738
Femtosecond spectroscopy of photosynthetic light-harvesting
systems
Graham R Fleming* and Rienk van Grondellet
Observing the elementary steps of light-harvesting in real
time is now possible using femtosecond spectroscopy.
This, combined with new structural data, has allowed a fairly
complete description of light-harvesting in purple bacteria and
substantial insights into higher plant antenna systems.
Addresses
~Department of Chemistry, University of California, Berkeley,
CA 94720-1460, USA; e-mail: fieming@cchem.berkeley.edu
tDepartment of Physics and Astronomy, Vrije Universiteit, De
Boelelaan 1081, NL-1081 HV, Amsterdam, The Netherlands;
e-mail: rienk@nat.vu.nl
peridinin-carotenoid protein of dinoflagellates [10°°] - - a l l
membrane-attached light-harvesting s y s t e m s - - w e now
have a multitude of structures available which exhibit
amazing variation which will allow us to greatly extend our
knowledge of the process of excitation energy transfer and
the underlying physics.
In this review, we describe the considerable recent
progress in understanding the purple bacterial antenna
system and outline the current views on green plant and
cyanobacterial systems, for which the structural data do not
yet allow for fully detailed modeling.
Current Opinion in Structural Biology 1997, 7:738-748
http://biomednet.com/elecref/O959440XO0700738
O Current Biology Ltd ISSN 0959-440X
Abbreviations
3PEPS three-pulse photon echo peak shift
BChl
bacteriochlorophyll
CD
circular dichroism
Chl
chlorophyll
RC
reactioncenter
Introduction
In order to harvest solar light, photosynthetic organisms
are equipped with a light-harvesting antenna system.
Photons absorbed by the antenna pigments are transferred
to the photosynthetic reaction center (Re) with great
speed. Once absorbed by the RC, the excitation energy
is efficiently converted into a stable charge separation.
Since the basic description of energy transfer and trapping
processes by Duysens [1,2] in the early 1950s, it has been
clear that the elementary steps of the light-harvesting
process are extremely rapid. Only recently, however, has it
become possible to make direct experimental observations
on the timescale of individual energy transfer steps; and
currently energy migration can be investigated in the
range of tens of femtoseconds to many nanoseconds.
Application of femtosecond laser spectroscopy has been
greatly stimulated by the remarkable successes in structure determination of several important light-harvesting
complexes in recent years. For example, the peripheral
light-harvesting complex (LHCII) of green plants [3], the
peripheral light-harvesting (LH2) complex of Rhodopseudomonas (Rps.) acidophila [4,5°°], the LH2 of RhodospiHllum
(Rs.) molischianum [6°°], and the core of Photosystem 1
(PSI) of cyanobactcria [7"°] have all been determined. All
these are intrinsic membrane proteins, and, together with
the known structures of the bacteriochlorophyll (BChl) a
protein of green sulphur bacteria [8], the phycobiliproteins [9], and the recently resolved structure of the
Disordered versus ordered light-harvesting
systems
Although the various structures now known exhibit a
wide spread in organizational motifs, one striking aspcct
stands out. Comparing bacterial and plant light-harvesting
systems, the bacterial peripheral, LH2, and core, LH1,
antenna are structures with a v e ~ high degree of symmetry (see Figure 1), whereas L H C I I and even more so
PSI appear spatially (i.e. positionally and orientationally)
much more disordered. One of the major reasons for
this variation is, of course, the size of the elementary
building block. In LH1 and LH2, this is a pair of small
transmembrane polypeptides, ot and 13, which carries two
and three BChls, respectively. Assembly into a larger
system will always lead to a structure with a high degree
of symmetry.
In contrast, the PS1 core consists of a single pair of large
polypeptides, the PsaA and PsaB gene products, which,
together with a large number of smaller subunits, forms
the PSI core that binds - 100 chlorophylls. L H C I I seems
to be an intermediate case. Although monomeric, LHCI1
still appears quite disordered in comparison with the LH1
and LH2 rings; the basic unit of L H C I I is a trinaer of
an - 2 5 kDa protein that exhibits perfect C3 symmetry.
The other apparent difference between plant and bacterial
light-harvesting systems is the pigment density. In LH1 of
purple bacteria, the density is two BChl ot-polypeptides
per 12kDa; in L H C I I of green plants, 12-14 Chls occur
per 2 5 k D a - - a factor of three more. A similar variation
applies in PSI. Thus, in plant light-harvesting systems,
the various opportunities to bind Chl molecules have been
exploited optimally.
Finall-y, although PS1 differs greatly from the LH1-RC
core of purple bacteria, it also has a fundamental similarity.
As should be apparent from Figure 1, in the LH1-RC
core the rate-limiting step for trapping photons is energy
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
pair of strongly interacting bacteriochlorophyll molecules)
in the RC. In this scenario, the trapping efficiency is
highest when as many antenna sites as possible are able to
transfer to the RC, and this is clearly optimized within a
ring. In PS1, crudely speaking, the pigments are organized
in a band around the electron transfer chain, and, on
average, they are all at a distance o f - 2-3 nm; however,
within this structure, the number of contact sites has
also been optimized, leading to efficient trapping. T h e
symmetry itself is not important, rather the avoidance of
quenching centers (e.g. stacked dimers) and the location
of the maximum number of pigments close to the site
where the primary charge separation occurs are important.
In order to avoid undesirable oxidation or reduction of the
antenna by the primary electron donor, however, the bulk
of the antenna molecules are kept at a distance > 2 n m
from the components involved in the electron transfer.
An additional feature of all Chl and BChl antenna
complexes resolved to date is the presence of carotenoid
molecules. T h e s e serve photo-protective, light-harvesting
and often structural roles.
Bacterial antennas
Energy transfer in the peripheral, LH2, and core, LH1, of
photosynthetic purple bacteria
T h e recently resolved structures of LH2 of Rps. acidophila
[4] and Rs. molischianum [6 °°] have revealed the highly
symmetric pigment-protein ring, displaying C9 symmetry
in the case of Rps. acidophila and C8 symmetry for
Rs. molischianum. Although only a low-resolution structure
is available for LH1 [11], it is evident that LH1 is also
organized as a ring, most probably with 16-fold symmetry.
T h e RC structure can be nicely fitted into the proposed
LH1 ring [11,12"].
Both for LH1 and LH2, the basic building block of
the structure is a heterodimer of two small (5-6kDa)
polypeptides, a and 13. Both consist of a single transmembrane helix with a highly conserved histidine that
ligates the BChl approximately one third of the way
along the a-helical stretch. Thus, in the LH1 and LH2
pigment-protein rings, the basic element is a BChl dimer.
For LH1, the o~13-BChl2 subunit can be purified; it is called
B820 after its absorption maximum and retains many of the
essential spectral properties of LH1 [13]. For LH2, such
740
Biophysicalmethods
a subunit cannot be obtained. In LH2, and most probably
in LH1, the heterodimeric subunits associate into a ring
with the 0t-polypeptides on the inside, the [3-polypeptides
at the outside, and the pigments sandwiched between the
two concentric rings of polypeptides. As a result of the
formation of the ring, the absorption shifts to - 8 7 0 nm for
LH1, and to - 8 5 0 n m for LH2, although in the latter case
other absorption maxima are also found for some species
(820 nm, 830 nm), depending on the presence or absence
of hydrogen bonds [14,15]. T h e [~-polypeptide of LH2
binds a second pigment, nearer to the cytosolic side of the
complex, and in LH2 of Rps. acidophila these BChls form
a nine-membered ring which absorbs at - 8 0 0 n m and is
positioned at a distance of - 1.7 nm from the B850 ring.
Electronic structure
Within the B850 ring of dimers, all distances between
the pigments are very similar--somewhat less than 1 rim.
Nevertheless, within the ctl3 subunit, the electron density
seems continuous, whereas electron density due to the two
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
the ct[3 subunit, overlap between the two BChls occurs
between chlorin tings I, as in the special pair of the RC,
where as overlap between BChls on adjacent subunits is
between chlorin rings III. As a consequence, one may
view LH2 and most probably LH1 as 'rings of interacting
directs'. This concept is supported by many experimental
observations (see below; and e.g. [16°]). The BChls in
the B850 ring of LH2 and in the B870 ring of LH1
all have their Qy transition dipole almost parallel and
their Qx transition dipole perpendicular to the membrane
plane. The estimated excitonic coupling between BChls
in a subunit is - 2 5 0 c m -1 [17"',18"]; the coupling is
somewhat less between BChls on adjacent subunits. In
contrast, the pigments in the B800 ring ate 'monomeric',
the distance between two neighbours is -2.1 nm, and the
corresponding dipole-dipole coupling is - 2 0 c m -1. T h e
interaction between pigments in the B800 ring and the
pigments in the B850 ring is of a similar magnitude. T h e
B800 rings in LH2 are almost flat in the plane of the
membrane, those in the LH2 of Po. molischianum are tilted
away from the membrane plane by - 3 0 °. Finallx; the LH2
rings of Rps. acidophila and Rs. mo/ischianum contain two
carotenoids per ot]3 subunit.
LH1 and LH2 have been subjected to a large number of spectroscopic studies, notably, polarized light
spectroscopy (circular dichroism [CD], linear dichroism)
[19], a variety of line-narrowing techniques (holeburning
[20,21,22"',23"1, site-selective fluorescence [24,25]), and
infrared and Raman spectroscopies. In addition, using
structural information, several of the spectroscopic features
have been modeled [17"',26°',27°']. From these studies,
two opposing views have emerged which we will discuss.
In the first view, LH1 and LH2 are considered to be
rings of interacting directs, in which many of the essential
spectroscopic features, including the dramatic red shift,
largely originate from within a dimer. T h e excitonic
interaction between neighbouring subunits within the ring
is considered to be a relatively weak perturbation, that
is, relative to the intradimer cxcitonic interaction, the
possible (and so far unknown) contribution from electron
exchange arising from ring I overlap, the intrinsic energetic
disorder and the electron-phonon or electron-vibration
coupling. T h e spectra of all photosynthetic pigmentproteins are now known to be strongly inhomogeneously
broadened, and estimates of the amount of inhomogeneous broadening range from 200-500cm -1. In addition,
the electron-phonon coupling is estimated to be of the
same order of magnitude. T h e general idea behind the
'ring of dimers' model is that, following excitation, any
phase relation between excitations on different dimers
is rapidly destroyed, either dynamically, because of the
coupling to vibrations or phonons, or as a consequence of
the interference of the pure eigenstates due to energetic
disorder.
In the alternative view, the spectroscopic features are
totally determined by the set of excitonic eigenstates
of the full ring. In this model, the excitonic interaction
between adjacent BChls is the dominant term that
completely determines the red shift observed upon
formation of the ring. The lowest state of the exciton
manifold is almost optically forbidden, because of the
inplane orientation of the Qy transition dipoles, and
all the oscillator strength is equally divided between
two orthogonal transitions slightly above the lowest one.
Experimental evidence to support this model includes
holeburning experiments [21,22"°,23"], the interpretation
of the low-temperature absorption spectrum [17"'], and
estimates of the absorption cross-section of the major
transition at 850nm or 870nm [28°',29]. We are of the
opinion that the latter view is less accurate, mainly because
it ignores all the nonexcitonic contributions that all have
the effect of destroying the fully delocalized coherent
states. In addition, as we show below, the ring of dimers
model provides a simple and elegant explanation for many
of the dynamic results.
Intraring energy transfer
From a variety of spectroscopic studies (for a review, see
van Grondelle eta/. [30]), the energy transfer dynamics
within LH1 and LH2 have been concluded to be ultrafast.
With the advent of femtosecond laser spectroscopy, in
particular using Ti:Sapphire lasers, many of the elementary
energy transfer steps have been resolved in time. T h e
B800--+B850 energy transfer at room temperature takes
- 7 0 0 - 8 0 0 f s for LH2 from Rb. sphaeroides, and this time
constant is not very species dependent [31-35,36°',37"].
The energy transfer time is only weakly dependent on
temperature, being - 1 ps at 77K and - 2 p s at 4K. The
B800--+B850 energy transfer has been modeled in terms
of a F6rster process [31,33,34]. T h e weak temperature
Femtosecond spectroscopy of photosynthetic light-harvesting systems Fleming and van Grondelle
dependence of this energy transfer step suggests the
involvement of some vibronic level of B850 or possibly
of the higher excitonic states of the B850 ring [22"°,37°].
Previously, efficient energy transfer was concluded to
occur within the B800 ring from fluorescence polarization
experiments [19]. More recently, a time constant of
-0.5-1 ps has been estimated for energy transfer between
neighbouring B800 rings from polarized pump-probe
spectroscopy [34,35,38°°].
For energy transfer within the B850 and B875 rings,
single-site lifetimes of the order of a few hundred
femtoseconds have been estimated, for example, from an
analysis of the efficiency of singlet-singlet annihilation
[39]. In addition, the observation that within a few
picoseconds transient absorption changes were almost
fully depolarized has been interpreted as subpicosecond
energy transfer among B850 rings in LH2 and B870
rings in LH1 [40,41]. T h e energy migration in B850
and B870 has been recorded directly using fluorescence
depolarization [42,43°°], and using equilibration of the
transient absorption spectrum [44]. Both studies used a
similar interpretation based on hopping between dimers
in a ring. The site energies of the dimers were taken at
random from an inhomogeneous distribution o f - 4 0 0 cm -1
width, and average hopping times o f - 1 0 0 f s
were
obtained. T h e fluorescence anisotropy decays faster in
LH2 than in LH1, and in this model this arises simply
from the smaller ring size of LH2 (larger angle change per
hop). T h e model could be extended to low temperatures
where the site energy variation impedes the energy
transfer over more than a few sites [45°°]. Remarkably,
Chachisvilis et al. [46] and Bradforth et al. [42] found that
oscillations at 105 cm -I, assigned to vibrational wavepacket
motion, dephased significantly slower than the observed
depolarization timescale, suggesting vibrational coherence
transfer [47] in the energy transfer process.
As discussed above, the extent of exciton delocalization
in LH1 and LH2 has been extensively debated. Key
quantities are the electronic coupling between the BChls,
the electron-phonon coupling (reorganization energy and
timescale), the temperature, and the disorder. From
the difference in position between the pump-induced
bleaching (ground state to one-exciton state) and pumpinduced absorption (one-exciton state to two-exciton
state), Sundstrtim and coworkers [48°,49 °] estimate a
delocalization length of 4 + 2 molecules in LH1 and LH2,
more or less independent of temperature. A measurement
of the superradiance in LH1 and LH2 gave an even
smaller number [50°°]. On the other hand, an ultrafast
decay in the transient absorption and emission of LH2
was taken as an indication for relaxation between fully
delocalized states [51°]. An incisive discussion of how
delocalization influences different observables has been
given by Leegwater [52 °] and more recently by Meier
et al. [53°°].
741
In an attempt to provide experimental characterization
of the electron-phonon coupling, Jimenez et al. [54 °°]
carried out three-pulse photon echo peak shift (3PEPS)
measurements on LH1 and LH2. T h e y concluded that
on a 50fs timescale fluctuations in the environment
and vibrations lead to the dynamic localization on a
dimeric subunit of LH1 and LH2. A similar conclusion
was drawn from the ultrafast reorganization, as observed
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
was interpreted as a loss in the rephasing capability of
the system due to energy transfer. During the energy
transfer, the system samples all the various environments
that contribute to the inhomogeneous broadening, and,
as a consequence, the information about the original
environment is lost. Again, the model that assumes
hopping on an inhomogeneously broadened ring of dimers
gave a fit to the results. T h e homogeneous broadening
was estimated to be - 2 0 0 c m -1, the inhomogeneous
b r o a d e n i n g - 5 0 0 c m -I, and the hopping time -100fs.
This interpretation is very much supported by a 3PEPS
experiment on the LH1 subunit, B820, in which the 100 fs
phase in the peak shift decay ascribed to energy transfer
was absent and replaced by a nondecaying component
arising from inhomogeneous broadening [56°°]. The
striking similarity between the 3PEPS of LH1 and B820
further supports the idea that excitations in these antenna
complexes are delocalized over only a dimer unit.
Carotenoids
Energy transfer from carotenoid to BChl in LH2 of
Rb. sphaeroides can occur on a timescale of a few
100 fs [57]. Recent fluorescence upconversion experiments
demonstrated that, in LH1 and LH2 of Rb. sphaeroides,
the $2 lifetime of sphaeroidene is shortened to - 5 5 f s
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
complex of Chromatium purpuratum, Gillbro and coworkers
[59 °°] report an S2--~BChl (Qx) transfer time of 100fs and
S1---)BChl (Qy) transfer times of 3.8 ps and 0.5 ps for the
carotenoid that transfers to B830 and the carotenoid that
transfers to B800, respectively.
Interring transfer
In the intact bacterial photosynthetic unit, the energy
transfer from one LH2 to another, and from LH2 to
LH1, takes place on a timescale of a few picoseconds
[60-62,63°]. Assuming Ftirster energy transfer between
BChls on neighbouring rings, this would imply a closest
distance o f - 3 nm between the two pigment-protein rings.
Long before the crystal structure of LH2 became available,
it was realized that the rate-limiting step in excitation
trapping was the step from the L f t l pigments to the
special pair in the RC. A transfer time o f - 3 5 p s was
obtained for the LH1 to special pair energy transfer fL64],
742
Biophysicalmethods
and this was interpreted as a distance of 4.5 nm between
the special pair and the LH1 ring, in good agreement
with models suggested for the L H 1 - R C core, assuming
that LH1 is organized as a ring of 16 ¢~-BChl 2 subunits
with a structure as in L H 2 of Rps. acidophila [12",65*°]. A
summary of the timescales is given in Figure 2.
Green p l a n t and cyanobacteria a n t e n n a s
LHCII
L H C I I is the major light-harvesting pigment-protein of
higher plants and algae and is responsible for the binding
o f - 5 0 % of all Chl on earth. It serves to feed excitation
energy into the minor light-harvesting complexes, CP29,
CP26, CP24, and into the core of Photosystem 2 (PS2)
which eventually is used for charge separation. L H C 2
is a m e m b e r of a family of light-harvesting complexes
which includes the various forms of L H C I I and the minor
light-harvesting complexes. T h e basic unit of all these
complexes is a membrane protein o f - 2 5 kDa, which is
known to fold into a structure with three transmembrane
helices: A, B and C.
In L H C I I , the monomeric subunit binds 7-8 Chl a,
5-6 Chl b, 2 luteins, 1 neoxanthin and substoichiometric
amounts of violaxanthin. In its native form L H C I I is a
trimer, and in 1994 the structure of the trimer of L H C I I b
was resolved to a resolution of 3 - 4 a by K0hlbrandt
and coworkers [3] using cryoelectron microscopy. At the
current resolution, Chl a and Chl b are indistinguishable.
In addition, the phytol tails of the Chls cannot be
observed, and, as a consequence, the orientation of the Qx
and Qy transition dipoles within each of the chlorin planes
is not known. In the proposed model, the assignment of
the Chl as and Chl bs is based on the following argument.
After excitation, there is a small but finite chance that a
triplet is formed selectively on one of the Chl as because of
the assumed fast Chl b to Chl a energy transfer. As one role
of the carotenoids is to quench these Chl a triplets with
high efficiency to prevent the formation of harmful oxygen
radicals, the Chl as must be positioned in van der Waals
contact with the luteins. T h e seven Chls in the core of
L H C I I , which all make close contact with the luteins, have
been therefore assigned to the seven Chl as: the remaining
Chls to Chl b. In view of recent reconstitution experiments
with L H C I I , it may be possible that some of the binding
sites are promiscuous and can be occupied by either a Chl
a or a Chl b. L H C I I exhibits intense C D spectra, indicative
of Chl a-Chl a and Chl b--Chl b cxcitonic interactions; the
interaction between Chl as and Chl bs is most probably
weak.
A variety of picosecond and femtosecond studies have
been performed to explore the dynamics of energy transfer
within L H C I I . In a pioneering fluorescence upconversion
study by Eads et al. [66], the dominant time constant for
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
LH1
LH2
~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
p u m p - p r o b e study, a slower Chl b--+Chl a energy transfer
time in the range of a few picoseconds was obtained in
addition to the ultrafast process [67]. Transient absorption
with shorter pulses [68] revealed Chl b--+Chl a energy
transfer times of 160fs and also the slow process of
5 + 2 p s similar to the results of Kwa et al. [67]. In a
fluorescence upconvetsion study by D u e t a/. [69], two
lifetimes in the rise in presumably Chl a fluorescence
were detected upon excitation at 650 nm, + 250 fs and 5 ps,
in rather good agreement with the p u m p - p r o b e results.
On the other hand, P~lsson et al. [70] using one-colour
pump-probe, detected a major 500 fs and a minor 2-3 ps
Chl b-+Chl a transfer time, and, despite the superior
time resolution, they could not distinguish any transfer
component faster than 500 fs. More recently, Visser et al.
[71"], and later Connelly et al. [72 °] resolved all three
phases in the Chl b--+Chl a energy transfer: 180fs, 600fs
and - 5 p s , with a relative anaplitude ratio of 40%, 40%
and 20%, respectivel'~: A study on L H C I I monomers
demonstrated that all three decay times are associated with
Chl b--+Chl a energy transfer within a monomeric unit of
L H C I I (FJ Kleima et al., unpublished data). According to
Visser e t a / . [7l*'], in the trimer all the energy transfer
occurred to the major red absorbing species at 676nm.
Connelly eta/. [72"] concluded from their data, which was
obtained with an excellent signal-to-noise ratio, that the
175 fs component probably partly reflected energy transfer
between 'blue' and 'red' Chl bs [73]. Measurements of
singlet-singlet and singlet-triplet annihilation suggest that
intermonomer energy transfer occurs on a timescale of
10-20 ps [68,71"].
"Very recently, Gradinaru et a/. (unpublished data) have
studied the Chl b--+Chl a transfer in one of the minor
light-harvesting complexes, CP29. In CP29, six of the
Chl a and two of the Chl b binding sites are conserved
[74"], suggesting a pigment stoichiometry of six Chl a : t w o
Chl b : one lutein : one neoxanthin : one violaxanthin. T h e
kinetics in CP29 contain many components similar to
those in L H C I I and most probably reflect the same
energy transfer processes. Specifically; Gradinaru e t a / .
(unpublished data) could assign a slow, 2 - 3 p s energy
transfer phase to a Chl b absorbing at 650 nm - - most
probably Chl b5 in the L H C I I a s s i g n m e n t - - a n d a fast,
0.2-0.3 ps energy transfer phase to a Chl b3.
Carotenoid to Chl energy transfer in L H C I I is highly
efficient. Recently, two conflicting reports appeared on the
dynamics and pathway of carotenoid to Chl a transfer.
Peterman et al. [75 *°] argued that no direct carotenoid to
Chl b transfer occurred, while carotenoid to Chl a energy
transfer took place in - 2 2 0 fs. In contrast, Connelly et al.
[76"'] claimed that the carotenoids exclusively transferred
energy to Chl b, followed by Chl b--+Chl a energy transfer.
T h e latter would be inconsistent with the assignment by
Ktihlbrandt and coworkers [3], where only close contacts
between Chl as and carotenoids exist.
743
PS1
T h e core of PS1 is the most complex photosynthetic
light-harvesting plus electron transfer system for which
a structure is now available [7"']. T h e functional unit
of the PSI core of Synechococcus elongatus consists of 11
subunits, including the two major subunits PSaA and
PSaB, each having a molecular weight o f - 8 0 k D a with
known sequcnce, and each binding - 1 0 0 Chl as, 10-25
carotenoids and three FeS clusters. T h e structure has been
resolved to 4 ~, and the positions o f - 90 Chls have been
determined. As in the case of L H C I I , no information
about the direction of the Qv and Qx transition dipoles
is available. T h e core of P~;1 is characterized by 22
transmembrane helices, 11 for each large subunit, which
exhibit C2 symmetry around an axis that passes through
the centrally located special pair of the electron transfer
chain, P700, and the FeS cluster E T h e electron transfer
chain is e m b e d d e d in a structure of ten transmembrane
helices, five from each subunit, the arrangement of which
is strongly reminiscent of that of the L and M subunits in
the purple bacterial RC. All the other 90 antenna Chl as
are dispersed in a band around this core and, for a large
part, are associated with the remaining six transmembrane
helices on each of the large subunits. For all the Chls
except two, the distance to any of the pigments in the
electron transfer chain exceeds 1.6nm, making energy
transfer slow (10-20 ps). Two chlorophylls are found that
seem to connect the antenna with the second and third
pair of Chls of the electron transfer system, and it has been
suggested that these form a special entry for excitation
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
been suggested that the pigment-protein arrangement of
the six outer transmembrane helices and their associated
Chls may be a good model for the PS2 core.
Trapping in PSI is fast (20-25 ps), and charge separation
is essentially irreversible [30,77,78]. Using ultrafast fluorescence depolarization, Du et al. [79] estimated that the
major hopping process within PS1 occurs on a timescale
of 100fs. On a timescale of a few picoseconds, the
excitation energy is seen to equilibrate between a pool
of very red pigments, absorbing at - 7 2 0 - 7 3 0 n m , and
the major PSI core pigments. T h e process of energy
transfer to P700 must occur at the same rate as this
equilibration between core and red pigments as, even at
very low temperatures where escape from the red states
is impossible, a reasonably high quantum yield for charge
separation is still observed upon excitation of the core
pigments [80,81"]. This has led to a model in which
essentially all sites within the PSI core are more or less
equally efficient in transferring their energy to P700 (or
any other pigment of the electron transfer chain) and
which may be viewed as the 3D version of the 2D ring to
special pair energy transfer model that seems to operate for
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
for excitation energy indeed have that role. T h e y have
more rapid energy transfer to the pigments in the electron
transfer chain but are simply outnumbered by all the other
Chls. A simple simulation of the trapping kinetics in PSI
shows that leaving out the pair of connecting Chls hardly
changes the trapping time.
T h e process of energy migration and charge separation
cannot be experimentally separated in PSI. Kumazaki et
al. [83•], Trinkunas and Holzwarth [84 °] and White et al.
[85 •°] have used modeling to extract the intrinsic electron
transfer rate. In a very recent study using a PSI mutant,
which seemed to affect the special pair P700 but not the
antenna spectra or dynamics, it was observed that the
excited state lifetime approximately doubled [86"]. This
was taken by Melkozernov et al. [86 °'] as evidence for a
model in which the charge separation rate by P700 is the
rate limiting step, in contrast to the 'transfer-to-the-trap'
limited model discussed above.
Conclusions
T h e combination of high-resolution structural data and
uhrafast spectroscopy has enabled the development of a
fairly complete picture of the light-harvesting process in
purple bacteria. T h e efficiency of the overall process is
based on individual energy transfer steps of 80-100fs.
In LH1, the core antenna surrounding the Re, several
hundred energy transfer steps occur before the final
transfer to the special pair (35ps) and the initiation of
charge separation. T h e observation of a sub 100fs energy
transfer, along with the retention of coherence and the
enhanced radiative rates in LH1 and LH2, raises many
challenging issues which will provide stimulus for theory
and experiment for years to come. Despite the high
symmetry and potential for strong intermolecular coupling,
it does not appear that extensive electronic delocalization
is necessary for achieving the near unit efficiency of the
light-harvesting process.
In green plant and cyanobacterial antennas, the structural
information is not yet sufficient for the most detailed
molecular modeling of energy migration. Enough is
known, however, to reveal both striking similarities and
differences with the purple bacteria. In particular, antenna
molecules are held away from close contact with the
primary electron donor, and efficiency is achieved by using
large numbers of antenna molecules with roughly similar
transfer rates to perform the final transfer step to the
primary donor.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
•
••
of special interest
of outstanding interest
Duysens LMN: Transfer of excitation energy in photosynthesis
[PhD Thesis]. Utrecht, The Netherlands: Utrecht University; 1952.
2.
Duysens LMN: Photosynthesis. ProgrBiophys 1964, 14:1-104.
3.
KOhlbrandt W, Wang DN, Fujiyoshi Y: Atomic model of plant
light-harvesting complex by electron crystallography. Nature
1994, 367:614-621.
4.
McDermott G, Prince SM, Freer AA, Hawthornthwaite-Lawless AM,
Papiz MZ, Cogdell PJ, Isaacs NW: Crystal structure of
an integral membrane light- harvesting complex from
photosynthetic bacteria. Nature 1995, 374:51 ?-521.
5.
o,
Freer AA, Prince S, Sauer K, Papiz MZ, HawthornthwaiteLawless AM, McDermott G, Cogdell RJ, Isaacs NW:
Pigment-pigment interaction and energy transfer in the
antenna complex of the photosynthetic bacterium Rps.
acidophile. Structure 1996, 4:449-462.
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
obtained high-resolution structure [4]. The FSrster dipole-dipole resonance
coupling is concluded to dominate the energy transfer from the B800 to
the B850 ring. Within the B850 ring, strong interactions exist between
nearest neighbour BChls partly because of a close to optimal alignment of
their transition moments, suggesting that delocalized excitonic states play a
role in the energy transfer. The orientations and distances of the rhodopin
molecules, the BS00 and B850 BChls, suggest that singlet-singlet energy
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.
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.
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
16 B850 and 8 B800 BChls in an eightfold symmetric ring. The 16 B850
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
in a ring with a diameter of 2.88 nm. The B6OOs are bound to Asp6 of the
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
to their position in LH2 of Rps. acidophila and are very much tilted away
from the plane of the membrane. Nevertheless, in this structure the B800 Q./
transition dipoles are more or less parallel to the B850 Qy transition dipoles.
6.
•.
7.
•.
Krauss N, Schubert W-D, Klukas O, Fromme P, Witt HT,
Saenger W: Photosystem I at 4 A resolution represents the first
structural model of a joint photosynthetic reaction centre and
core antenna system. Nat Struct Bio/1996, 3:965-973.
The structure of PS1 from Synechococcus e/ongatus is determined to 4 A
resolution using X-ray crystallographic methods. The arrangement of the 22
transmembrane and 4 surface helices displays a twofold symmetry for the
large subunits PsaA and PsaB. The central part of the structure, which is
proposed to carry the electron transfer chain, including PTO0, the acceptor
A 0, and the three FeS clusters, shows a striking resemblance to the LM core
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
continuous with the electron transfer chain via the second and third Chl pairs
of the electron transfer system. This suggests a dual role for these Chl as
both in excitation transfer and electron transfer.
8.
Fenna RE, Matthews BW: Chlorophyll arrangement in a
bacteriochlorophyll protein from Chlorobium limicola. Nature
1975, 258:573-577.
9.
Brejc K, Ficner R, Huber R, Steinbacher S: Isolation,
crystallization, crystal structure analysis and refinement of
allophycocyanin from the cyanobacterium Spirulina platensis
at 2.3 A resolution. J Mol Bio/1995, 249:424-440.
10.
•.
Hoffmann E, Wrench PM, Sharpies FP, Hiller RG, Welte W,
Diederichs K: Structural basis of light-harvesting by
carotenoids: peridinin-chlorophyll*protein from Amphinidium
carterae. Science 1996, 272:1 788-1791.
The structure of the peridinin-chlorophyll-protein (PCP) is solved to a resolution 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
the N-terminal and C-terminal domains of each polypeptide is related by
a twofold symmetry axis, and it surrounds a hydrophobic cavity filled with
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
peridinins at van der Waals distances around the Chls.
11.
Karrasch S, Bullough PA, Ghosh R: The 8.5/~ projection map
of the light-harvesting complex I from Rhodospirillum rubrum
reveals a ring composed of 16 subunits. EMBO J 1995,
14:631-638.
Femtosecond spectroscopy of photosynthetic light-harvesting systems Fleming and van Grondelle
12.
•
Papiz MZ, Prince SM, Hawthornthwaite-Lawless AM,
McDermott G, Freer AA, Isaacs NW, Cogdell PJ: A model for the
photosynthetic apparatus of purple bacteria. Trends P/ant Sci
1996, 1:198-206.
A model is produced for the whole photosynthetic unit of purple bacteria
based on the crystal structure of the LH2 peripheral light-harvesting complex of Rps. acidophila. To model the (x16~1s projection map of Karrasch
et aL [11 ] the (x9139structure of LH2 is increased to produce the larger ring.
The known structure of the reaction center is found to fit in this LH1 ring.
Precisely six LH2s can be fitted in a circle around this LH1 core, thereby
reproducing the typical stoichiometry of LH1 to LH2.
13.
Visschers RW, Chang MC, van Mourik F, Parkes-Loach PS,
Heller BA, Loach PA, van Grondelle R: Fluorescence polarization
and low-temperature absorption spectroscopy of a subunit
form of light-harvesting complex I from purple photosynthetic
bacteria. Biochemistry 1991, 30:5734-5?42.
14.
Fowler GJS, Visschers RW, Grief GG, van Grondelle R,
Hunter CN: Genetically modified photosynthetic antenna
complexes with blue-shifted absorbance bands. Nature 1992,
355:848-850.
15.
Fowler GJS, Sockalingum GD, Robert B, Hunter CN: Blue shifts
in becteriochlorophyll ebsorbance correlate with changed
hydrogen bonding patterns in light-harvesting LH2 mutants
of Rhodobacter spheeroides with alterations at c(Tyr44 and 45.
Biochem J 1 g94, 299:695-700.
16.
•
Beekman LMP, Steffen M, Stokkum IHM, van Olsen JD, Hunter
CN, Boxer SG, van Grondelle R: Characterization of the light
harvesting antennas of photosynthetic purple bacteria by Stark
spectroscopy. 1. LH1 antenna complex and the B820 subunit
from Rhodospiri/lum rubrum. J Phys Chem 1997, in press.
The response of the optical absorption spectrum to an externally applied
electrical field (Stark effect) is measured for LH1, the B820 dimeric subunit
of LH1, and the reconstituted LH1 of purple photosynthetic bacteria. The
Stark effect for LH1 is strongly reminiscent of that measured for the special
pair of the purple bacterial reaction center, it is dominated by a large change
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
subunit of LH1.
17.
°•
Sauer K, Cogdell RJ, Prince, SM, Freer A, Isaacs NW, Scheer H:
Structure-based calculations of the optical spectra of the LH2
bacteriochlorophyll-protein complex from Rhodopseudomonas
acidophila. Photochem Photob~o11996, 64:564-576.
The molecular structure of the LH2 complex of Rps. acidophila is used
to provide orientations and distances for the 27 BChls in the complex as
the input parameters for an excitonic hamiltonian. Assuming dipole-dipole
interactions among all the chromophores, the ring of 18 closely coupled
¢hromophores is assigned to B850, whereas the parallel ring of nine weakly
¢oup4ed BChls is assigned to B800. The pairwise excitonic interactio,1 betweetn the B850s is estimated to be - 2 5 0 - 3 0 0 cm -1. The general trends
obaervable in the CD spectrum of LH2 are tentatively explained. The calculations predict strong CD features at - 7 9 0 nm which await experimental
verification.
Sturgis JN, Robert B: The role of chromophore coupling in
tuning the spectral properties of the peripheral light-harvesting
protein of purple bacteria, Photosynth Res 1996, 50:5-10.
The authors calculate the excitonic contribution to the absorption spectrum
for LH2 of photosynthetic purple bacteria and conclude that this contribution
is not very sensitive to small variations in the LH2 structure. For that reason,
they propose that in LH2 of Rps. acidophi/a the redshift to 850 nm originates
roughly equally from pigment-pigment and pigment-protein interactions. In
BS00-B820, a related but slightly different LH2, the redshift is largely due
to pigment-pigment interaction. As a consequence, the total amount of excitonic interaction between neighbouring pigments can not be much larger
than 200 cm -1.
18.
•
745
antenna complex of Rhodopseudomonas ecidophila (strain
10050), J Phys Chem 1996, 100:12022-12033.
The transfer of excitation energy in the LH2 complex of Rps. acidophila is studied using holeburning and femtosecond laser spectroscopy.
B S 0 0 ~ B 8 5 0 energy transfer is observed to occur with monophasic kinetics with a time constant ranging from 1.6 ps (lgK) to 1.1 ps (130K), while
holeburning at 4.2K yields 1.8 ps. Holeburning with applied pressure shows
no effect on the energy transfer rate. Together with the weak temperature
dependence, this suggests that the B800 emission overlaps with a weak
vibronic band of B850. Time-domain and holebuming spectroscopy show
that there is an additional relaxation channel for B800 excitations when
the excitation is to the blue of the B800 band. Two possible processes,
intra-B800 transfer and coupling with the quasi degenerate upper exciton
manifold of B850, are discussed.
23.
•
Wu HM, Reddy NRS, Cogdell RJ, Muenke C, Michel H, Small G J:
A comparison of the LH2 antenna complex of three purple
bacteria by hole-burning and absorption spectroscopies. Mo/
Cryst Liq Cryst 1996, 291:163-173.
The LH2 complexes of Rps. acidophila, Rb. sphaeroides and Rs. molischianum 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
process in the other species. The absorption spectra of Rs. molischianum
and Rps. acidophila undergo a dramatic redshift and thermal narrowing upon
cooling from room temperature to 4.2K; for LH2 of Rb. sphaeroides, these
effects are much smaller. This is interpreted as a smaller excitonic coupling
in the latter species.
24.
Van Mourik F, Visschers RW, van Grondelle R: Energy transfer
and aggregate size effects in the inhomogeneously broadened
core light-harvesting complex of Rhodobacter sphaeroides.
Chem Phys Lett 1992, 193:1-7.
25.
Monshouwer RM, Visschers RW, van Mourik F, Freiberg A,
van Grondelle R: Low-temperature absorption and siteselected fluorescence of the light-harvesting antenna of
Rhodopseudomonas v/r/dis. Evidence for heterogeneity.
Biochim Biophys Acta 1995, 1229:373-380.
26.
•o
Alden RG, Johnson E, Nagarajan V, Parson WW, Law C J,
Cogdell RJ: Calculations of spectroscopic properties of
the LH2 bacteriochlorophyll-protein antenna complex from
Rhodopseudomonas acidophila. J Phys Chern B 199'7,
101:4667-4680.
This paper describes the calculation of absorption and CD spectra of a
photosynthetic bacterial antenna complex based on the crystal structure of
the LH2 complex from Rps. acidophila. Molecular orbitals for the three different BChl structures in the complex are obtained by semiempirical quantum
mechanical calculations. Exciton and charge transfer interactions are introduced at the level of configuration interactions. Absorption bandshapes are
treated with vibronic parameters as obtained from holeburning experiments,
whereas inhomogeneous broadening is included by a Monte Carlo method.
Calculations reproduce the measured absorption and CD spectra. The resuits support the idea that excitations are rather delocalized in LH2.
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
LH2 antenna complex of Rhodopseudomonas acidophila: a
structure based study. J Phys Chem 1997, in press.
The published crystal structure of LH2 of Rps. acidophi/a is used to calculate
absorption and CD spectra of the complex. It is shown that the relative
position of the CD zero crossing with respect to the absorption maximum
is an important parameter that is rather sensitive to structural changes. It
is demonstrated that the experimentally observed CD spectrum can only
be explained if the whole ring is considered and if the o(-polypeptide and
I~-polypeptide BChls are allowed to have different excitation energies.
28.
°°
Leupold D, Stiel H, Teuchner K, Nowak F, Sandner W, 0cker B,
Scheer H: Size enhancement of transition dipoles to one and
two-exciton bands in a photosynthetic antenna. Phys Rev Lett
1996, 77:4675-46"78.
From a measurement of the nonlinear absorption, the differential absorption density spectrum, and the fluorescence for LH2 from Rb. sphaeroides,
the dipole moments associated with the ground state-->one-exciton and
one-exciton-->two-exciton transition are estimated to be 25.5 D and 21.5 D,
respectively. These values are seen as an indication that in LH2 the exciton
is delocalized over 16 +4 BChl molecules, corresponding to the full physical
length of the circular aggregate.
19.
Kramer HJM, van Grondelle R, Hunter CN, Westerhuis WHJ,
Amesz J: Pigment organization of the B800-850 antenna
complex of Rhodopseudomonas sphaeroides. Biochim Biophys
Acta 1984, 765:156-165.
20.
De Caro C, Visschers RW, van Grondelle R, VSIker S: Inter- and
intraband energy transfer in LH2-antenna complexes of purple
bacteria. A fluorescence line-narrowing and hole-burning
study. J Phys Chem 1994, 98:10584-10590.
21.
Reddy NRS, Picorel R, Small G J: B896 and B870 components
of the Rhodobacter sphaeroides antenna: a hole-burning study.
J Phys Chem 1992, 96:6458-6464.
29.
Novoderezhkin VI, Razjivin AP: Exciton dynamics in circular
aggregates: application to antenna of photosynthetic purple
bacteria. Biophys J 1995, 68:1089-1100.
22.
••
Wu HM, Savikhin S, Reddy NRS. Jankowiak R, Cogdell RJ,
Struve WS, Small G J: Femtosecond and hole-burning studies
of B8OO's excitation energy relaxation dynamics in the LH2
30.
van Grondelle R, Dekker JP, Gillbro T, SundstrSm V: Energy
transfer and trapping in photosynthesis. Biochim Biophys Acta
~994, 1187:1-65.
746
Biophysical methods
31.
van Grondelle R, Kramer HJM, Rijgersberg CP: Energy transfer
in the B800-850-carotenoid light-harvesting complex of
various mutants of Rhodopseudomonas sphaeroides and of
Rhodopseudomonas capsulatus. Biochim Biophys Acta 1982,
682:208-215.
32.
Trautman JK, Shreve AP, Violette CA, Frank HA, Owens TG,
Albrecht AC: Femtesecond dynamics of energy transfer
in B800-850 light-harvesting complexes of Rhodobacter
sphaeroides. Proc Nat/Acad Sci USA 1990, 87:215-219.
33.
Laan H, Schmidt Th, Visschers RW, Visscher KJ, van Grondelle R,
VSIker S: Energy transfer in the B800-850 antenna complex
of the purple bacterium Rhodobacter sphaeroides: a study by
spectral hole-burning. Chem Phys Lett 1990, 170:231-238.
34.
Monshouwer R, Ortiz de Zarate I, van Mourik F, van Grondelle R:
Low-intensity pump-probe spectroscopy on the B800 to B850
transfer in the light harvesting 2 complex of Rhodobacter
sphaeroides. Chem Phys Lett 1995, 246:341-346.
35.
Hess S, ~kesson E, Cogdell RJ, Pullerits T, Sundstr6m V: Energy
transfer in spectrally inhomogeneous light-harvesting pigmentprotein complexes of purple bacteria. Biophys J 1995, 69:22112225.
36.
••
Joe 1", Jia Y, Yu, J-Y, Jonas DM, Fleming GR: Dynamics in isolated
bacterial light harvesting antenna (LH2) of Rhodobacter
sphaeroides at room temperature. J Phys Chem 1996,
100:2399-2409.
Transient absorption, transient grating and photon echoes are measured on
the B800 band of LH2 of Rb. sphaeroides using 30fs pulses. B800--~B850
energy transfer occurs in 800fs. The three-pulse photon echo peak shift
experiment identifies important contributions to the 6800 lineshape and
thereby the dynamics of the system involved: low-frequency intramotecular
vibrations; ultrafast bath (solvent plus protein) responses; and static inhomogeneity on a timescale longer than the energy transfer time. The transient
absorption is observed to decay nonexponentially. The authors argue that
the fast phase is vibrational relaxation within the B800 band.
3?.
•
Ma Y-Z, Cogdell RJ, Gillbro T: Energy transfer and exciton
annihilation in the B800-850 antenna complex of the
photosynthetic bacterium Rhodopseudomonas ecidophila
(strain 10050). A transient femtosecond absorption study.
J Phys Chem B 1997, 101:1087-1095.
This paper describes femtosecond pump-probe experiments on LH2 of
Rps. acidophi/a. At room temperature, B800--~B850 energy transfer is
- 0 . 8 ps; at 77K, - 1 . 3 p s . Anisotropy kinetics measured within 6800 band
indicate that relaxation within the B800 band is wavelength dependent. A
fast depolarization time of 210fs observed at 77K is thought to originate
from exciton relaxation. From a dramatic energy dependence of the B800
kinetics, it is speculated that several high-lying excitonic states of B850 exist
that show good spectral overlap with the B800 band and thus could serve
as excellent accepters for the energy transfer from B800 to B850.
38.
••
Monshouwer R, van Grondelle R: Excitations and excitons in
bacterial light-harvesting complexes. Biochim Biophys Acta
1996, 1275:70-75
The localization versus delocalization of excitations in bacterial lightharvesting complexes is discussed. It is argued that a 'ring-of-dimers' model
is adequate to explain most of the spectroscopic and time-resolved data.
Furthermore, two-colour pump-probe experiments in the B800 band of Rb.
sphaeroides reveal 'blue-to-red' energy transfer on a timescale of 400fs
within the B800 band.
39.
Bakker JGC, van Grondelle R, den Hollander WTF: Trapping, loss
and annihilation of excitations in a photosynthetic system. II.
Experiments with the purple bacteria Rhodospirillum rubrum
and Rhodopseudomonas capsulatus. Biochim Biophys Acta
1 g83, 725:508-518.
40.
Sundstr6m V, van Grondelle R, BergstrSm H, ,~kesson E, Gillbro T:
Excitation-energy transport in the bacteriochlorophyll antenna
systems of
Rhodospirillum rubrum and Rhodobacter sphaeroides studied
by low-intensity picosecond absorption spectroscopy. Biochim
Biophys Acta 1986, 851:431-446.
41.
van Grondelle R, Bergstr6m H, SundstrSm V, Gillbro T: Energy
transfer within the bacteriochlorophyll antenna of purple
bacteria at 77K studied by picosecond absorption recovery.
Biochim Biophys Acta 1987, 894:313-326.
42.
Bradforth SE, Jiminez R, van Mourik F, van Grondelle R, Fleming
GR: Excitation transfer in the core light-harvesting complex
(LH-1) of Rhodobacter sphaeroides: an ultrafast fluorescence
depolarization and annihilation study. J Phys Chem 1995,
99:16179-16191.
43.
•.
Jiminez R, Dikshit SN, Bradforth SE, Fleming GR: Electronic
excitation transfer in the LH2 complex of Rhodobactar
sphaeroides. J Phys Chem 1996, 100:6825-6834.
Fluorescence upconversion experiments on LH-2 yield two time constants in
the anisotropy decay: 5 0 - 9 0 f s and 400-500fs. 6800-->B850 transfer occurs within 650fs. Depolarization is modeled using a model with inhomogeneous broadening (250 cm -1) and a dimer-to-dimer hopping time of 100fs.
A calculation of the spectrum using an intradimer coupling of 230cm -1, ~and
coupling between adjacent chromophores on different subunits of 110 cm -1
yields an average deloealization length of - 5 molecules in the middle of the
band.
44.
Visser HM, Somsen OJG, van Mourik F, Lin S, van Stokkum IHM,
van Grondelle R: Direct observation of sub-picosecond
equilibration of excitation energy in the light-harvesting
antenna of Rhodospirillum rubrum. Biophys J 1995, 69:10831099.
45.
,,
Visser HM, Somsen OJG, van Mourik F, van Grondelle R: Excitedstate energy equilibration via sub-picosecond energy transfer
within the inhomogeneously broadened light-harvesting
antenna of the LH-1 only Rhodobacter sphaeroides mutant
M2192 at room temperature and 4.2K. J Phys Chem 1996,
100:18859-18867.
The spectral evolution following an ultrafast laser flash is studied for the core
antenna of photosynthetic purple bacteria. The authors conclude that ultrafast energy transfer occurs, resulting in a net shift of the excitation distribution
to the low-energy pigments. At room and low temperature, the major part of
the spectral shift takes less than a picosecond. The energy transfer dynamics
at room temperature are fitted assuming a hopping time of - 100 fs between
dimers on a ring and an inhomogeneous width of 400cm -1. At 4K hopping
is somewhat slower, - 0 . 4 ps, and the inhomogeneous width has decreased
to - 200cm -1.
46.
Chachisvilis M, Pullerits T, Jones MR, Hunter CN, Sundstr~m V:
Vibrational dynamics in the light harvesting complexes of the
photosynthetic bacterium Rhodobacter sphaeroides. Chem
Phys Lett 1994, 224:345-351.
47.
Jean JM, Fleming GR: Competition between energy and phase
relaxation in electronic curve crossing processes. J Chem Phys
1995, 103:2092-2101.
48.
•
Pullerits 1", Chaehisvilis M, Sundstr6m V: Exciton delocalization
length in the B850 antenna of Rhodobacter sphaeroides.
J Phys Chem 1996, 100:10787-10792.
Excitation transfer dynamics in LH2 of Rb. sphaeroides is investigated using
transient absorption spectroscopy. In LH2, the anisotropy decays in 130 fs,
whereas the isotropic decay occurs in ?0fs. For a ninefeld symmetric ring,
with the orientation of the transition dipoles as in LH2, a factor of 3 between
the two time constants is expected. As this is not observed, the authors conclude that the energy migration is (partially) coherent. From an analysis of the
transient absorption difference spectrum, they conctude that the excitation
is delocalized over 4_+2 monomers.
49.
•
Chachisvilis M, K(Jhn O, Pullerits T, Sundstr6m V: Excitons in
photosynthetic purple bacteria. Wavelike motion or incoherent
hopping? J Phys Chem 1997, in press.
From a comparison of isotropic and anisotropic pump probe signals measured for LH1 and LH2 of Rb. sphaeroides, the anisotropy decay is observed
to be always about twofold slower. Modeling using a hopping model predicts
a much more dramatic difference between isotropic and anisotropy decays.
As a consequence, the authors believe that the excitation transfer is partly
coherent. From a fit of the pump-probe spectrum, they conclude that the
delocalization length is - 4 BChls and is not stongly dependent on temperature,
50.
••
Monshouwer R, Abrahamsson M, van Mourik F, van Grondelle R:
Superradiance and exciton delocalisation in bacterial
photosynthetic light-harvesting systems. J Phys Chem 1997,
in press.
The radiative rate of LH1 and LH2 of Rb. sphaeroides are measured as a
function of temperature. For LH2, the radiative rate is about threefold larger
compared with that of monomeric BChl a and is independent of temperature. LH1 is very similar to LH2 at room temperature, but the radiative rate
increases about 2.4-fold upon lowering the temperature to 4K. The results
are interpreted in terms of a model that includes both the coupling between
all the pigments and the inhomogeneous broadening and suggests that the
ratio between coupling and inhomogeneous broadening is - 2 . The results
suggest that in LH2 the excitation is rather localized at all temperatures. The
degree of delocalization in LH1 may be somewhat larger, certainly at low
temperature.
51.
•
Nagarajan V, Alden RG, Williams JC, Parson WW: Ultrafast
exciton relaxation in the B850 antenna complex of
Femtosecond spectroscopy of photosynthetic light-harvesting systems Fleming and van Grondelle
Rhodobacter sphaeroides. Proc Nat/Acad Sci USA 1996,
93:13774-13779.
The femtosecond response of LH2 of Rb. sphaeroides at room temperature
is measured. The authors observe a 35fs relaxation phase in absorption and
emission spectra of the excited state, and a 20fs anisotropy decay. They
ascribe these dynamics to interlevel relaxation and dephasing, respectively,
of extensively delocalized exciton states of the circular bacteriochlorophyll
aggregate.
52.
•
Leegwater JA: Coherent versus incoherent energy transfer and
trapping in photosynthetic antenna complexes. J Phys Chem
1996, 100:14403-14409.
A model is described which has an arbitrary ratio of homogeneous broadening versus interaction energy. This allows the study of the crossover from
hopping dynamics to exciton dynamics. For the survival time, the hopping
approach is shown to yield a surprisingly accurate estimate, even when the
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.
Meier T, Chernyak V, Mukamel S: Multiple exciton coherence
sizes in photosynthetic antenna complexes viewed by
pump-probe spectroscopy. J Phys Chem 199"7, in press.
From an analysis of the pump-probe signal from the LH2 light-harvesting
antenna of purple bacteria, the localization size is determined to be 15 at
4.2K. The analysis of the difference in frequency between positive and negative peaks in the pump-probe spectrum yields an estimate for the exciton
mean free path (or the exciton dephasing lengthscale) of - 11.
light-harvesting antenna complexes from the purple bacterium
Chromatium purpuratum. Chem Phys 1996, 210:195-217.
Energy transfer from the carotenoid okenone to BChl a in the light-harvesting
complex B 8 0 0 - 8 3 0 and in chromatophores of Chromatium purpuratum was
studied by steady state fluorescence and femtosecond transient absorption spectroscopy. The overall efficiency for energy transfer from okenone
to BChl a is 95+5%. There is a fast (<200fs) transfer from at least one
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 mechanism. On a longer timescale, the okenone S 1 state transfers energy to B830
in 3.8 ps, whereas a second carotenoid transfers its energy via the S t state
to B800 in - 0.5 ps.
60.
Freiberg A, Godik VI, Pullerits T, Timpmann K: Directed
picosecond excitation transport in purple photosynthetic
bacteria. Chem Phys 1988, 128:227-235.
61.
Zhang FG, van Grondelle R, Sundstr6m V: Pathways of
energy flow through the light-harvesting antenna of the
photosynthetic purple bacterium Rhodobacter spheeroides.
Biophys J 1992, 61:911-920.
62.
Hess S, Chachisvilis M, Timpmann K, Jones MR, Fowler GJC,
Hunter CN, Sundstr6m V: Temporally and spectrally resolved
subpicosecond energy transfer within LH2 and from LH2 to
LH1 in photosynthetic purple bacteria. Proc Nat/Acad Sci USA
1995, 92:12333-1233'7.
53.
•.
54.
•.
Jimenez R, van Mourik F, Fleming GR: Three pulse echo
peak shift measurements on LH1 and LH2 complexes of
Rhodobacter sphaeroides: a nonlinear spectroscopic probe of
energy transfer. J Phys Chem 1997, in press.
Three pulse photon echo peak shift measurements are performed on the
B875 and B850 bands of detergent-isolated LH1 and LH2 complexes at
room temperature. The peak shifts are rnuch larger and decay much faster
than those typically observed for dye molecules in solution. The peak shift
decay is simulated on the basis of the optical frequency correlation function,
M(t), which includes contributions from rapid fluctuations of the protein, vibrational motion and energy transfer. The 90 fs and 130 fs exponential components in M(t) observed for LH1 and LH2, respectively, are ascribed to
energy transfer. A simulation based on a model that assumes hopping in a
ring of dimers, with each dimer randomly selected from an inhomogeneous
distribution, explains the results.
55.
•
Kumble R, Palese S, Visschers RW, Dutton PL, Hochstrasser RM:
Ultrafast dynamics within the B820 subunit from the core
(LH-1) antenna complex of Rs. rubrum. Chem Phys Let/1996,
261:396-401.
This paper describes polarized femtosecond transient absorption experiments on B820, the oc~-BChl 2 subunit of LH1, and on its reaggregated form,
B873. In B820, the timescale of the Stokes' shift is sub 50fs, as reflected
by a shift of the nuclear wavepacket and a fast component in the anisotropy
decay. In similar experiments on reassociated B873, the anisotropy is observed to decay from 0.24 to 0.07 with two time constants: 70fs and 400 fs.
The authors suggest that, following fast scattering, the excitation transfer
proceeds between states of dimeric subunits.
56.
•.
Yu J-Y, Nagasawa Y, van Grondelle R, Fleming GR: Three pulse
echo peak shift measurements on B820 subunit of LH1 of
Rhodospirillum rubrum. Chem Phys Let/1997, in press.
This paper describes the measurement cf the three pulse photon echo peak
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
phase that was ascribed to energy transfer in LH1 and is now present as a
nondecaying component. The experiment strongly supports the idea that, in
LH1, the excitation is also localized on s BChl dimer.
5?.
Shreve AP, Trautman JK, Frank HA, Owens TG, Albrecht AC:
Femtosecond energy-transfer processes in the B800-850
light-harvesting complex of Rhodobacter sphaeroides 2.4.1.
Biochim Biophys Acta 1991, 1058:280-288.
58.
•
Ricci M, Bradforth SE, Jiminez R, Fleming GR: Internal
conversion and energy transfer dynamics of spheroidene in
solution and in the LH-1 and LH-2 light-harvesting complexes.
Chem Phys Lett 1996, 259:381-390.
The lifetime of the 1Bu+ state of spheroidene is measured in vitro in various polar and nonpolar solvents and in LH1 and LH2 of Rb. sphaeroides.
The 1Bu+-->2Ag-internal
conversion time varies from 150fs to 250fs in
.
the solvents studied and depends on the polarizability of the surrounding
environment. Estimated internal conversion time is 150fs within LH2, and
170fs within LH1. The 1Bu + lifetime inside LH1 and LH2 is 60fs and 80fs,
respectively, and suggests fast energy transfer from the 1Bu + state.
59.
••
Andersson PO, Cogdell ILl, Gillbro T: Femtosecond dynamics
of carotenoid-to-bacteriochlorophyll a energy transfer in the
747
63.
•
Nagarajan V, Parson WW: Excitation energy transfer between
the B850 and B875 antenna complexes of Rhodobacter
sphaeroides. Biochemistry 199'7, 36:2300-2306.
In membrane of Rb. sphaeroides, energy transfer from B850 (LH2) to B875
(LH1) proceeds with two time constants, 4.6 ps and 26.3 ps, but a significant
fraction of the excitations remain in B850 for considerably longer times. The
fast step is ascribed to hopping from LH2 to LH1, the slow step to migration
within the LH2 pool. Back transfer from LH1 to LH2 could not be detected.
64.
Beekman LMP, van Mourik F, Jones MR, Visser HM, Hunter CN,
van Grondelle R: Trapping kinetics in mutants of the
photosynthetic purple bacterium Rhodobacter sphaeroides:
influence of the charge separation rate and consequences
for the rate-limiting step in the light-harvesting process.
Biochemistry 1994, 33:3143-3147.
65.
o.
Pullerits T, Sundstr~m V: Photosynthetic light-harvesting
pigment-proteins: toward understanding how and why. Acc
Chem Res 1996, 29:381-389.
The energy transfer and trapping dynamics in photosynthetic purple bacteria
is reviewed: a model is proposed for the LH2-LH1 association based on the
measured LH2--~LH1 energy transfer time of 3.3ps at room temperature.
Modeling this step gives a 3 nm distance of closest approach between the
LH2 and LH1 rings.
66.
Eads DD, Castner EW Jr, Alberte RS, Mets L, Fleming GR: Direct
observation of energy transfer in a photosynthetic membrane:
chlorophyll b to chlorophyll a transfer in LHCII. J Phys Chem
1990, 93:8271-8275.
67.
Kwa SLS, van Amerongen H, Lin S, Dekker J P, van Grondelle R,
Struve WS: Ultrafast energy transfer in LHC-II trimers from the
Chl a/b light-harvesting antenna of Photosystem II. Biochim
Biophys Acts 1992, 1102:202-212.
68.
Bittner T, Irrgang KD, Renger G, Wasielewski MR: Ultrafast
excitation energy transfer and exciton-exciton annihilation
processes in isolated light harvesting complexes of
Photosystem II (LHCII) from spinach. J Phys Chem 1994,
98:11821-11826.
69.
Du M, Xie X, Mets L, Fleming GR: Direct observation of ultrafast
energy-transfer processes in light-harvesting complex II.
J Phys Chem 1994, 98:4736-4741.
70.
71.
•,
P~lsson LO, Spangfort MD, Gulbinas V, Gillbro T: Ultrafast
chlorophyll ~-chlorophyll a excitation energy transfer in the
isolated light harvesting complex, LHCII, of green plants.
Implications for the organization of chlorophylls. FEBS Let/
1994, 339:134-138.
Visser HM, Kleima FJ, van Stokkum IHM, van Grondelle R,
van Amerongen H: Probing the many energy-transfer processes
in the photosynthetic light-harvesting complex II at 77K
using energy-selective sub-picosecond transient absorption
spectroscopy. Chem Phys 1996, 210:297-312.
The ultrafast energy transfer dynamics in LHCII is measured using transient
absorption spectroscopy. Three phases in the Chl b--~Chl a energy transfer
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
the major red absorbing species at 676 nm. Measurements of singlet-singlet
and singlet-triplet annihilation suggest that intermonomer energy transfer
occurs on a timescale of 10-20 ps.
72.
•
Connelly JP, M~ller MG, Hucke M, Gatzen G, Mullineaux CW,
Ruban AV, Horton P, Holzwarth AR: Ultrafast spectroscopy of
trimeric light-harvesting complex II from higher plants. J Phys
Chem B 1997, 101:1902-1909.
These authors perform a highly sensitive transient absorption experiment to
resolve all three phases in the Chl b-->Chl a energy transfer and find time
constants of 175 fs, 600 fs and - 5 ps. Furthermore, they conclude from their
data that, probably, the 175fs component partly reflected energy transfer
between 'blue' and 'red' Chl bs.
73.
Trinkunas G, Connelly JP, Mi.iller MG, Valkunas L, Holzwarth AR:
A model for the excitation dynamics in the light-harvesting
complex II from higher plants. J Phys Chern B 1997, in press.
Giuffra E, Cugini D, Croce R, Bassi R: Reconstitution and
pigment-binding properties of recombinant CP29. Eur J
Biochern 1996, 238:112-120.
This paper describes the reconstitution of the minor chlorophyll a/b-binding
protein CP29, overexpressed in Escherichia coil. The recombinant pigmentprotein shows biochemical and spectroscopic properties identical to the
native CP29 complex, with a Chl a:Chl b ratio ef three. Also other stoichiometries yielded stable complexes.
74.
•
75.
•-
Peterman EJG, Monshouwer R, van Stokkum IHM,
van Grondelle R, van Amerongen H: Ultrafast singlet excitation
transfer from carotenoids to chlorophylls via different
pathways in light-harvesting complex II of higher plants. Chern
Phys Lett 199"7, 264:279-284.
Energy transfer from the xanthophylls to the chlorophylls is studied in trimeric
LHCII at 77K. No evidence for direct energy transfer from the xanthophylls to
Chl b is found, whereas efficient xanthophyll to Chl a energy transfer occurs
in 220fs. With preferential violaxanthin excitation (514 nrn) relative to lutein
excitation (500 nm), energy transfer to Chl as absorbing at - 6 7 0 nm is more
pronounced, compared with transfer to Chl as absorbing at - 6 7 6 nm. Following 514 nm excitation, the transfer from 670 to 676 nm occurs in 2.1 ps.
76.
•.
Connelly JP, MiJIler MG, Bassi R, Croce R, Holzwarth AR:
Femtosecond transient absorption study of carotenoid to
chlorophyll energy transfer in the light-harvesting complex II
of Photosystem II. Biochemistry 10gT, 36:281-287.
Energy transfer from the xanthophylls to the chlorophylls is studied in LHCII
trimers from Arabidopsis thafiana at room temperature. At 475 and 490 nm
excitation, energy transfer is mainly from xanthophyll to Chl b.
7"7.
Holzwarth AR, Schatz G, Brock H, Bittersmann E: Energy transfer
and charge separation kinetics in Photosystem I. Part 1:
Picosecond transient absorption and fluorescence study
of cyanobacterial Photosystem I particles. Biophys J 1993,
64:1813-1822.
78.
Hastings G, Hoshina S, Webber AN, Blankenship RE: Universality
of energy and electron transfer processes in Photosystem I.
Biochemistry 1995, 34:15512-15522.
79.
Du M, Xie X, Jia Y, Mets L, Fleming GR: Direct observation of
ultrafast energy transfer in PS1 core antenna. Chern Phys Lett
1993, 201:535-542.
80.
Gobets B, van Amerongen H, Monshouwer R, Kruip J,
R6gner M, van Grondelle R, Dekker JP: Polarized site-selection
spectroscopy of isolated Photosystem 1 particles. Biochim
Biophys Acta 1994, 1188:75-85.
81.
•
P&lsson LO, Dekker JP, Schlodder E, Monshouwer R,
van Gronclelle R: Polarized site-selective fluorescence
spectroscopy of the long-wavelength emitting chlorophylls in
isolated Photosystem I particles of Synechococcuselongatus.
Photosynth Res 1996, 48:239-246.
Isolated trimeric PS1 complexes of Synechococcus e/ongatus have been
studied in absorption and polarized fluorescence. Two types of long wavelength pigments are distinguished: C708 and C719. Their contribution to the
absorption spectrum corresponds to ~ 4 - 5 C708 and 5 - 6 C? 19 per PT00.
From low-temperature energy-selective polarized fluorescence experiments,
it is concluded that at ultra low temperatures C708 still is able to transfer excitation energy to C719 and furthermore that energy transfer among C719s
occurs.
82.
Valkunas L, Liuolia V, Dekker JP, van Grondelle R: Description of
energy migration and trapping in Photosystem I by a model
with two distance scaling parameters. Photosynth Res 1995,
43:149-154.
Kumazaki S, Ikegami I, Yoshihara K: Excitation and electron
transfer from selectively excited primary donor chlorophyll
(P700) in a Photosystem I reaction center. J Phys Chem A
1997, 101:597-604.
Primary processes in a Photosystem 1 reaction center are studied using subpicosecond fluorescence upconversion. In these enriched reaction centers
there are - 14 Chl as per PTO0. The 1 ps fluorescence anisotropy decay following selective PT00 excitation is ascribed to equilibration between P?00
and the surrounding antenna Chls. In the isotropic fluorescence decay, at
least two components can be distinguished: 2.2 ps (35%) and 15 ps (55o/o).
The fast and slow phases are interpreted in terms of charge separation
before and after full equilibration of the excited state, respectively. From
kinetic modeling, the intrinsic time constant for charge separation from PT00
is concluded to be < 4 ps.
83.
•
Trinkunas G, Holzwarth AR: Kinetic modeling of exciton
migration in photosynthetic systems. 3. Application of genetic
algorithms to simulations of excitation dynamics in threedimensional Photosystem I core antenna/reaction center
complexes. Biophys J 1996, 71:351-364.
This paper describes calculations of energy transfer and trapping in Photosystem 1 using a genetic algorithm. Various 3D models for the pigment
arrangement and the corresponding energy transfer dynamics are tested
for Photosystem 1. It is concluded that: the red pigments never are close
to P700; the red pigments are also never far away from P700 and tend
to cluster; the charge separation time is shorter than 1.2 ps; and the total
energy transfer time within the main antenna pool is < 1 ps.
84.
•
85.
o-
White NTH, Beddard GS, Thorne JRG, Feehan TM, Keyes TE,
Heathcote P: Primary charge separation and energy transfer
in the Photosystem I reaction center of higher plants. J Phys
Chern 1996, 100:12086-12099.
A detailed analysis is given of the Photosystem 1 trapping kinetics in a
Photosystem 1 core particle from plants. The A0--A 0 difference spectrum
takes 3 ps to form upon ?08 nm excitation reflecting the equilibration time
between the first charge separated state and the antenna excitation. The
equilibrated state decays in 2 0 - 2 0 ps. From extensive modeling, the intrinsic
rate of electron transfer is concluded to be 0.7 ps -1.
86.
•°
Melkozernov AN, Su H, Lin $, Bingham S, Webber AN,
Blankenship RE: Specific mutation near the primary donor
in Photosystem 1 from Chlamydamonasreinhardtii alters
the trapping time and spectroscopic properties of P7ooBiochemistry 1997, 36:2898-2907
Time-resolved absorption and fluorescence spectroscopy are used to investigate the energy and electron transfer processes in the detergent-isolated
Photosystem I core particles from the site directed mutant of Ch/arnydarnonas reinhardtiiwith the His656 of PsaB replaced by asparagine. There is
no indication that the mutation affects the spectral distribution in the antenna;
however, the excited state lifetime increases from - 3 0 ps to - 6 5 ps. It is
proposed that the excited state decay is limited by charge separation.