen F, 1 Urbanus ML, 1 Borst HC, 1 Peterman EJG, 1 van Grondelle R, 1 van Amerongen H, 1 Hobe S, 2 Paulsen H. 2 1 Dept. of Physics and Astronomy and Inst. for Molecular Biological Sciences, Vrije Univ. Amsterdam (NL), 2 Botanisches Inst. III

P-E2-17 Long-wavelength antenna pigments in photosystem I: Pålsson L-O, 1 Gobets B 1, Fleming C, 2 Ericsson L-M, 1 van Grondelle R, 1 Schlodder E, 2 Dekker JP 1 1 Dept. of Physics and Astronomy, Vrije Universiteit, Amsterdam (NL), 2 Max-Volmer-Institut, Technische Universität Berlin (D)

P-E2-17 Long-wavelength antenna pigments in photosystem I: Pålsson L-O, 1 Gobets B 1, Fleming C, 2 Ericsson L-M, 1 van Grondelle R, 1 Schlodder E, 2 Dekker JP 1 1 Dept. of Physics and Astronomy, Vrije Universiteit, Amsterdam (NL), 2 Max-Volmer-Institut, Technische Universität Berlin (D)

P-E2-19 Low temperature stark spectroscopy on trimeric and aggregated LHC II complexes: Gradinaru C.C., Frese R., van Amerongen H., van Grondelle R. Dept. of Physics and Astronomy, Vrije Universiteit, Amsterdam (NL)

P-E2-19 Low temperature stark spectroscopy on trimeric and aggregated LHC II complexes: Gradinaru C.C., Frese R., van Amerongen H., van Grondelle R. Dept. of Physics and Astronomy, Vrije Universiteit, Amsterdam (NL)

P-E2-20 Spectroscopy and structure of antenna complexes; differences and similarities of LH1 and LH2: Koolhaas MHC, 1 van der Zwan G, 1 van Mourik F, 2 van Grondelle R. 2 1 Fac. of Chemistry, 2 Fac. of Physics and Astronomy, Vrije Universiteit, Amsterdam (NL)

P-E2-20 Spectroscopy and structure of antenna complexes; differences and similarities of LH1 and LH2: Koolhaas MHC, 1 van der Zwan G, 1 van Mourik F, 2 van Grondelle R. 2 1 Fac. of Chemistry, 2 Fac. of Physics and Astronomy, Vrije Universiteit, Amsterdam (NL)

P-E1-14 Spectroscopic investigation of the reaction center of the green-sulfur bacteria Chlorobium limicola: Frese R N 1, Albouy D 2, Robert B 2, Van Grondelle R 1 1 Dept. of Physics and Astronomy, Vrije Universiteit, Amsterdam (NL). 2 Centre d'Etudes de Saclay (F)

P-E1-14 Spectroscopic investigation of the reaction center of the green-sulfur bacteria Chlorobium limicola: Frese R N 1, Albouy D 2, Robert B 2, Van Grondelle R 1 1 Dept. of Physics and Astronomy, Vrije Universiteit, Amsterdam (NL). 2 Centre d'Etudes de Saclay (F)

P-E2-22 Sub picosecond two colour absorption spectroscopy of the LH-1 antenna of Rps. viridis at room temperature : Monshouwer, R., Baltuska, A., van Mourik, F., van Grondelle, R. Faculty of Physics and Astronomy, Vrije Universiteit, Amsterdam (NL)

P-E2-22 Sub picosecond two colour absorption spectroscopy of the LH-1 antenna of Rps. viridis at room temperature : Monshouwer, R., Baltuska, A., van Mourik, F., van Grondelle, R. Faculty of Physics and Astronomy, Vrije Universiteit, Amsterdam (NL)

P-E3-17 Subpicosecond spectroscopy of the photoactive yellow protein: Baltuska A, 1 Van Stokkum IHM, 1 Kroon A, 2 Monshouwer R, 1 Hellingwerf KJ, 2 Van Grondelle R. 1 1 Faculty of Physics and Astronomy, Vrije Universiteit, Amsterdam (NL), 2 Dept. of Microbiology, Univ. of Amsterdam, Amsterdam (NL)

P-E3-17 Subpicosecond spectroscopy of the photoactive yellow protein: Baltuska A, 1 Van Stokkum IHM, 1 Kroon A, 2 Monshouwer R, 1 Hellingwerf KJ, 2 Van Grondelle R. 1 1 Faculty of Physics and Astronomy, Vrije Universiteit, Amsterdam (NL), 2 Dept. of Microbiology, Univ. of Amsterdam, Amsterdam (NL)

Excitation Transfer in the Core Light-Harvesting Complex (LH-1) of Rhodobacter sphaeroides: An Ultrafast Fluorescence Depolarization and Annihilation Study

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTExcitation Transfer in the Core Light-Harvesting Complex (LH-1) of Rhodobacter sphaeroides: An Ultrafast Fluorescence Depolarization and Annihilation StudyStephen E. Bradforth, Ralph Jimenez, Frank van Mourik, Rienk van Grondelle, and Graham R. FlemingCite this: J. Phys. Chem. 1995, 99, 43, 16179–16191Publication Date (Print):October 1, 1995Publication History Published online1 May 2002Published inissue 1 October 1995https://doi.org/10.1021/j100043a071RIGHTS & PERMISSIONSArticle Views826Altmetric-Citations273LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (2 MB) Get e-Alerts

Energy migration in Rhodobacter sphaeroides mutants altered by mutagenesis of the peripheral LH2 complex or by removal of the core LH1 complex

The photosynthetic apparatus of the purple bacterium Rhodobacter sphaeroides is organised so that light energy absorbed by the peripheral antenna (LH2) complexes migrates towards the core (LH1) complex, before being trapped by the reaction centre (RC). This migration and trapping process has been studied in mutants where the energy levels of the LH2 BChls have been raised by mutagenesis of the C-terminal aromatic residues (Fowler, G.J.S., Visschers, R.W., Grief, G.G., Van Grondelle, R. and Hunter, C.N. (1992) Nature 355, 848–850), and in a mutant which lacks the core complex. In the former case, the alterations to the LH2 complexes did not prevent efficient energy transfer to the LHI-RC complex, but fluorescence emission spectra indicated that the equilibrium of energy within the system was affected so that back transfer from the LH1-RC core is minimised. This mimics the situation found in some other bacteria such as Rhodopseudomonas acidophila and Rps. cryptolactis. In the mutant lacking LH1, energy is transferred from LH2 directly to the RC, despite the absence of the core antenna. Energy transfer efficiencies for carotenoids and LH2 to LH1 were measured for the blue-shifted LH2 mutants, and were found to be high (70%) in each case. These data, together with measurements of excitation annihilation as a function of incident excitation energy, were used to estimate the domain sizes for energy transfer in these mutants. In the LH2 mutants, domains of about 50 to 170 core BChls were found, depending on the type of mutation. One effect of the removal of LH1 appears to be the reorganisation of the peripheral LH2 antenna to form domains of at least 250 BChls.