Femtosecond Spectroscopy of Photosynthetic Light-Harvesting Systems Graham R Fleming* and Rienk Van Grondellet

738

Femtosecond spectroscopy of photosynthetic light-harvesting systems Graham R Fleming* and Rienk van Grondellet

Observing the elementary steps of light-harvesting in real peridinin-carotenoid protein of dinoflagellates [10 °°] --all time is now possible using femtosecond spectroscopy. membrane-attached light-harvesting systems--we now This, combined with new structural data, has allowed a fairly have a multitude of structures available which exhibit complete description of light-harvesting in purple bacteria and amazing variation which will allow us to greatly extend our substantial insights into higher plant antenna systems. knowledge of the process of excitation energy transfer and the underlying physics.

Addresses In this review, we describe the considerable recent ~Department of Chemistry, University of California, Berkeley, CA 94720-1460, USA; e-mail: [email protected] progress in understanding the purple bacterial antenna tDepartment of Physics and Astronomy, Vrije Universiteit, De system and outline the current views on green plant and Boelelaan 1081, NL-1081 HV, Amsterdam, The Netherlands; cyanobacterial systems, for which the structural data do not e-mail: [email protected] yet allow for fully detailed modeling. Current Opinion in Structural Biology 1997, 7:738-748 http://biomednet.com/elecref/O959440XO0700738 Disordered versus ordered light-harvesting systems O Current Biology Ltd ISSN 0959-440X Although the various structures now known exhibit a Abbreviations wide spread in organizational motifs, one striking aspcct 3PEPS three-pulse photon echo peak shift stands out. Comparing bacterial and plant light-harvesting BChl bacteriochlorophyll CD circular dichroism systems, the bacterial peripheral, LH2, and core, LH1, Chl chlorophyll antenna are structures with a ve~ high degree of sym- RC reactioncenter metry (see Figure 1), whereas LHCII and even more so PSI appear spatially (i.e. positionally and orientationally) much more disordered. One of the major reasons for Introduction this variation is, of course, the size of the elementary In order to harvest solar light, photosynthetic organisms building block. In LH1 and LH2, this is a pair of small are equipped with a light-harvesting antenna system. transmembrane polypeptides, ot and 13, which carries two Photons absorbed by the antenna pigments are transferred and three BChls, respectively. Assembly into a larger to the photosynthetic reaction center (Re) with great system will always lead to a structure with a high degree speed. Once absorbed by the RC, the excitation energy of symmetry. is efficiently converted into a stable charge separation. In contrast, the PS1 core consists of a single pair of large Since the basic description of energy transfer and trapping polypeptides, the PsaA and PsaB gene products, which, processes by Duysens [1,2] in the early 1950s, it has been together with a large number of smaller subunits, forms clear that the elementary steps of the light-harvesting the PSI core that binds - 100 chlorophylls. LHCII seems process are extremely rapid. Only recently, however, has it to be an intermediate case. Although monomeric, LHCI1 become possible to make direct experimental observations still appears quite disordered in comparison with the LH1 on the timescale of individual energy transfer steps; and and LH2 rings; the basic unit of LHCII is a trinaer of currently energy migration can be investigated in the an -25 kDa protein that exhibits perfect C3 symmetry. range of tens of femtoseconds to many nanoseconds. The other apparent difference between plant and bacterial Application of femtosecond laser spectroscopy has been light-harvesting systems is the pigment density. In LH1 of greatly stimulated by the remarkable successes in struc- purple bacteria, the density is two BChl ot-polypeptides ture determination of several important light-harvesting per 12kDa; in LHCII of green plants, 12-14 Chls occur complexes in recent years. For example, the peripheral per 25kDa--a factor of three more. A similar variation light-harvesting complex (LHCII) of green plants [3], the applies in PSI. Thus, in plant light-harvesting systems, peripheral light-harvesting (LH2) complex of Rhodopseu- the various opportunities to bind Chl molecules have been domonas (Rps.) acidophila [4,5°°], the LH2 of RhodospiHllum exploited optimally. (Rs.) molischianum [6°°], and the core of Photosystem 1 (PSI) of cyanobactcria [7 "°] have all been determined. All Finall-y, although PS1 differs greatly from the LH1-RC these are intrinsic membrane proteins, and, together with core of purple bacteria, it also has a fundamental similarity. the known structures of the bacteriochlorophyll (BChl) a As should be apparent from Figure 1, in the LH1-RC protein of green sulphur bacteria [8], the phycobilipro- core the rate-limiting step for trapping photons is energy teins [9], and the recently resolved structure of the transfer from any of the light-harvesting pigments to the Femtosecond spectroscopy of photosynthetic light-harvesting systems Fleming and van Grondelte 739

Figure 1

B8o0

,c 1997 Current Opinion in Structural Biology

A model for the light-harvesting and photon trapping machinery in the photosynthetic membrane of a purple bacterium. The view is along the membrane plane and only the bacteriochlorophyll pigments are shown. The primary electron donor (the special pair, P) of the RC is indicated by an arrow. The LH2 (smaller gray rings) and RC structures are based on crystallography. The LH1 structure (large b~ack ring) is modeled truing the size of the RC protein (not shown) and the c~I] unit of the LH2 structure. Note that no special orientation requirements are needed for effective transfer from LH2 to LH1. Carotenoids present in both structures are not shown. special pair (the primal" electron donor, consisting of a Bacterial antennas pair of strongly interacting bacteriochlorophyll molecules) Energy transfer in the peripheral, LH2, and core, LH1, of in the RC. In this scenario, the trapping efficiency is photosynthetic purple bacteria highest when as many antenna sites as possible are able to The recently resolved structures of LH2 of Rps. acidophila transfer to the RC, and this is clearly optimized within a [4] and Rs. molischianum [6 °°] have revealed the highly ring. In PS1, crudely speaking, the pigments are organized symmetric pigment-protein ring, displaying C9 symmetry in a band around the electron transfer chain, and, on in the case of Rps. acidophila and C8 symmetry for average, they are all at a distance of- 2-3 nm; however, Rs. molischianum. Although only a low-resolution structure within this structure, the number of contact sites has is available for LH1 [11], it is evident that LH1 is also also been optimized, leading to efficient trapping. The organized as a ring, most probably with 16-fold symmetry. symmetry itself is not important, rather the avoidance of The RC structure can be nicely fitted into the proposed quenching centers (e.g. stacked dimers) and the location LH1 ring [11,12"]. of the maximum number of pigments close to the site where the primary charge separation occurs are important. Both for LH1 and LH2, the basic building block of In order to avoid undesirable oxidation or reduction of the the structure is a heterodimer of two small (5-6kDa) antenna by the primary electron donor, however, the bulk polypeptides, a and 13. Both consist of a single trans- of the antenna molecules are kept at a distance >2nm membrane helix with a highly conserved histidine that from the components involved in the electron transfer. ligates the BChl approximately one third of the way along the a-helical stretch. Thus, in the LH1 and LH2 An additional feature of all Chl and BChl antenna pigment-protein rings, the basic element is a BChl dimer. complexes resolved to date is the presence of carotenoid For LH1, the o~13-BChl2 subunit can be purified; it is called molecules. These serve photo-protective, light-harvesting B820 after its absorption maximum and retains many of the and often structural roles. essential spectral properties of LH1 [13]. For LH2, such 740 Biophysicalmethods

a subunit cannot be obtained. In LH2, and most probably spectroscopic features, including the dramatic red shift, in LH1, the heterodimeric subunits associate into a ring largely originate from within a dimer. The excitonic with the 0t-polypeptides on the inside, the [3-polypeptides interaction between neighbouring subunits within the ring at the outside, and the pigments sandwiched between the is considered to be a relatively weak perturbation, that two concentric rings of polypeptides. As a result of the is, relative to the intradimer cxcitonic interaction, the formation of the ring, the absorption shifts to -870 nm for possible (and so far unknown) contribution from electron LH1, and to -850nm for LH2, although in the latter case exchange arising from ring I overlap, the intrinsic energetic other absorption maxima are also found for some species disorder and the electron-phonon or electron-vibration (820 nm, 830 nm), depending on the presence or absence coupling. The spectra of all photosynthetic pigment- of hydrogen bonds [14,15]. The [~-polypeptide of LH2 proteins are now known to be strongly inhomogeneously binds a second pigment, nearer to the cytosolic side of the broadened, and estimates of the amount of inhomoge- complex, and in LH2 of Rps. acidophila these BChls form neous broadening range from 200-500cm -1. In addition, a nine-membered ring which absorbs at -800nm and is the electron-phonon coupling is estimated to be of the positioned at a distance of - 1.7 nm from the B850 ring. same order of magnitude. The general idea behind the 'ring of dimers' model is that, following excitation, any Electronic structure phase relation between excitations on different dimers Within the B850 ring of dimers, all distances between is rapidly destroyed, either dynamically, because of the the pigments are very similar--somewhat less than 1 rim. coupling to vibrations or phonons, or as a consequence of Nevertheless, within the ctl3 subunit, the electron density the interference of the pure eigenstates due to energetic seems continuous, whereas electron density due to the two disorder. neighbouring BChls on adjacent subunits is discontinuous. The reason for this is that the ]3-polypeptide BChl is clearly bent. One further important point is that within In the alternative view, the spectroscopic features are the ct[3 subunit, overlap between the two BChls occurs totally determined by the set of excitonic eigenstates between chlorin tings I, as in the special pair of the RC, of the full ring. In this model, the excitonic interaction where as overlap between BChls on adjacent subunits is between adjacent BChls is the dominant term that between chlorin rings III. As a consequence, one may completely determines the red shift observed upon view LH2 and most probably LH1 as 'rings of interacting formation of the ring. The lowest state of the exciton directs'. This concept is supported by many experimental manifold is almost optically forbidden, because of the observations (see below; and e.g. [16°]). The BChls in inplane orientation of the Qy transition dipoles, and the B850 ring of LH2 and in the B870 ring of LH1 all the oscillator strength is equally divided between all have their Qy transition dipole almost parallel and two orthogonal transitions slightly above the lowest one. their Qx transition dipole perpendicular to the membrane Experimental evidence to support this model includes plane. The estimated excitonic coupling between BChls holeburning experiments [21,22"°,23"], the interpretation in a subunit is -250cm -1 [17"',18"]; the coupling is of the low-temperature absorption spectrum [17"'], and somewhat less between BChls on adjacent subunits. In estimates of the absorption cross-section of the major contrast, the pigments in the B800 ring ate 'monomeric', transition at 850nm or 870nm [28°',29]. We are of the the distance between two neighbours is -2.1 nm, and the opinion that the latter view is less accurate, mainly because corresponding dipole-dipole coupling is -20cm -1. The it ignores all the nonexcitonic contributions that all have the effect of destroying the fully delocalized coherent interaction between pigments in the B800 ring and the pigments in the B850 ring is of a similar magnitude. The states. In addition, as we show below, the ring of dimers model provides a simple and elegant explanation for many B800 rings in LH2 are almost flat in the plane of the membrane, those in the LH2 of Po. molischianum are tilted of the dynamic results. away from the membrane plane by -30 °. Finallx; the LH2 rings of Rps. acidophila and Rs. mo/ischianum contain two Intraring energy transfer carotenoids per ot]3 subunit. From a variety of spectroscopic studies (for a review, see LH1 and LH2 have been subjected to a large num- van Grondelle eta/. [30]), the energy transfer dynamics ber of spectroscopic studies, notably, polarized light within LH1 and LH2 have been concluded to be ultrafast. spectroscopy (circular dichroism [CD], linear dichroism) With the advent of femtosecond laser spectroscopy, in [19], a variety of line-narrowing techniques (holeburning particular using Ti:Sapphire lasers, many of the elementary [20,21,22"',23"1, site-selective fluorescence [24,25]), and energy transfer steps have been resolved in time. The infrared and Raman spectroscopies. In addition, using B800--+B850 energy transfer at room temperature takes structural information, several of the spectroscopic features -700-800fs for LH2 from Rb. sphaeroides, and this time have been modeled [17"',26°',27°']. From these studies, constant is not very species dependent [31-35,36°',37"]. two opposing views have emerged which we will discuss. The energy transfer time is only weakly dependent on temperature, being -1 ps at 77K and -2ps at 4K. The In the first view, LH1 and LH2 are considered to be B800--+B850 energy transfer has been modeled in terms rings of interacting directs, in which many of the essential of a F6rster process [31,33,34]. The weak temperature Femtosecond spectroscopy of photosynthetic light-harvesting systems Fleming and van Grondelle 741

dependence of this energy transfer step suggests the In an attempt to provide experimental characterization involvement of some vibronic level of B850 or possibly of the electron-phonon coupling, Jimenez et al. [54 °°] of the higher excitonic states of the B850 ring [22"°,37°]. carried out three-pulse photon echo peak shift (3PEPS) Previously, efficient energy transfer was concluded to measurements on LH1 and LH2. They concluded that occur within the B800 ring from fluorescence polarization on a 50fs timescale fluctuations in the environment experiments [19]. More recently, a time constant of and vibrations lead to the dynamic localization on a -0.5-1 ps has been estimated for energy transfer between dimeric subunit of LH1 and LH2. A similar conclusion neighbouring B800 rings from polarized pump-probe was drawn from the ultrafast reorganization, as observed spectroscopy [34,35,38°°]. by the formation of the Stokes' shift in a few tens of femtoseconds [55°]. In the peak shift decay, this initial phase was followed by an exponential phase that For energy transfer within the B850 and B875 rings, was interpreted as a loss in the rephasing capability of single-site lifetimes of the order of a few hundred the system due to energy transfer. During the energy femtoseconds have been estimated, for example, from an transfer, the system samples all the various environments analysis of the efficiency of singlet-singlet annihilation that contribute to the inhomogeneous broadening, and, [39]. In addition, the observation that within a few as a consequence, the information about the original picoseconds transient absorption changes were almost environment is lost. Again, the model that assumes fully depolarized has been interpreted as subpicosecond hopping on an inhomogeneously broadened ring of dimers energy transfer among B850 rings in LH2 and B870 gave a fit to the results. The homogeneous broadening rings in LH1 [40,41]. The energy migration in B850 was estimated to be -200cm -1, the inhomogeneous and B870 has been recorded directly using fluorescence broadening-500cm -I, and the hopping time -100fs. depolarization [42,43°°], and using equilibration of the This interpretation is very much supported by a 3PEPS transient absorption spectrum [44]. Both studies used a experiment on the LH1 subunit, B820, in which the 100 fs similar interpretation based on hopping between dimers phase in the peak shift decay ascribed to energy transfer in a ring. The site energies of the dimers were taken at was absent and replaced by a nondecaying component random from an inhomogeneous distribution of-400 cm -1 arising from inhomogeneous broadening [56°°]. The width, and average hopping times of-100fs were striking similarity between the 3PEPS of LH1 and B820 obtained. The fluorescence anisotropy decays faster in further supports the idea that excitations in these antenna LH2 than in LH1, and in this model this arises simply complexes are delocalized over only a dimer unit. from the smaller ring size of LH2 (larger angle change per hop). The model could be extended to low temperatures where the site energy variation impedes the energy Carotenoids transfer over more than a few sites [45°°]. Remarkably, Energy transfer from carotenoid to BChl in LH2 of Chachisvilis et al. [46] and Bradforth et al. [42] found that Rb. sphaeroides can occur on a timescale of a few oscillations at 105 cm -I, assigned to vibrational wavepacket 100 fs [57]. Recent fluorescence upconversion experiments motion, dephased significantly slower than the observed demonstrated that, in LH1 and LH2 of Rb. sphaeroides, depolarization timescale, suggesting vibrational coherence the $2 lifetime of sphaeroidene is shortened to -55fs transfer [47] in the energy transfer process. for the former and 80fs for the latter. This should be compared with a 150-250 fs internal conversion time from $2---)S 1, dependent on the solvent [58°]. For a B800-830 As discussed above, the extent of exciton delocalization complex of Chromatium purpuratum, Gillbro and coworkers in LH1 and LH2 has been extensively debated. Key [59 °°] report an S2--~BChl (Qx) transfer time of 100fs and quantities are the electronic coupling between the BChls, S1---)BChl (Qy) transfer times of 3.8 ps and 0.5 ps for the the electron-phonon coupling (reorganization energy and carotenoid that transfers to B830 and the carotenoid that timescale), the temperature, and the disorder. From transfers to B800, respectively. the difference in position between the pump-induced bleaching (ground state to one-exciton state) and pump- induced absorption (one-exciton state to two-exciton Interring transfer state), Sundstrtim and coworkers [48°,49 °] estimate a In the intact bacterial photosynthetic unit, the energy delocalization length of 4+2 molecules in LH1 and LH2, transfer from one LH2 to another, and from LH2 to more or less independent of temperature. A measurement LH1, takes place on a timescale of a few picoseconds of the superradiance in LH1 and LH2 gave an even [60-62,63°]. Assuming Ftirster energy transfer between smaller number [50°°]. On the other hand, an ultrafast BChls on neighbouring rings, this would imply a closest decay in the transient absorption and emission of LH2 distance of- 3 nm between the two pigment-protein rings. was taken as an indication for relaxation between fully Long before the crystal structure of LH2 became available, delocalized states [51°]. An incisive discussion of how it was realized that the rate-limiting step in excitation delocalization influences different observables has been trapping was the step from the Lftl pigments to the given by Leegwater [52 °] and more recently by Meier special pair in the RC. A transfer time of-35ps was et al. [53°°]. obtained for the LH1 to special pair energy transfer fL64], 742 Biophysicalmethods

and this was interpreted as a distance of 4.5 nm between current resolution, Chl a and Chl b are indistinguishable. the special pair and the LH1 ring, in good agreement In addition, the phytol tails of the Chls cannot be with models suggested for the LH1-RC core, assuming observed, and, as a consequence, the orientation of the Qx that LH1 is organized as a ring of 16 ¢~-BChl 2 subunits and Qy transition dipoles within each of the chlorin planes with a structure as in LH2 of Rps. acidophila [12",65*°]. A is not known. In the proposed model, the assignment of summary of the timescales is given in Figure 2. the Chl as and Chl bs is based on the following argument. After excitation, there is a small but finite chance that a Green plant and cyanobacteria antennas triplet is formed selectively on one of the Chl as because of LHCII the assumed fast Chl b to Chl a energy transfer. As one role LHCII is the major light-harvesting pigment-protein of of the carotenoids is to quench these Chl a triplets with higher plants and algae and is responsible for the binding high efficiency to prevent the formation of harmful oxygen of-50% of all Chl on earth. It serves to feed excitation radicals, the Chl as must be positioned in van der Waals energy into the minor light-harvesting complexes, CP29, contact with the luteins. The seven Chls in the core of CP26, CP24, and into the core of Photosystem 2 (PS2) LHCII, which all make close contact with the luteins, have which eventually is used for charge separation. LHC2 been therefore assigned to the seven Chl as: the remaining is a member of a family of light-harvesting complexes Chls to Chl b. In view of recent reconstitution experiments which includes the various forms of LHCII and the minor with LHCII, it may be possible that some of the binding light-harvesting complexes. The basic unit of all these sites are promiscuous and can be occupied by either a Chl complexes is a membrane protein of-25 kDa, which is a or a Chl b. LHCII exhibits intense CD spectra, indicative known to fold into a structure with three transmembrane of Chl a-Chl a and Chl b--Chl b cxcitonic interactions; the helices: A, B and C. interaction between Chl as and Chl bs is most probably weak. In LHCII, the monomeric subunit binds 7-8 Chl a, 5-6 Chl b, 2 luteins, 1 neoxanthin and substoichiometric A variety of picosecond and femtosecond studies have amounts of violaxanthin. In its native form LHCII is a been performed to explore the dynamics of energy transfer trimer, and in 1994 the structure of the trimer of LHCIIb within LHCII. In a pioneering fluorescence upconversion was resolved to a resolution of 3-4a by K0hlbrandt study by Eads et al. [66], the dominant time constant for and coworkers [3] using cryoelectron microscopy. At the energy was estimated to be 0.5+0.2 ps. In a low-intensity

Figure 2

B875

3 ps 100 fs B850

0.7 ps

B800

8o fs LH2 LH1 ~c 1997 Current Opinion in Structural Biology

A summary of the timescales for energy transfer in purple bacteria. Note the slow, final step (35 ps) from LH1 to the special pair in the RC. Not shown are carotenoid to BOhl transfer times which are of the order of 100fs in LH2. The B875 and B850 molecules are shown as dimers (ovals), whereas the B800 molecules are shown as monomers (diamonds). Femtosecond spectroscopy of photosynthetic light-harvesting systems Fleming and van Grondelle 743

pump-probe study, a slower Chl b--+Chl a energy transfer PS1 time in the range of a few picoseconds was obtained in The core of PS1 is the most complex photosynthetic addition to the ultrafast process [67]. Transient absorption light-harvesting plus electron transfer system for which with shorter pulses [68] revealed Chl b--+Chl a energy a structure is now available [7"']. The functional unit transfer times of 160fs and also the slow process of of the PSI core of Synechococcus elongatus consists of 11 5+2ps similar to the results of Kwa et al. [67]. In a subunits, including the two major subunits PSaA and fluorescence upconvetsion study by Duet a/. [69], two PSaB, each having a molecular weight of-80kDa with lifetimes in the rise in presumably Chl a fluorescence known sequcnce, and each binding -100 Chl as, 10-25 were detected upon excitation at 650 nm, + 250 fs and 5 ps, carotenoids and three FeS clusters. The structure has been in rather good agreement with the pump-probe results. resolved to 4 ~, and the positions of- 90 Chls have been On the other hand, P~lsson et al. [70] using one-colour determined. As in the case of LHCII, no information pump-probe, detected a major 500 fs and a minor 2-3 ps about the direction of the Qv and Qx transition dipoles Chl b-+Chl a transfer time, and, despite the superior is available. The core of P~;1 is characterized by 22 time resolution, they could not distinguish any transfer transmembrane helices, 11 for each large subunit, which component faster than 500 fs. More recently, Visser et al. exhibit C2 symmetry around an axis that passes through [71"], and later Connelly et al. [72 °] resolved all three the centrally located special pair of the electron transfer phases in the Chl b--+Chl a energy transfer: 180fs, 600fs chain, P700, and the FeS cluster E The electron transfer and -5ps, with a relative anaplitude ratio of 40%, 40% chain is embedded in a structure of ten transmembrane and 20%, respectivel'~: A study on LHCII monomers helices, five from each subunit, the arrangement of which demonstrated that all three decay times are associated with is strongly reminiscent of that of the L and M subunits in Chl b--+Chl a energy transfer within a monomeric unit of the purple bacterial RC. All the other 90 antenna Chl as LHCII (FJ Kleima et al., unpublished data). According to are dispersed in a band around this core and, for a large Visser eta/. [7l*'], in the trimer all the energy transfer part, are associated with the remaining six transmembrane occurred to the major red absorbing species at 676nm. helices on each of the large subunits. For all the Chls Connelly eta/. [72"] concluded from their data, which was except two, the distance to any of the pigments in the obtained with an excellent signal-to-noise ratio, that the electron transfer chain exceeds 1.6nm, making energy 175 fs component probably partly reflected energy transfer transfer slow (10-20 ps). Two chlorophylls are found that between 'blue' and 'red' Chl bs [73]. Measurements of seem to connect the antenna with the second and third singlet-singlet and singlet-triplet annihilation suggest that pair of Chls of the electron transfer system, and it has been intermonomer energy transfer occurs on a timescale of suggested that these form a special entry for excitation 10-20 ps [68,71"]. energy. A remarkable sequence analogy exists between the antenna part of the large PSI subunits and the core proteins of PS2, CP47 and CP43, and for that reason it has "Very recently, Gradinaru et a/. (unpublished data) have been suggested that the pigment-protein arrangement of studied the Chl b--+Chl a transfer in one of the minor the six outer transmembrane helices and their associated light-harvesting complexes, CP29. In CP29, six of the Chls may be a good model for the PS2 core. Chl a and two of the Chl b binding sites are conserved [74"], suggesting a pigment stoichiometry of six Chl a:two Chl b : one lutein : one neoxanthin : one violaxanthin. The Trapping in PSI is fast (20-25 ps), and charge separation kinetics in CP29 contain many components similar to is essentially irreversible [30,77,78]. Using ultrafast fluo- those in LHCII and most probably reflect the same rescence depolarization, Du et al. [79] estimated that the energy transfer processes. Specifically; Gradinaru eta/. major hopping process within PS1 occurs on a timescale (unpublished data) could assign a slow, 2-3ps energy of 100fs. On a timescale of a few picoseconds, the transfer phase to a Chl b absorbing at 650 nm -- most excitation energy is seen to equilibrate between a pool probably Chl b5 in the LHCII assignment--and a fast, of very red pigments, absorbing at -720-730nm, and 0.2-0.3 ps energy transfer phase to a Chl b3. the major PSI core pigments. The process of energy transfer to P700 must occur at the same rate as this Carotenoid to Chl energy transfer in LHCII is highly equilibration between core and red pigments as, even at efficient. Recently, two conflicting reports appeared on the very low temperatures where escape from the red states dynamics and pathway of carotenoid to Chl a transfer. is impossible, a reasonably high quantum yield for charge Peterman et al. [75 *°] argued that no direct carotenoid to separation is still observed upon excitation of the core Chl b transfer occurred, while carotenoid to Chl a energy pigments [80,81"]. This has led to a model in which transfer took place in -220 fs. In contrast, Connelly et al. essentially all sites within the PSI core are more or less [76"'] claimed that the carotenoids exclusively transferred equally efficient in transferring their energy to P700 (or energy to Chl b, followed by Chl b--+Chl a energy transfer. any other pigment of the electron transfer chain) and The latter would be inconsistent with the assignment by which may be viewed as the 3D version of the 2D ring to Ktihlbrandt and coworkers [3], where only close contacts special pair energy transfer model that seems to operate for between Chl as and carotenoids exist. purple bacteria [82]. In our view, it is highly unlikely that 744 Biophysical methods

the two Chls that were proposed to act as a special entry 2. Duysens LMN: Photosynthesis. ProgrBiophys 1964, 14:1-104. for excitation energy indeed have that role. They have 3. KOhlbrandt W, Wang DN, Fujiyoshi Y: Atomic model of plant more rapid energy transfer to the pigments in the electron light-harvesting complex by electron crystallography. Nature transfer chain but are simply outnumbered by all the other 1994, 367:614-621. Chls. A simple simulation of the trapping kinetics in PSI 4. McDermott G, Prince SM, Freer AA, Hawthornthwaite-Lawless AM, Papiz MZ, Cogdell PJ, Isaacs NW: Crystal structure of shows that leaving out the pair of connecting Chls hardly an integral membrane light- harvesting complex from changes the trapping time. photosynthetic bacteria. Nature 1995, 374:51 ?-521. 5. Freer AA, Prince S, Sauer K, Papiz MZ, Hawthornthwaite- The process of energy migration and charge separation o, Lawless AM, McDermott G, Cogdell RJ, Isaacs NW: Pigment-pigment interaction and energy transfer in the cannot be experimentally separated in PSI. Kumazaki et antenna complex of the photosynthetic bacterium Rps. al. [83•], Trinkunas and Holzwarth [84 °] and White et al. acidophile. Structure 1996, 4:449-462. [85 •°] have used modeling to extract the intrinsic electron This paper discusses the pathways of excitation energy transfer in the LH2 peripheral antenna complex of Rps. acidophi/a, in the light of the recently transfer rate. In a very recent study using a PSI mutant, obtained high-resolution structure [4]. The FSrster dipole-dipole resonance which seemed to affect the special pair P700 but not the coupling is concluded to dominate the energy transfer from the B800 to the B850 ring. Within the B850 ring, strong interactions exist between antenna spectra or dynamics, it was observed that the nearest neighbour BChls partly because of a close to optimal alignment of excited state lifetime approximately doubled [86"]. This their transition moments, suggesting that delocalized excitonic states play a role in the energy transfer. The orientations and distances of the rhodopin was taken by Melkozernov et al. [86 °'] as evidence for a molecules, the BS00 and B850 BChls, suggest that singlet-singlet energy model in which the charge separation rate by P700 is the transfer from carotenoid to BChl involves mainly transfer to B850 and occurs predominantly from the S 2 state of the carotenoid to the Qx state of the BChl. rate limiting step, in contrast to the 'transfer-to-the-trap' limited model discussed above. 6. Koepke J, Hu X, Muenke C, Schulten K, Michel H: The crystal • . structure of the light-harvesting complex II (B800-850) from Rhodospirillum molischienum. Structure 1996, 4:581-597. Conclusions The crystal structure of LH2 of Rs. mo/ischianum is obtained via a molecular replacement method at a resolution of 2.4]k. It is an (c(~)8 complex with The combination of high-resolution structural data and 16 B850 and 8 B800 BChls in an eightfold symmetric ring. The 16 B850 uhrafast spectroscopy has enabled the development of a BChls, sandwiched between the two polypeptide rings, are in a ring with a radius of 2.3 nm. The BSO0 BChls are situated between the 13-polypeptides fairly complete picture of the light-harvesting process in in a ring with a diameter of 2.88 nm. The B6OOs are bound to Asp6 of the purple bacteria. The efficiency of the overall process is c(-polypeptide. The eight lycopenes span the membrane and are held in place by aromatic sidechains. The B800 chlorin planes are rotated by - 90" relative based on individual energy transfer steps of 80-100fs. to their position in LH2 of Rps. acidophila and are very much tilted away In LH1, the core antenna surrounding the Re, several from the plane of the membrane. Nevertheless, in this structure the B800 Q./ hundred energy transfer steps occur before the final transition dipoles are more or less parallel to the B850 Qy transition dipoles. transfer to the special pair (35ps) and the initiation of 7. Krauss N, Schubert W-D, Klukas O, Fromme P, Witt HT, • . Saenger W: Photosystem I at 4A resolution represents the first charge separation. The observation of a sub 100fs energy structural model of a joint photosynthetic reaction centre and transfer, along with the retention of coherence and the core antenna system. Nat Struct Bio/1996, 3:965-973. The structure of PS1 from Synechococcus e/ongatus is determined to 4 A enhanced radiative rates in LH1 and LH2, raises many resolution using X-ray crystallographic methods. The arrangement of the 22 challenging issues which will provide stimulus for theory transmembrane and 4 surface helices displays a twofold symmetry for the and experiment for years to come. Despite the high large subunits PsaA and PsaB. The central part of the structure, which is proposed to carry the electron transfer chain, including PTO0, the acceptor symmetry and potential for strong intermolecular coupling, A 0, and the three FeS clusters, shows a striking resemblance to the LM core it does not appear that extensive electronic delocalization of the bacterial reaction center. The 90 densely packed antenna Chls form an oval clustered net, relatively distant from the heart of the complex and only is necessary for achieving the near unit efficiency of the continuous with the electron transfer chain via the second and third Chl pairs light-harvesting process. of the electron transfer system. This suggests a dual role for these Chl as both in excitation transfer and electron transfer. In green plant and cyanobacterial antennas, the structural 8. Fenna RE, Matthews BW: Chlorophyll arrangement in a bacteriochlorophyll protein from Chlorobium limicola. Nature information is not yet sufficient for the most detailed 1975, 258:573-577. molecular modeling of energy migration. Enough is 9. Brejc K, Ficner R, Huber R, Steinbacher S: Isolation, known, however, to reveal both striking similarities and crystallization, crystal structure analysis and refinement of differences with the purple bacteria. In particular, antenna allophycocyanin from the cyanobacterium Spirulina platensis at 2.3 A resolution. J Mol Bio/1995, 249:424-440. molecules are held away from close contact with the primary electron donor, and efficiency is achieved by using 10. Hoffmann E, Wrench PM, Sharpies FP, Hiller RG, Welte W, • . Diederichs K: Structural basis of light-harvesting by large numbers of antenna molecules with roughly similar carotenoids: peridinin-chlorophyll*protein from Amphinidium transfer rates to perform the final transfer step to the carterae. Science 1996, 272:1 788-1791. The structure of the peridinin-chlorophyll-protein (PCP) is solved to a resolu- primary donor. tion of 2.0/~ using X-ray diffraction. PCP is a water-soluble light-harvesting complex, which has a blue-green absorbing carotenoid as its major pigment and which is present in most photosynthetic dinoflagellates. The fold of References and recommended reading the N-terminal and C-terminal domains of each polypeptide is related by Papers of particular interest, published within the annual period of review, a twofold symmetry axis, and it surrounds a hydrophobic cavity filled with have been highlighted as: two lipid, eight peridinin and two Chl a molecules. The structural basis for efficient energy transfer from peridinin to Chl is found in the clustering of • of special interest peridinins at van der Waals distances around the Chls. • • of outstanding interest 11. Karrasch S, Bullough PA, Ghosh R: The 8.5/~ projection map of the light-harvesting complex I from Rhodospirillum rubrum Duysens LMN: Transfer of excitation energy in photosynthesis reveals a ring composed of 16 subunits. EMBO J 1995, [PhD Thesis]. Utrecht, The Netherlands: Utrecht University; 1952. 14:631-638. Femtosecond spectroscopy of photosynthetic light-harvesting systems Fleming and van Grondelle 745

12. Papiz MZ, Prince SM, Hawthornthwaite-Lawless AM, antenna complex of Rhodopseudomonas ecidophila (strain • McDermott G, Freer AA, Isaacs NW, Cogdell PJ: A model for the 10050), J Phys Chem 1996, 100:12022-12033. photosynthetic apparatus of purple bacteria. Trends P/ant Sci The transfer of excitation energy in the LH2 complex of Rps. aci- 1996, 1:198-206. dophila is studied using holeburning and femtosecond laser spectroscopy. A model is produced for the whole photosynthetic unit of purple bacteria BS00~B850 energy transfer is observed to occur with monophasic kinet- based on the crystal structure of the LH2 peripheral light-harvesting com- ics with a time constant ranging from 1.6 ps (lgK) to 1.1 ps (130K), while plex of Rps. acidophila. To model the (x16~1s projection map of Karrasch holeburning at 4.2K yields 1.8 ps. Holeburning with applied pressure shows et aL [11 ] the (x9139 structure of LH2 is increased to produce the larger ring. no effect on the energy transfer rate. Together with the weak temperature The known structure of the reaction center is found to fit in this LH1 ring. dependence, this suggests that the B800 emission overlaps with a weak Precisely six LH2s can be fitted in a circle around this LH1 core, thereby vibronic band of B850. Time-domain and holebuming spectroscopy show reproducing the typical stoichiometry of LH1 to LH2. that there is an additional relaxation channel for B800 excitations when the excitation is to the blue of the B800 band. Two possible processes, 13. Visschers RW, Chang MC, van Mourik F, Parkes-Loach PS, intra-B800 transfer and coupling with the quasi degenerate upper exciton Heller BA, Loach PA, van Grondelle R: Fluorescence polarization manifold of B850, are discussed. and low-temperature absorption spectroscopy of a subunit form of light-harvesting complex I from purple photosynthetic 23. Wu HM, Reddy NRS, Cogdell RJ, Muenke C, Michel H, Small G J: bacteria. Biochemistry 1991, 30:5734-5?42. • A comparison of the LH2 antenna complex of three purple bacteria by hole-burning and absorption spectroscopies. 14. Fowler GJS, Visschers RW, Grief GG, van Grondelle R, Mo/ 1996, 291:163-173. Hunter CN: Genetically modified photosynthetic antenna Cryst Liq Cryst The LH2 complexes of Rps. acidophila, Rb. sphaeroides and Rs. molischi- complexes with blue-shifted absorbance bands. Nature 1992, 355:848-850. anum are investigated using holeburning. B800--)B850 energy transfer at 4.2K takes 1.9 ps, very similar to the time constant observed for the same 15. Fowler GJS, Sockalingum GD, Robert B, Hunter CN: Blue shifts process in the other species. The absorption spectra of Rs. molischianum in becteriochlorophyll ebsorbance correlate with changed and Rps. acidophila undergo a dramatic redshift and thermal narrowing upon hydrogen bonding patterns in light-harvesting LH2 mutants cooling from room temperature to 4.2K; for LH2 of Rb. sphaeroides, these of Rhodobacter spheeroides with alterations at c(Tyr44 and 45. effects are much smaller. This is interpreted as a smaller excitonic coupling Biochem J 1 g94, 299:695-700. in the latter species. 16. Beekman LMP, Steffen M, Stokkum IHM, van Olsen JD, Hunter 24. Van Mourik F, Visschers RW, van Grondelle R: Energy transfer • CN, Boxer SG, van Grondelle R: Characterization of the light and aggregate size effects in the inhomogeneously broadened harvesting antennas of photosynthetic purple bacteria by Stark core light-harvesting complex of Rhodobacter sphaeroides. spectroscopy. 1. LH1 antenna complex and the B820 subunit Chem Phys Lett 1992, 193:1-7. from Rhodospiri/lum rubrum. J Phys Chem 1997, in press. The response of the optical absorption spectrum to an externally applied 25. Monshouwer RM, Visschers RW, van Mourik F, Freiberg A, electrical field (Stark effect) is measured for LH1, the B820 dimeric subunit van Grondelle R: Low-temperature absorption and site- of LH1, and the reconstituted LH1 of purple photosynthetic bacteria. The selected fluorescence of the light-harvesting antenna of Stark effect for LH1 is strongly reminiscent of that measured for the special Rhodopseudomonas v/r/dis. Evidence for heterogeneity. pair of the purple bacterial reaction center, it is dominated by a large change Biochim Biophys Acta 1995, 1229:373-380. in polarizability between ground and excited states-most probably due to the mixing of charge transfer states into the lower excited states of a dimeric 26. Alden RG, Johnson E, Nagarajan V, Parson WW, Law C J, subunit of LH1. • o Cogdell RJ: Calculations of spectroscopic properties of the LH2 bacteriochlorophyll-protein antenna complex from 17. Sauer K, Cogdell RJ, Prince, SM, Freer A, Isaacs NW, Scheer H: Rhodopseudomonas acidophila. J Phys Chern B 199'7, °• Structure-based calculations of the optical spectra of the LH2 101:4667-4680. bacteriochlorophyll-protein complex from Rhodopseudomonas This paper describes the calculation of absorption and CD spectra of a acidophila. Photochem Photob~o11996, 64:564-576. photosynthetic bacterial antenna complex based on the crystal structure of The molecular structure of the LH2 complex of Rps. acidophila is used the LH2 complex from Rps. acidophila. Molecular orbitals for the three differ- to provide orientations and distances for the 27 BChls in the complex as ent BChl structures in the complex are obtained by semiempirical quantum the input parameters for an excitonic hamiltonian. Assuming dipole-dipole mechanical calculations. Exciton and charge transfer interactions are intro- interactions among all the chromophores, the ring of 18 closely coupled duced at the level of configuration interactions. Absorption bandshapes are ¢hromophores is assigned to B850, whereas the parallel ring of nine weakly treated with vibronic parameters as obtained from holeburning experiments, ¢oup4ed BChls is assigned to B800. The pairwise excitonic interactio,1 be- whereas inhomogeneous broadening is included by a Monte Carlo method. tweetn the B850s is estimated to be -250-300 cm -1. The general trends Calculations reproduce the measured absorption and CD spectra. The re- obaervable in the CD spectrum of LH2 are tentatively explained. The cal- suits support the idea that excitations are rather delocalized in LH2. culations predict strong CD features at -790 nm which await experimental verification. 27. Koolhaas MHC, van der Zwan G, Frese RN, van Grondelle R: • - The red shift of the zero-crossing in the CD spectra of the 18. Sturgis JN, Robert B: The role of chromophore coupling in LH2 antenna complex of Rhodopseudomonas acidophila: a • tuning the spectral properties of the peripheral light-harvesting structure based study. J Phys Chem 1997, in press. protein of purple bacteria, Photosynth Res 1996, 50:5-10. The published crystal structure of LH2 of Rps. acidophi/a is used to calculate The authors calculate the excitonic contribution to the absorption spectrum absorption and CD spectra of the complex. It is shown that the relative for LH2 of photosynthetic purple bacteria and conclude that this contribution position of the CD zero crossing with respect to the absorption maximum is not very sensitive to small variations in the LH2 structure. For that reason, is an important parameter that is rather sensitive to structural changes. It they propose that in LH2 of Rps. acidophi/a the redshift to 850 nm originates is demonstrated that the experimentally observed CD spectrum can only roughly equally from pigment-pigment and pigment-protein interactions. In be explained if the whole ring is considered and if the o(-polypeptide and BS00-B820, a related but slightly different LH2, the redshift is largely due I~-polypeptide BChls are allowed to have different excitation energies. to pigment-pigment interaction. As a consequence, the total amount of excitonic interaction between neighbouring pigments can not be much larger 28. Leupold D, Stiel H, Teuchner K, Nowak F, Sandner W, 0cker B, than 200 cm -1. °° Scheer H: Size enhancement of transition dipoles to one and two-exciton bands in a photosynthetic antenna. Phys Rev Lett 19. Kramer HJM, van Grondelle R, Hunter CN, Westerhuis WHJ, 1996, 77:4675-46"78. Amesz J: Pigment organization of the B800-850 antenna From a measurement of the nonlinear absorption, the differential absorp- complex of Rhodopseudomonas sphaeroides. Biochim Biophys tion density spectrum, and the fluorescence for LH2 from Rb. sphaeroides, Acta 1984, 765:156-165. the dipole moments associated with the ground state-->one-exciton and 20. De Caro C, Visschers RW, van Grondelle R, VSIker S: Inter- and one-exciton-->two-exciton transition are estimated to be 25.5 D and 21.5 D, intraband energy transfer in LH2-antenna complexes of purple respectively. These values are seen as an indication that in LH2 the exciton bacteria. A fluorescence line-narrowing and hole-burning is delocalized over 16 +4 BChl molecules, corresponding to the full physical study. J Phys Chem 1994, 98:10584-10590. length of the circular aggregate. 21. Reddy NRS, Picorel R, Small G J: B896 and B870 components 29. Novoderezhkin VI, Razjivin AP: Exciton dynamics in circular of the Rhodobacter sphaeroides antenna: a hole-burning study. aggregates: application to antenna of photosynthetic purple J Phys Chem 1992, 96:6458-6464. bacteria. Biophys J 1995, 68:1089-1100. 22. Wu HM, Savikhin S, Reddy NRS. Jankowiak R, Cogdell RJ, 30. van Grondelle R, Dekker JP, Gillbro T, SundstrSm V: Energy • • Struve WS, Small G J: Femtosecond and hole-burning studies transfer and trapping in photosynthesis. Biochim Biophys Acta of B8OO's excitation energy relaxation dynamics in the LH2 ~994, 1187:1-65. 746 Biophysical methods

31. van Grondelle R, Kramer HJM, Rijgersberg CP: Energy transfer 43. Jiminez R, Dikshit SN, Bradforth SE, Fleming GR: Electronic in the B800-850-carotenoid light-harvesting complex of • . excitation transfer in the LH2 complex of Rhodobactar various mutants of Rhodopseudomonas sphaeroides and of sphaeroides. J Phys Chem 1996, 100:6825-6834. Rhodopseudomonas capsulatus. Biochim Biophys Acta 1982, Fluorescence upconversion experiments on LH-2 yield two time constants in 682:208-215. the anisotropy decay: 50-90fs and 400-500fs. 6800-->B850 transfer occurs within 650fs. Depolarization is modeled using a model with inhomoge- 32. Trautman JK, Shreve AP, Violette CA, Frank HA, Owens TG, neous broadening (250 cm -1) and a dimer-to-dimer hopping time of 100fs. Albrecht AC: Femtesecond dynamics of energy transfer A calculation of the spectrum using an intradimer coupling of 230cm -1, ~and in B800-850 light-harvesting complexes of Rhodobacter coupling between adjacent chromophores on different subunits of 110 cm -1 sphaeroides. Proc Nat/Acad Sci USA 1990, 87:215-219. yields an average deloealization length of - 5 molecules in the middle of the band. 33. Laan H, Schmidt Th, Visschers RW, Visscher KJ, van Grondelle R, VSIker S: Energy transfer in the B800-850 antenna complex 44. Visser HM, Somsen OJG, van Mourik F, Lin S, van Stokkum IHM, of the purple bacterium Rhodobacter sphaeroides: a study by van Grondelle R: Direct observation of sub-picosecond spectral hole-burning. Chem Phys Lett 1990, 170:231-238. equilibration of excitation energy in the light-harvesting 34. Monshouwer R, Ortiz de Zarate I, van Mourik F, van Grondelle R: antenna of Rhodospirillum rubrum. Biophys J 1995, 69:1083- Low-intensity pump-probe spectroscopy on the B800 to B850 1099. transfer in the light harvesting 2 complex of Rhodobacter sphaeroides. Chem Phys Lett 1995, 246:341-346. 45. Visser HM, Somsen OJG, van Mourik F, van Grondelle R: Excited- ,, state energy equilibration via sub-picosecond energy transfer 35. Hess S, ~kesson E, Cogdell RJ, Pullerits T, Sundstr6m V: Energy within the inhomogeneously broadened light-harvesting transfer in spectrally inhomogeneous light-harvesting pigment- antenna of the LH-1 only Rhodobacter sphaeroides mutant protein complexes of purple bacteria. Biophys J 1995, 69:2211- M2192 at room temperature and 4.2K. J Phys Chem 1996, 2225. 100:18859-18867. The spectral evolution following an ultrafast laser flash is studied for the core 36. Joe 1", Jia Y, Yu, J-Y, Jonas DM, Fleming GR: Dynamics in isolated antenna of photosynthetic purple bacteria. The authors conclude that ultra- • • bacterial light harvesting antenna (LH2) of Rhodobacter fast energy transfer occurs, resulting in a net shift of the excitation distribution sphaeroides at room temperature. J Phys Chem 1996, to the low-energy pigments. At room and low temperature, the major part of 100:2399-2409. the spectral shift takes less than a picosecond. The energy transfer dynamics Transient absorption, transient grating and photon echoes are measured on at room temperature are fitted assuming a hopping time of - 100 fs between the B800 band of LH2 of Rb. sphaeroides using 30fs pulses. B800--~B850 dimers on a ring and an inhomogeneous width of 400cm -1. At 4K hopping energy transfer occurs in 800fs. The three-pulse photon echo peak shift is somewhat slower, -0.4 ps, and the inhomogeneous width has decreased experiment identifies important contributions to the 6800 lineshape and to - 200cm -1. thereby the dynamics of the system involved: low-frequency intramotecular vibrations; ultrafast bath (solvent plus protein) responses; and static inho- 46. Chachisvilis M, Pullerits T, Jones MR, Hunter CN, Sundstr~m V: mogeneity on a timescale longer than the energy transfer time. The transient Vibrational dynamics in the light harvesting complexes of the absorption is observed to decay nonexponentially. The authors argue that photosynthetic bacterium Rhodobacter sphaeroides. Chem the fast phase is vibrational relaxation within the B800 band. Phys Lett 1994, 224:345-351. 3?. Ma Y-Z, Cogdell RJ, Gillbro T: Energy transfer and exciton • annihilation in the B800-850 antenna complex of the 47. Jean JM, Fleming GR: Competition between energy and phase photosynthetic bacterium Rhodopseudomonas ecidophila relaxation in electronic curve crossing processes. J Chem Phys (strain 10050). A transient femtosecond absorption study. 1995, 103:2092-2101. J Phys Chem B 1997, 101:1087-1095. This paper describes femtosecond pump-probe experiments on LH2 of 48. Pullerits 1", Chaehisvilis M, Sundstr6m V: Exciton delocalization Rps. acidophi/a. At room temperature, B800--~B850 energy transfer is • length in the B850 antenna of Rhodobacter sphaeroides. -0.8 ps; at 77K, -1.3ps. Anisotropy kinetics measured within 6800 band J Phys Chem 1996, 100:10787-10792. indicate that relaxation within the B800 band is wavelength dependent. A Excitation transfer dynamics in LH2 of Rb. sphaeroides is investigated using fast depolarization time of 210fs observed at 77K is thought to originate transient absorption spectroscopy. In LH2, the anisotropy decays in 130 fs, from exciton relaxation. From a dramatic energy dependence of the B800 whereas the isotropic decay occurs in ?0fs. For a ninefeld symmetric ring, kinetics, it is speculated that several high-lying excitonic states of B850 exist with the orientation of the transition dipoles as in LH2, a factor of 3 between that show good spectral overlap with the B800 band and thus could serve the two time constants is expected. As this is not observed, the authors con- as excellent accepters for the energy transfer from B800 to B850. clude that the energy migration is (partially) coherent. From an analysis of the transient absorption difference spectrum, they conctude that the excitation 38. Monshouwer R, van Grondelle R: Excitations and excitons in is delocalized over 4_+2 monomers. • • bacterial light-harvesting complexes. Biochim Biophys Acta 1996, 1275:70-75 49. Chachisvilis M, K(Jhn O, Pullerits T, Sundstr6m V: Excitons in The localization versus delocalization of excitations in bacterial light- • photosynthetic purple bacteria. Wavelike motion or incoherent harvesting complexes is discussed. It is argued that a 'ring-of-dimers' model hopping? J Phys Chem 1997, in press. is adequate to explain most of the spectroscopic and time-resolved data. From a comparison of isotropic and anisotropic pump probe signals meas- Furthermore, two-colour pump-probe experiments in the B800 band of Rb. ured for LH1 and LH2 of Rb. sphaeroides, the anisotropy decay is observed sphaeroides reveal 'blue-to-red' energy transfer on a timescale of 400fs to be always about twofold slower. Modeling using a hopping model predicts within the B800 band. a much more dramatic difference between isotropic and anisotropy decays. As a consequence, the authors believe that the excitation transfer is partly 39. Bakker JGC, van Grondelle R, den Hollander WTF: Trapping, loss coherent. From a fit of the pump-probe spectrum, they conclude that the and annihilation of excitations in a photosynthetic system. II. delocalization length is -4 BChls and is not stongly dependent on temper- Experiments with the purple bacteria Rhodospirillum rubrum ature, and Rhodopseudomonas capsulatus. Biochim Biophys Acta 1 g83, 725:508-518. 50. Monshouwer R, Abrahamsson M, van Mourik F, van Grondelle R: • • Superradiance and exciton delocalisation in bacterial 40. Sundstr6m V, van Grondelle R, BergstrSm H, ,~kesson E, Gillbro T: photosynthetic light-harvesting systems. J Phys Chem 1997, Excitation-energy transport in the bacteriochlorophyll antenna in press. systems of The radiative rate of LH1 and LH2 of Rb. sphaeroides are measured as a Rhodospirillum rubrum and Rhodobacter sphaeroides studied function of temperature. For LH2, the radiative rate is about threefold larger by low-intensity picosecond absorption spectroscopy. Biochim compared with that of monomeric BChl a and is independent of tempera- Biophys Acta 1986, 851:431-446. ture. LH1 is very similar to LH2 at room temperature, but the radiative rate 41. van Grondelle R, Bergstr6m H, SundstrSm V, Gillbro T: Energy increases about 2.4-fold upon lowering the temperature to 4K. The results transfer within the bacteriochlorophyll antenna of purple are interpreted in terms of a model that includes both the coupling between bacteria at 77K studied by picosecond absorption recovery. all the pigments and the inhomogeneous broadening and suggests that the Biochim Biophys Acta 1987, 894:313-326. ratio between coupling and inhomogeneous broadening is -2. The results suggest that in LH2 the excitation is rather localized at all temperatures. The 42. Bradforth SE, Jiminez R, van Mourik F, van Grondelle R, Fleming degree of delocalization in LH1 may be somewhat larger, certainly at low GR: Excitation transfer in the core light-harvesting complex temperature. (LH-1) of Rhodobacter sphaeroides: an ultrafast fluorescence depolarization and annihilation study. J Phys Chem 1995, 51. Nagarajan V, Alden RG, Williams JC, Parson WW: Ultrafast 99:16179-16191. • exciton relaxation in the B850 antenna complex of Femtosecond spectroscopy of photosynthetic light-harvesting systems Fleming and van Grondelle 747

Rhodobacter sphaeroides. Proc Nat/Acad Sci USA 1996, light-harvesting antenna complexes from the purple bacterium 93:13774-13779. Chromatium purpuratum. Chem Phys 1996, 210:195-217. The femtosecond response of LH2 of Rb. sphaeroides at room temperature Energy transfer from the carotenoid okenone to BChl a in the light-harvesting is measured. The authors observe a 35fs relaxation phase in absorption and complex B800-830 and in chromatophores of Chromatium purpuratum was emission spectra of the excited state, and a 20fs anisotropy decay. They studied by steady state fluorescence and femtosecond transient absorp- ascribe these dynamics to interlevel relaxation and dephasing, respectively, tion spectroscopy. The overall efficiency for energy transfer from okenone of extensively delocalized exciton states of the circular bacteriochlorophyll to BChl a is 95+5%. There is a fast (<200fs) transfer from at least one aggregate. carotenoid to B830, and this occurs from the S 2 state of the carotenoid to the ex transition dipole of BChl a, probably employing the F~rster mecha- 52. Leegwater JA: Coherent versus incoherent energy transfer and nism. On a longer timescale, the okenone S 1 state transfers energy to B830 • trapping in photosynthetic antenna complexes. J Phys Chem in 3.8 ps, whereas a second carotenoid transfers its energy via the S t state 1996, 100:14403-14409. to B800 in - 0.5 ps. A model is described which has an arbitrary ratio of homogeneous broadening versus interaction energy. This allows the study of the crossover from 60. Freiberg A, Godik VI, Pullerits T, Timpmann K: Directed hopping dynamics to exciton dynamics. For the survival time, the hopping picosecond excitation transport in purple photosynthetic approach is shown to yield a surprisingly accurate estimate, even when the bacteria. Chem Phys 1988, 128:227-235. dynamics is excitonic. For LH1, it is estimated that the excitation is, on the average, delocalized over two dimers. The excitation is localized by phonons. 61. Zhang FG, van Grondelle R, Sundstr6m V: Pathways of energy flow through the light-harvesting antenna of the 53. Meier T, Chernyak V, Mukamel S: Multiple exciton coherence photosynthetic purple bacterium Rhodobacter spheeroides. • . sizes in photosynthetic antenna complexes viewed by Biophys J 1992, 61:911-920. pump-probe spectroscopy. J Phys Chem 199"7, in press. From an analysis of the pump-probe signal from the LH2 light-harvesting 62. Hess S, Chachisvilis M, Timpmann K, Jones MR, Fowler GJC, antenna of purple bacteria, the localization size is determined to be 15 at Hunter CN, Sundstr6m V: Temporally and spectrally resolved 4.2K. The analysis of the difference in frequency between positive and neg- subpicosecond energy transfer within LH2 and from LH2 to ative peaks in the pump-probe spectrum yields an estimate for the exciton LH1 in photosynthetic purple bacteria. Proc Nat/Acad Sci USA mean free path (or the exciton dephasing lengthscale) of - 11. 1995, 92:12333-1233'7. 54. Jimenez R, van Mourik F, Fleming GR: Three pulse echo 63. Nagarajan V, Parson WW: Excitation energy transfer between • . peak shift measurements on LH1 and LH2 complexes of • the B850 and B875 antenna complexes of Rhodobacter Rhodobacter sphaeroides: a nonlinear spectroscopic probe of sphaeroides. Biochemistry 199'7, 36:2300-2306. energy transfer. J Phys Chem 1997, in press. In membrane of Rb. sphaeroides, energy transfer from B850 (LH2) to B875 Three pulse photon echo peak shift measurements are performed on the (LH1) proceeds with two time constants, 4.6 ps and 26.3 ps, but a significant B875 and B850 bands of detergent-isolated LH1 and LH2 complexes at fraction of the excitations remain in B850 for considerably longer times. The room temperature. The peak shifts are rnuch larger and decay much faster fast step is ascribed to hopping from LH2 to LH1, the slow step to migration than those typically observed for dye molecules in solution. The peak shift within the LH2 pool. Back transfer from LH1 to LH2 could not be detected. decay is simulated on the basis of the optical frequency correlation function, 64. Beekman LMP, van Mourik F, Jones MR, Visser HM, Hunter CN, M(t), which includes contributions from rapid fluctuations of the protein, vi- van Grondelle R: Trapping kinetics in mutants of the brational motion and energy transfer. The 90 fs and 130 fs exponential com- photosynthetic purple bacterium Rhodobacter sphaeroides: ponents in M(t) observed for LH1 and LH2, respectively, are ascribed to influence of the charge separation rate and consequences energy transfer. A simulation based on a model that assumes hopping in a for the rate-limiting step in the light-harvesting process. ring of dimers, with each dimer randomly selected from an inhomogeneous Biochemistry 1994, 33:3143-3147. distribution, explains the results. 65. Pullerits T, Sundstr~m V: Photosynthetic light-harvesting 55. Kumble R, Palese S, Visschers RW, Dutton PL, Hochstrasser RM: o. pigment-proteins: toward understanding how and why. Acc • Ultrafast dynamics within the B820 subunit from the core Chem Res 1996, 29:381-389. (LH-1) antenna complex of Rs. rubrum. Chem Phys Let/1996, The energy transfer and trapping dynamics in photosynthetic purple bacteria 261:396-401. is reviewed: a model is proposed for the LH2-LH1 association based on the This paper describes polarized femtosecond transient absorption experi- measured LH2--~LH1 energy transfer time of 3.3ps at room temperature. ments on B820, the oc~-BChl 2 subunit of LH1, and on its reaggregated form, Modeling this step gives a 3 nm distance of closest approach between the B873. In B820, the timescale of the Stokes' shift is sub 50fs, as reflected LH2 and LH1 rings. by a shift of the nuclear wavepacket and a fast component in the anisotropy decay. In similar experiments on reassociated B873, the anisotropy is ob- 66. Eads DD, Castner EW Jr, Alberte RS, Mets L, Fleming GR: Direct served to decay from 0.24 to 0.07 with two time constants: 70fs and 400 fs. observation of energy transfer in a photosynthetic membrane: The authors suggest that, following fast scattering, the excitation transfer chlorophyll b to chlorophyll a transfer in LHCII. J Phys Chem proceeds between states of dimeric subunits. 1990, 93:8271-8275. 56. Yu J-Y, Nagasawa Y, van Grondelle R, Fleming GR: Three pulse 67. Kwa SLS, van Amerongen H, Lin S, Dekker J P, van Grondelle R, • . echo peak shift measurements on B820 subunit of LH1 of Struve WS: Ultrafast energy transfer in LHC-II trimers from the Rhodospirillum rubrum. Chem Phys Let/1997, in press. Chl a/b light-harvesting antenna of Photosystem II. Biochim This paper describes the measurement cf the three pulse photon echo peak Biophys Acts 1992, 1102:202-212. shift for the LH1 subunit, B820, a protein bound BChl dimer. The major difference between B820 and LH1 is the absence of the 100fs exponential 68. Bittner T, Irrgang KD, Renger G, Wasielewski MR: Ultrafast phase that was ascribed to energy transfer in LH1 and is now present as a excitation energy transfer and exciton-exciton annihilation nondecaying component. The experiment strongly supports the idea that, in processes in isolated light harvesting complexes of LH1, the excitation is also localized on s BChl dimer. Photosystem II (LHCII) from spinach. J Phys Chem 1994, 98:11821-11826. 5?. Shreve AP, Trautman JK, Frank HA, Owens TG, Albrecht AC: Femtosecond energy-transfer processes in the B800-850 69. Du M, Xie X, Mets L, Fleming GR: Direct observation of ultrafast light-harvesting complex of Rhodobacter sphaeroides 2.4.1. energy-transfer processes in light-harvesting complex II. Biochim Biophys Acta 1991, 1058:280-288. J Phys Chem 1994, 98:4736-4741. 58. Ricci M, Bradforth SE, Jiminez R, Fleming GR: Internal 70. P~lsson LO, Spangfort MD, Gulbinas V, Gillbro T: Ultrafast • conversion and energy transfer dynamics of spheroidene in chlorophyll ~-chlorophyll a excitation energy transfer in the solution and in the LH-1 and LH-2 light-harvesting complexes. isolated light harvesting complex, LHCII, of green plants. Chem Phys Lett 1996, 259:381-390. Implications for the organization of chlorophylls. FEBS Let/ The lifetime of the 1Bu+ state of spheroidene is measured in vitro in vari- 1994, 339:134-138. ous polar and nonpolar solvents and in LH1 and LH2 of Rb. sphaeroides. The 1Bu+-->2Ag -internal . conversion time varies from 150fs to 250fs in 71. Visser HM, Kleima FJ, van Stokkum IHM, van Grondelle R, the solvents studied and depends on the polarizability of the surrounding • , van Amerongen H: Probing the many energy-transfer processes environment. Estimated internal conversion time is 150fs within LH2, and in the photosynthetic light-harvesting complex II at 77K 170fs within LH1. The 1Bu + lifetime inside LH1 and LH2 is 60fs and 80fs, using energy-selective sub-picosecond transient absorption respectively, and suggests fast energy transfer from the 1Bu + state. spectroscopy. Chem Phys 1996, 210:297-312. The ultrafast energy transfer dynamics in LHCII is measured using transient 59. Andersson PO, Cogdell ILl, Gillbro T: Femtosecond dynamics absorption spectroscopy. Three phases in the Chl b--~Chl a energy transfer • • of carotenoid-to-bacteriochlorophyll a energy transfer in the are resolved: 200 fs, 600fs and - 5 ps, with relative amplitude ratios of 40%, 748 Biophysical methods

40% and 20%, respectively. In the trimer, all the energy transfer occurs to spectroscopy of the long-wavelength emitting chlorophylls in the major red absorbing species at 676 nm. Measurements of singlet-singlet isolated Photosystem I particles of Synechococcus elongatus. and singlet-triplet annihilation suggest that intermonomer energy transfer Photosynth Res 1996, 48:239-246. occurs on a timescale of 10-20 ps. Isolated trimeric PS1 complexes of Synechococcus e/ongatus have been studied in absorption and polarized fluorescence. Two types of long wave- 72. Connelly JP, M~ller MG, Hucke M, Gatzen G, Mullineaux CW, length pigments are distinguished: C708 and C719. Their contribution to the • Ruban AV, Horton P, Holzwarth AR: Ultrafast spectroscopy of absorption spectrum corresponds to ~ 4-5 C708 and 5-6 C? 19 per PT00. trimeric light-harvesting complex II from higher plants. J Phys From low-temperature energy-selective polarized fluorescence experiments, Chem B 1997, 101:1902-1909. it is concluded that at ultra low temperatures C708 still is able to transfer ex- These authors perform a highly sensitive transient absorption experiment to citation energy to C719 and furthermore that energy transfer among C719s resolve all three phases in the Chl b-->Chl a energy transfer and find time occurs. constants of 175 fs, 600 fs and - 5 ps. Furthermore, they conclude from their data that, probably, the 175fs component partly reflected energy transfer 82. Valkunas L, Liuolia V, Dekker JP, van Grondelle R: Description of between 'blue' and 'red' Chl bs. energy migration and trapping in Photosystem I by a model with two distance scaling parameters. Photosynth Res 1995, 73. Trinkunas G, Connelly JP, Mi.iller MG, Valkunas L, Holzwarth AR: 43:149-154. A model for the excitation dynamics in the light-harvesting complex II from higher plants. J Phys Chern B 1997, in press. 83. Kumazaki S, Ikegami I, Yoshihara K: Excitation and electron 74. Giuffra E, Cugini D, Croce R, Bassi R: Reconstitution and • transfer from selectively excited primary donor chlorophyll (P700) in a Photosystem I reaction center. • pigment-binding properties of recombinant CP29. Eur J J Phys Chem A 1997, 101:597-604. Biochern 1996, 238:112-120. This paper describes the reconstitution of the minor chlorophyll a/b-binding Primary processes in a Photosystem 1 reaction center are studied using sub- picosecond fluorescence upconversion. In these enriched reaction centers protein CP29, overexpressed in Escherichia coil. The recombinant pigment- there are - 14 Chl as per PTO0. The 1 ps fluorescence anisotropy decay fol- protein shows biochemical and spectroscopic properties identical to the native CP29 complex, with a Chl a:Chl b ratio ef three. Also other stoi- lowing selective PT00 excitation is ascribed to equilibration between P?00 and the surrounding antenna Chls. In the isotropic fluorescence decay, at chiometries yielded stable complexes. least two components can be distinguished: 2.2 ps (35%) and 15 ps (55o/o). 75. Peterman EJG, Monshouwer R, van Stokkum IHM, The fast and slow phases are interpreted in terms of charge separation • - van Grondelle R, van Amerongen H: Ultrafast singlet excitation before and after full equilibration of the excited state, respectively. From transfer from carotenoids to chlorophylls via different kinetic modeling, the intrinsic time constant for charge separation from PT00 pathways in light-harvesting complex II of higher plants. Chern is concluded to be < 4 ps. Phys Lett 199"7, 264:279-284. Energy transfer from the xanthophylls to the chlorophylls is studied in trimeric 84. Trinkunas G, Holzwarth AR: Kinetic modeling of exciton LHCII at 77K. No evidence for direct energy transfer from the xanthophylls to • migration in photosynthetic systems. 3. Application of genetic Chl b is found, whereas efficient xanthophyll to Chl a energy transfer occurs algorithms to simulations of excitation dynamics in three- in 220fs. With preferential violaxanthin excitation (514 nrn) relative to lutein dimensional Photosystem I core antenna/reaction center excitation (500 nm), energy transfer to Chl as absorbing at -670 nm is more complexes. Biophys J 1996, 71:351-364. pronounced, compared with transfer to Chl as absorbing at -676 nm. Fol- This paper describes calculations of energy transfer and trapping in Pho- lowing 514 nm excitation, the transfer from 670 to 676 nm occurs in 2.1 ps. tosystem 1 using a genetic algorithm. Various 3D models for the pigment arrangement and the corresponding energy transfer dynamics are tested 76. Connelly JP, MiJIler MG, Bassi R, Croce R, Holzwarth AR: for Photosystem 1. It is concluded that: the red pigments never are close • . Femtosecond transient absorption study of carotenoid to to P700; the red pigments are also never far away from P700 and tend chlorophyll energy transfer in the light-harvesting complex II to cluster; the charge separation time is shorter than 1.2 ps; and the total of Photosystem II. Biochemistry 10gT, 36:281-287. energy transfer time within the main antenna pool is < 1 ps. Energy transfer from the xanthophylls to the chlorophylls is studied in LHCII trimers from Arabidopsis thafiana at room temperature. At 475 and 490 nm 85. White NTH, Beddard GS, Thorne JRG, Feehan TM, Keyes TE, excitation, energy transfer is mainly from xanthophyll to Chl b. o- Heathcote P: Primary charge separation and energy transfer in the Photosystem I reaction center of higher plants. J Phys 7"7. Holzwarth AR, Schatz G, Brock H, Bittersmann E: Energy transfer Chern 1996, 100:12086-12099. and charge separation kinetics in Photosystem I. Part 1: A detailed analysis is given of the Photosystem 1 trapping kinetics in a Picosecond transient absorption and fluorescence study Photosystem 1 core particle from plants. The A0--A 0 difference spectrum of cyanobacterial Photosystem I particles. Biophys J 1993, takes 3 ps to form upon ?08 nm excitation reflecting the equilibration time 64:1813-1822. between the first charge separated state and the antenna excitation. The equilibrated state decays in 20-20 ps. From extensive modeling, the intrinsic 78. Hastings G, Hoshina S, Webber AN, Blankenship RE: Universality rate of electron transfer is concluded to be 0.7 ps-1. of energy and electron transfer processes in Photosystem I. Biochemistry 1995, 34:15512-15522. 86. Melkozernov AN, Su H, Lin $, Bingham S, Webber AN, • ° Blankenship RE: Specific mutation near the primary donor 79. Du M, Xie X, Jia Y, Mets L, Fleming GR: Direct observation of in Photosystem 1 from Chlamydamonas reinhardtii alters ultrafast energy transfer in PS1 core antenna. Chern Phys Lett the trapping time and spectroscopic properties of P7oo- 1993, 201:535-542. Biochemistry 1997, 36:2898-2907 80. Gobets B, van Amerongen H, Monshouwer R, Kruip J, Time-resolved absorption and fluorescence spectroscopy are used to inves- R6gner M, van Grondelle R, Dekker JP: Polarized site-selection tigate the energy and electron transfer processes in the detergent-isolated spectroscopy of isolated Photosystem 1 particles. Biochim Photosystem I core particles from the site directed mutant of Ch/arnyda- Biophys Acta 1994, 1188:75-85. rnonas reinhardtiiwith the His656 of PsaB replaced by asparagine. There is no indication that the mutation affects the spectral distribution in the antenna; 81. P&lsson LO, Dekker JP, Schlodder E, Monshouwer R, however, the excited state lifetime increases from -30 ps to -65 ps. It is • van Gronclelle R: Polarized site-selective fluorescence proposed that the excited state decay is limited by charge separation.