Single Molecule Detection in Solution. Edited by Christoph Zander, JoÈrg Enderlein, Richard A. Keller
Copyright c 2002 Wiley-VCH Verlag GmbH & Co. KGaA
ISBNs: 3-527-40310-8 (Hardback); 3-527-60080-9 (Electronic)
6
Spectroscopy of Individual Photosynthetic Pigment-Protein
Complexes
J. Wrachtrup, T. J. Aartsma, J. KoÈhler, M. Ketelaars, A. M. van Oijen, M. Matsushita,
J. Schmidt, C. Tietz and F. Jelezko
6.1
Introduction
Nature provides many examples of highly organized systems in which finely tuned
intermolecular interactions govern complex chains of events, where each link
in the chain is of vital importance for the living organisms. Detailed structural
information of these systems is becoming available at an ever increasing rate.
In many cases these structures are designed to facilitate ingenious, efficient
and effective mechanistic steps which are often intimately related to fundamental concepts of physics. One of the current challenges is to discover how the
basic principles of physics govern life processes. The foremost example of a
biological process which builds on fundamental physical concepts is the process of photosynthesis which sustains all life on earth. Photosynthesis is carried
out by many different organisms, i. e., plants, algae and photosynthetic bacteria
which all have developed highly efficient and optimized systems to collect
the light of the sun and to use the light energy as the driving force for
their metabolic reactions. Photosynthesis requires synergism in the interaction
between tens of different proteins. The structure of many of these proteins has
been solved or will be solved in the near future. This structural information
provides a strong basis for detailed studies of structure function relationships
of these proteins.
The photosynthetic light reactions basically consist of four primary steps: 1. absorption of (sun)light by a pigment, 2. ultrafast transfer of the excitation energy to
a `photoactive' pigment, 3. oxidation of this excited photoactive pigment, and 4. stabilization of the charge-separated state by secondary electron-transfer reactions.
The protein pigment complexes involved in the first two processes are often called
antenna or light-harvesting (LH) complexes. These complexes permit the organism
to increase its absorption cross-section for sunlight significantly. The primary electron transfer steps (processes 3 and 4) usually occur in a special protein pigment
complex which is referred to as the reaction center (RC).
185
186
6.1 Introduction
All photosynthetic light reactions take place in specialized proteins, which bind
the pigment and/or electron transfer cofactors at optimized binding sites and thus
ensure the high efficiency of the photosynthetic light reactions in the various organisms. The function of photosynthetic protein pigment complexes is determined not only by the intrinsic properties of the pigments involved, but also to
a large extent by the intermolecular interactions between those pigments [1,2]:
the interactions between the chromophores are directly responsible for the primary
processes of energy transfer and charge separation. These interactions depend
strongly on relative orientations and distances, and specific protein pigment interactions may also play a role. In `primitive' photosynthetic organisms, such as the
well-studied photosynthetic purple bacteria, the light reactions are carried out
by only 5 7 different types of proteins, whereas in green plants more than
50 types of proteins are involved in two different light reactions, carried out by
the so-called photosystems I and II (PSI and PSII). This large number of proteins
reflects the complexity of the organism, and is needed for efficient photosynthesis
under a wide variety of different environmental conditions.
6.1.1
Bacterial Photosynthesis
Photosynthetic bacteria show a more simplified structure of the antenna and core
complexes than green plants. For example, in most purple bacteria, the photosynthetic unit (PSU) present in the membrane contains, besides the RC, only two
types of antenna complexes, i. e., light-harvesting complex 1 (LH1) and light-harvesting complex 2 (LH2). LH1 is known to form a ring-like structure which encloses the RC [3, 4], whereas LH2 is not in direct contact with the RC, but transfers
the energy to the RC via the LH1 complex [5, 6]. Figure 6.1 shows a model of the
bacterial PSU of the purple bacteria Rb. sphaeroides where the organization of the
different protein pigment complexes is depicted.
Figure 6.2 shows the low-temperature absorption spectrum of membrane fragments of the purple bacterium Rhodopseudomonas (Rps.) acidophila. In the nearinfrared it shows broad bands at 800 and 870 nm and a small band at 910 nm.
The first two bands are assigned to the LH2 antenna, whereas the third band is
assigned to the LH1 antenna. Since the LH2 complex absorbs at shorter wavelengths, the light-harvesting system functions as an energy funnel towards the
RC. This transfer of energy from LH2 to LH1 and subsequently to the RC occurs
in vivo on a timescale of 5 to 50 ps [1], very fast compared to the excited state decay
of isolated LH2 complexes, which corresponds to a lifetime of about 1 ns.
6 Spectroscopy of Individual Photosynthetic Pigment-Protein Complexes
Figure 6.1. Model for the photosynthetic unit
(PSU) of the purple bacterium Rhodobacter
sphaeroides. The PSU consist of two types of
protein pigment complexes: the photosynthetic reaction center (RC) and the light-harvesting complexes (LH). The main function of
the light-harvesting complexes is to gather light
energy and to transfer this energy to the
reaction centers for the photo-induced redox
processes. In most purple bacteria, the photosynthetic membranes contain two types of lightharvesting complexes: light-harvesting complex
1 (LH1) and light-harvesting complex 2 (LH2).
While LH1 is tightly bound to the RC, LH2 is
not directly associated with it, but transfers
energy to the RC via LH1. Figure reproduced
with permission from Ref. [6].
1
absorbance (A.U.)
LH2
LH1
0
750
800
850
900
950
wavelength (nm)
Figure 6.2. Absorption spectrum of a solution of membrane fragments of Rps. acidophila.
The membrane fragments were diluted in a standard LH2 buffer in the presence of 67 % glycerol.
The spectrum was taken at 10 K.
187
188
6.1 Introduction
The light-harvesting complex II
The LH2 complex is a peripheral antenna complex which serves to absorb light and
transfer the excited state energy to the LH1 RC complex. The high-resolution
X-ray structure of the LH2 complex [7], and the lower resolution structural information for LH1 [4], show a remarkable symmetry in the arrangement of the
light-absorbing pigments in their protein matrix. The basic building block of
LH2 is a protein heterodimer (ab), which binds three BChl a pigments and one
carotenoid molecule. The LH2 complex of Rps. acidophila consists of nine such
ab polypeptide heterodimers. The 3-dimensional structure is depicted in Fig.
6.3a, where the upper part of the figure shows the pigment protein complex as
a whole while the lower part shows only the BChl a pigments for clarity. A very
pronounced feature of the complex is that, at least in the crystalline form, it has
a C9, nine-fold rotational symmetry [7]. Two BChl a pigment pools can be distinguished and are labeled B800 and B850, according to their room-temperature absorption maxima in the near-infrared. The B800 ring consists of nine pigments,
which have their molecular plane perpendicular to the symmetry axis. The B850
ring consists of nine repeating pairs of a- and b-bound pigments, which are tightly
organized as the blades of a turbine with their molecular planes parallel to the symmetry axis.
Upon excitation, energy is transferred from the B800 to B850 pigments in 1 to
2 ps [8 12], while energy transfer among the B850 molecules is an order of magnitude faster [13 15]. Much information about the intermolecular interactions can
be obtained from the electronic structure and excited-state dynamics of the pigment-molecules in isolated antenna complexes. For this reason, all forms of optical
spectroscopy are very highly instrumental in elucidating the electronic structure
and the dynamics of the optically excited states. Nevertheless, even isolated protein pigment complexes of photosynthetic systems are rather complex, and it
has proven difficult to analyze the excited state properties of these systems in detail. This is mainly caused by the pronounced disorder in these types of systems,
which masks details in the steady-state optical spectra, even at low temperature
(Fig.6.3b). To eliminate this complication, we have successfully applied the technique of single-molecule spectroscopy to study the absorption spectrum of LH2.
We were able to resolve details which are not visible in the bulk spectra, and the
results offer a new view on the electronic structure of LH2 [16 21]. Here we review
the progress that has been made in this work. It is shown that the LH2 complex is
an ideal model system for studying the basic principles of exciton interactions in
the strong and weak coupling limit, and how they affect the observed optical
spectra.
6.1.1.1
6 Spectroscopy of Individual Photosynthetic Pigment-Protein Complexes
a
y
z
x
x
10 Å
absorbance (A.U.)
b
780
800
Figure 6.3. (a) Structure of the LH2 complex
from Rps. acidophila as determined with X-ray
diffraction [7]. The left part of the figure displays a top view of the assembly and the right
part a side view. The upper part shows the
whole protein pigment complex, the lower part
only the BChl a pigments. The pigments are
820
840
860
wavelength (nm)
880
arranged in two concentric rings commonly
termed B800 (yellow) and B850 (red).
(b) Fluorescence-detected absorption spectrum
of an ensemble of LH2 complexes at 1.2 K. The
spectrum was measured at 20 W cm 2 with
LH2 dissolved in polyvenyl alcohol (PVA)-buffer
solution and spin-coated on a LiF substrate.
189
190
6.1 Introduction
6.1.2
The Photosynthetic Unit of Green Plants
Higher plants and algae, in contrast to purple bacteria, generate oxygen. To do this
they utilize two photosystems, termed PSI and PSII. Both PSII and PSI contain,
among others, two chlorophyll A molecules, and both undergo light-driven transport of an electron across the thylakoid membrane. These chlorophylls in the two
reaction centers differ in their light-absorption maxima (680 nm for PSII, 700 nm
for PSI) because of differences in the protein environments, and thus the reactioncenter chlorophylls are often denoted P680 and P700. More importantly, the two
photosystems differ significantly in their functions see Fig. 6.4. PSII splits
water: the absorption of a photon causes an electron to move from P680 to an
acceptor quinone on the stromal surface, and the resultant positive charge on
the P680 strips electrons from the highly unwilling donor H2O, forming O2
and protons that remain in the thylakoid lumen and form part of the proton motive force. The electrons in the acceptor quinone move via a series of carriers, to
the electron-donor site on the luminal surface of the PSI; during this process
additional protons are transported into the thylacoid lumen. PSI uses the energy
of an absorbed photon to transfer the electron to ferridoxin, an Fe S-containing
acceptor protein on the stromal surface. From there, electrons are passed to the
ultimate acceptor, NADP‡, forming NADPH. Associated with both PSI and
PSII are light-harvesting complexes that absorb light and transfer the absorbed
energy to the reaction center chlorophylls.
Figure 6.4. Simplified functional organization of the protein complexes involved in energy,
electron and proton transfer in oxygenic photosynthesis.
6 Spectroscopy of Individual Photosynthetic Pigment-Protein Complexes
Photosystem II and light-harvesting complex II
The PSII complex consists of approximately 25 different proteins, referred to as
PsbA-W or Lhcb1-6, according to the genes that encode them. More than 20 of
these are integral membrane proteins, with an estimated total of about 50 transmembrane helices. At the heart of this multisubunit complex is the PSII reaction
center composed of D1 and D2 proteins. These two proteins bind the aforementioned cofactors that are involved in the light-driven primary and secondary electron transfer processes. Upon illumination P680 is initially excited to its first singlet state where it rapidly donates the energized electron to a pheophytin (Phe) molecule to form the radical pair state P680‡Phe . Phe then passes an electon to a
bound plastoquinone molecule (QA) within 200 ps, while P680‡ is reduced in
nanoseconds by a redox-active tyrosine. Within milliseconds QA reduces a second
plastoquinone, QB, and the tyrosine is reduced by a four-atom manganese oxide
cluster located on the lumenal surface of the PSII complex. This manganese cluster is positioned at the center of the oxygen evolving complex of PSII. Surrounding
the D1 and D2 reaction center proteins are the other PSII subunits. These include
the chlorophyll-a-binding proteins, CP43, CP47 internal antenna, cytochrome b559
and the minor subunits, form the PSII core complex. In higher plants and green
algae, an additional outer light-harvesting system is composed of proteins that bind
both chlorophyll A and B. These proteins make up the majority of the light-harvesting system and are known as LHCII. LHCII is organized as a trimer and its structure has been solved to 3.4 AÊ. The other chlorophyll A/B binding proteins, Lhc4,5
and 6, also known as CP29,CP26 and CP24, respectively, bind less chlorophyll B
than LHCII and are thought to exist as monomers. They seem to function as a conduit for the transfer of excitation energy form the LHCII trimers to the reaction
center via CP47 and CP43. The remaining proteins either serve as chlorophyll
carriers (PsbS) or may protect the reaction center from photodamage (PsbE
and F) or have masses less than 10 kDa with unknown function. Although a
high-resolution structure of PSII has not yet been determined, considerable progress towards this goal has been made recently by cryoelectron microscopy of
two-dimensional crystals.
LHCII is the most abundant protein in chloroplasts of green plants and green
algae. It binds about 50 % of all chlorophyll in these organisms. The protein has
a mass of 25 kDa and is isolated as a trimeric complex. The resolution achieved
in cryoelectron microscopy is sufficient to determine the position of 80 % of
the aminoacid residues, 12 chlorophyll and 2 carotenoid molecules [22]. Figure
6.5 shows an LHCII trimer in the top view. A monomeric subunit is made up
of one protein spanning the membrane three times.
A short a-helical piece is parallel to the membrane. The carotenoids (luteins) not
shown in Fig. 6.5 are located in the center of the complex and also span the
complex from the lumenal to the stromal side. Because of their central position
the luteins are essential for the complex. A reconstruction of the complex without luteins is not possible. The carotenoids extend the absorption of the LHCII
complex to the blue. However their major role most probably is to protect the
chlorophylls against photochemical degradation. Currently the resolution of
6.1.2.1
191
192
6.1 Introduction
Figure 6.5.
Structure of LHC2 from electron microscopy [22].
structural data is not sufficient to distinguish between Chl A and Chl B molecules
or to determine the orientation of the chlorophyll molecules in the plane. The
attribution of Chl A and B molecules is thus based on phenomenological considerations. Because of fast energy transfer from Chl B to A, Chl A molecules are
populated with the highest probability. They thus need to be protected against
photodestruction more efficiently than Chl B molecules. Hence the seven Chl
sites closest to the carotenoids have been attributed to Chl A. There is considerable
discussion on this point in the literature. Recently, it has been proposed that one or
two pairs of Chl A/B molecules may have to be exchanged. A variety of picosecond
and femtosecund studies have been performed to explore the energy transfer
dynamics in LHCII.
The first available structure data led to a wealth of spectroscopic investigations to
unravel the complicated interplay among the Chls in monomers and trimers of the
complex [23 28]. The Qy-region of the bulk absorption spectrum of LHC-II is relatively unstructured showing two broad bands around 650 nm attributed to the
absorption of Chl B and 675 nm attributed to Chl A. However, several room and
low temperature techniques such as linear and circular dichroism,[29] or non-linear
polarization spectroscopy in the frequency domain [30] reveal up to 11 spectral subbands within the Qy-absorption band. A satisfying interpretation of this sub-structure as a result of Chl protein and/or Chl Chl interaction is still missing. At
low temperature (I 5 K) the energetically lowest band lies at 680.4 nm and hole
burning reveals a line width of 0.037 cm 1 [27] for this transition.
From femtosecond spectroscopy it is known that the Chl B Chl A energy transfer occurs within the monomeric subunit of LHC-II trimers [24]. This is consistent
with the weak coupling between Chl molecules among different sub-units [31]
6 Spectroscopy of Individual Photosynthetic Pigment-Protein Complexes
owing to the relatively large distance between the Chls in different monomers. The
coupling strength between Chl molecules of different subunits is thought to be less
than 5 cm 1 [27].
Photosystem I
PSI is the most complex photosynthetic light-harvesting plus electron transfer system for which a structure is now available. The functional unit of the PSI core of
Synechococuss elongatus consists of 11 subunits, including the two major subunits
PsaA and PsaB, each having a molecular weight of around 80 kD with known
sequence, and each binding roughly 100 chlorophyll A and 10 25 carotenoid molecules plus 3 FeS clusters. The structure has been resolved to 4 AÊ, and the position
of 90 Chls have been determined. As in the case of LHCII no information about
the direction of the transitions dipoles is available. The core of PSI contains 22
transmembrane helices which exhibit C2 symmetry. The electron transfer chain
is embedded in a structure of 10 transmembrane helices with an arrangement
close to that of the L and M subunits in the purple bacteria reaction center. All
other 90 antenna Chl A molecules are dispersed in a band around this core and,
for a large part, are associated with the remaining 6 transmembrane helices on
each of the large subunits. A pair of chlorophylls located in the central area of
the photosynthetic unit was assigned to the primary electron donor P700. The electronic transitions of all major Chl antenna molecules are at higher transition energies than those of P700 and form an absorption band around 680 nm (C680).
However there are also a considerable number of chlorophyll molecules absorbing
at wavelength longer than 680 nm. These chlorophylls are often referred to as the
red pool. Different spectroscopic methods have been successfully applied to unravel the energy pathway in PSI aggregates. Time resolved absorption measurements
indicate that the energy equilibration between the main C680 and the red pool of
pigments takes place on a sub-picosecond timescale. Several models have been
applied to describe energy trapping dynamics in PSI. A trap-limited model predicts
that the overall kinetics in PSI is determined by the rate of charge separation in the
reaction center part. An alternative mechanism was proposed by van Grondelle et
al. [1] and several other authors. They claimed that energy trapping in the low-energy Chl pool is dominating in PSI kinetics (transfer-to-trap limited model). Under
ambient conditions energy captured by low-energy Chls can be efficiently transferred to P700 via thermally populated vibrational levels. Recent results based on
charge-transfer induced absorption changes show high efficiency of this process
at physiological temperatures [32]. The photophysical properties of red-pool Chls
have been subject to intense spectroscopic studies during the last few years (for
a recent review see Ref. [33]). The amount of PSI low-energy pigments and their
spectral forms depend on the type of photosynthetic unit. Deconvolution of Synechocystis mutant absorption spectra shows that the red pigments pool cannot be
associated with peripheral protein subunits [34]. Recently reported absorption spectra of Synechococcus elongatus show that low-energy Chls may belong to the connecting domain of the monomeric PSI within the trimer [32]. It was estimated
6.1.2.2
193
194
6.2 Fluorescence-excitation Spectroscopy
that five Chls C708 and six chlorophylls C719 contribute to the absorption in this
spectral region [35]. The spectral assignment of the red-most pigments is tentative
because of the strong overlap of spectral bands [35].
For all the chlorophylls except two, the distance to any of the pigments in the
electron transfer chain exceeds 1.6 nm, 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 port for excitation energy. Trapping in PS1 is fast
(20 25 ps) and charge separation is irreversible. Using fluorescence depolarization
measurements it has been estimated that the major hopping process within PS1
occurs on a timescale of 100 fs. Equilibration between pool and very red chlorophyll molecules, absorbing between 720 and 730 nm, seems to occur on a timescale of a few ps. The process of energy transfer to P700 must occur at the
same rate as this equilibration between core and red pigments. Even at very low
temperatures where escape from the red states is impossible, a reasonably high
quantum yield for charge separation is still observed. This has led to a model in
which essentially all sites within the PS1 core are more or less equally efficient
in transferring their energy to P700 which may be viewed as a 3D model version
of the 2D model operating in purple bacteria.
Absorption and fluorescence emission bands of long-wavelength pigments are
strongly inhomogeneously broadened. Hole-burning spectroscopy has proven to
be a powerful method for probing the electronic structure and photophysical properties of many photosynthetic complexes [36]. Hole-burning data provide evidence
for strong electron phonon coupling in a reaction center containing photosynthetic units if compared to light-harvesting antenna (LH2, LHCII)[37].
6.2
Fluorescence-excitation Spectroscopy of Individual Light-Harvesting II Complexes
of Rhodopseudomonas acidophila
6.2.1
Experimental
Sample preparation
The LH2 complexes were isolated from the membranes of the purple non-sulfur
bacterium Rps. acidophila (strain 10050) as described elsewhere [38]. Hydrolyzed
polyvinyl alcohol (PVA) (BDH; Mwˆ125,000) was purified over a mixed resin in
order to remove ionic impurities [39]. A polymer-LH2 solution was prepared by
adding 1 % (wt/wt) purified PVA to a solution of 5x10 11 M LH2 in buffer
(10 mM Tris, 0.1 % LDAO1), 1 mM EDTA2), pH 8.0). A drop of this solution was
spin-coated onto a LiF substrate by spinning it for 15 s at 500 rpm and 60 s at
2000 rpm, producing a high quality film with a thickness of less than 1 mm. The
sample was mounted in a liquid-helium bath cryostat and cooled to 1.2 K.
6.2.1.1
1) LDAO ± Lauryldimethylamine oxide
2) EDTA ± Ethylenediaminetetraacetic acid
6 Spectroscopy of Individual Photosynthetic Pigment-Protein Complexes
Set-up
The sample was excited at a wavelength of 800 nm by focusing the output of a continuous wave, tunable Ti-Sapphire laser (Spectra Physics 3900S) onto the sample
through a simple planoconvex lens with large focal length (f ˆ 250 mm) which creates a 100 mm large spot on the polymer film (Fig. 6.6a, top). The fluorescence
emitted by the LH2 complexes was collected by an objective consisting of an aspheric singlet lens (NA 0.55, working distance 850 mm) mounted in the cryostat close
to the sample, and focused on a red-sensitive, back-illuminated CCD camera (Princeton Instruments, 512SB). Residual laser light was blocked by bandpass filters,
which only transmitted a 20 nm wide spectral window around 890 nm, where
the fluorescence intensity of LH2 is maximal. From the wide-field fluorescence
images obtained by the CCD camera (Fig. 6.6a, bottom) a spatially isolated LH2
complex was selected for further spectroscopic investigation. After recording a
wide-field image, the microscope was switched to the confocal mode by simply rearranging a mirror (Fig. 6.6b, top). In this mode the excitation light is passed
through the objective lens in the cryostat, illuminating a diffraction-limited excitation volume (less than 1 mm3) of the sample. The scan mirror in the confocal microscope was then adjusted such that this excitation volume exactly coincided with
the position of a selected LH2 complex observed in the wide-field image. The fluorescence, collected by the same objective lens that illuminated the excitation spot,
was focussed on a confocally placed avalanche photodiode, for single-photon count6.2.1.2
a
b
TiSa
pump laser
pump laser
TiSa
sample
sample
objective lens
objective lens
scan mirror
f = 250 mm
cut-off filter
CCD
cryostat with
liquid helium
(~1.2 K)
cut-off filter
excitation pinhole
detection pinhole
10 mm
fluorescence (cps)
avalanche
photo diode
100
780
800
820
840
860
880
excitation wavelength
Figure 6.6. Schematic representation of the
two experimental arrangements.
(a) Wide-field. In the lower part a typical
wide-field image is depicted with an excitation
wavelength of 800 nm and detection at
890 nm.
(b) Confocal. In the lower part a typical fluorescence-excitation spectrum is shown.
195
196
6.2 Fluorescence-excitation Spectroscopy
ing (EG&G, SPM 200). A fluorescence-excitation spectrum was obtained by scanning the excitation wavelength, while detecting the fluorescence at 890 nm (Fig.
6.6b, bottom). In the confocal-detection mode the superior background suppression allowed recording of fluorescence-excitation spectra with high signal-to-background ratios [18].
To minimize light-induced fluctuations of the fluorescence intensity on a timescale of a single scan, the spectra were obtained by rapidly scanning the whole
spectral range and storing the different traces separately [17,19]. With a scan
speed of the laser of 3 nm s 1 and an acquisition time of 10 ms per data point,
this yields a nominal resolution of 0.5 cm 1 ensuring that the spectral resolution
is limited by the spectral bandwidth of the laser (1 cm 1). Most spectra presented
here were obtained by the summation of about 70 scans.
To examine the polarization dependence of the spectra, a 1¤2 l plate was put in
the confocal excitation path. For each individual complex six spectra were obtained
at polarization intervals of 30 h.
6.2.2
Results and Discussion
In Fig. 6.7 the fluorescence-excitation spectra of several individual LH2 complexes
are shown for comparison. The upper trace shows the fluorescence-excitation spectrum taken from a bulk sample (dashed line) together with the spectrum that results from the summation of the spectra of 19 individual LH2 complexes (solid
line). The 2 spectra are in excellent agreement and both feature two broad structureless bands around 800 and 860 nm corresponding to the absorptions of the
B800 and B850 pigments of the complex. By measuring the fluorescence-excitation
spectra of the individual complexes remarkable features become visible which are
obscured in the ensemble average. In particular, a striking difference between the
B800 and B850 bands becomes evident. The spectra around 800 nm show a distribution of narrow absorption bands, whereas in the B850 spectral region 2 3 broad
bands are present. Before we discuss these observations in detail, a theoretical
framework will be provided in order to understand the different features that characterize the individual LH2 spectra.
Figure 6.7. Fluorescence-excitation spectra of LH2 complexes of Rps. acidophila. The
top traces show the comparison between an ensemble
spectrum (dashed line) and the
sum of spectra recorded from
19 individual complexes (solid
line). For each complex this
included 6 individual spectra
that were obtained at different
excitation polarizations. The
lower 5 traces display spectra
from single LH2 complexes. All
spectra were measured at 1.2 K
at 20 W cm 2 with LH2 dissolved in a PVA-buffer solution
[20].
fluorescence (cps)
6 Spectroscopy of Individual Photosynthetic Pigment-Protein Complexes
200
780 800
820
840
860
880
wavelength (nm)
Localized vs. delocalized excitations
One of the main functions of the LH2 complex is to transfer energy in the form of
an optically excited state. This transfer of energy originates from the electronic
interaction between the BChl a pigments. In the simplest approximation of the
Coulomb interaction between the electrons of neighboring pigments, the BChl a
may be described as interacting point-dipoles having specific mutual orientations
and center to center distances [40]. Experimental values for the orientation and distances can be deduced from the crystal structure.
In such a simple model there are three main factors that determine the electronic structure and the resulting spectroscopic properties of the LH2 complex:
6.2.2.1
1. The transition energy or site-energy of each pigment.
2. The strength of the electronic interaction between the pigments.
3. The direction and magnitude of the transition dipole moments.
197
198
6.2 Fluorescence-excitation Spectroscopy
These factors can be combined into the following Hamiltonian:
Hˆ
N
P
nˆ1
…E0 ‡ DEn †jnihnj ‡
N
P
nˆ1
…V0 ‡ DVn †‰jnihn ‡ 1j ‡ H.c.Š ,
…1†
where E0 denotes the site-energy of each pigment and V0 the nearest-neighbor interaction in a perfect circular system. H. c. stands for the Hermitian conjugate. The
energy levels corresponding to the overall system are now determined by the
strength of the coupling and the site-energies of the system, while the distribution
of oscillator strength of the transitions connecting the energy levels depends also
on the orientation of the dipole moments.
Local variations in the protein environment of the binding sites give rise to static
disorder in the site-energies of the pigments, represented by a random shift of DEn.
This disorder is referred to as diagonal disorder and is usually modeled by a Gaussian distribution of site-energies [41]. Any structural disorder or deformation in the
ring will cause deviations from the perfect C9 symmetry and results in a variation
of the interactions, represented by DVn. This disorder is referred to as off-diagonal
disorder.
Two limits of the dipolar interaction can be distinguished, that of the weak and
the strong coupling. This is depicted schematically in Fig. 6.8 for two interacting
dipoles, which corresponds to N ˆ 2 in Eq. (1). In the weak coupling case the interaction between the transition dipoles is much smaller than the difference in siteenergies of the two pigments (DE1,2 ˆ jDE1 ± DE2j). As a result the excitations are
mainly localized on individual pigments. In the extreme limit where V1 II DE1,2 a
complete localization of excitation occurs. The spread of the absorption bands reflects directly the spread in site-energies or diagonal disorder.
In the strong coupling limit the interaction is much larger than the difference in
site-energies of the two pigments. As a result the wavefunctions of the individual
pigments are not eigenfunctions of the Hamiltonian (Eq. (1)). Instead, linear
superpositions of the individual excited state wavefunctions form the set of eigenfunctions, or Frenkel exciton states, leading to a manifold of energy levels as indicated in Fig. 6.8b. In the case of degeneracy where V1 ii DE1,2, the excitation is
completely delocalized, i. e., the wavefunctions have equal amplitudes on both pigments. The splitting between the two exciton states is twice the electronic interaction between the monomer pigments. The distribution of oscillator strength over
the two exciton levels depends on the mutual orientation of the transition dipole
moments. If the dipolar coupling and the site-energy difference are similar in magnitude, the behavior is expected to be intermediate between the extremes of weak
and strong coupling.
6 Spectroscopy of Individual Photosynthetic Pigment-Protein Complexes
a
weak coupling:
V1 << DE1, 2
E0, 2
DE1, 2
E0, 1
0
2
1
2
1
absorbance
b
strong coupling:
V1 >> DE1, 2
exciton
V1
E0+V1
E0, 2
E0, 1
2V1
E0-V1
0
1
2
Figure 6.8. Effects of the interaction (V1) and
site-energy difference (DE1,2 ˆ jDE1 ± DE2j) on
the energy levels and spectra of two monomer
pigments 1 and 2.
(a) In the limiting case of weak coupling the
interaction between the pigments is relatively
small and the resulting absorption spectrum is
independent of the mutual orientation of the
pigments.
1+2
absorbance
(b) In the limiting case of strong coupling there
is a relatively small difference in site-energies
and as a result two new, delocalized exciton
states are created belonging to the dimer
(1‡2). Spectra are shown for two different
orientations of the transition dipole moments,
indicated by small arrows.
Exciton model for a circular aggregate
For a ring of closely interacting pigments, like the B850 band of LH2 it is possible
to calculate the energies of the excited states (exciton states) for an unperturbed
B850 ring using Eq. (1) with N ˆ 18 and DEn ˆ DVn ˆ 0. The energy levels are
depicted in Fig. 6.9, manifold 2. It consists of 16 pairwise degenerate states, labeled
by their quantum number k (k ˆ e1, e2, e3, e4), and two nondegenerate states
(k ˆ 0). The manifold is separated into a lower and upper branch owing to the interaction within the dimer (Fig. 6.9, manifold 1). Due to the circular symmetry and
orientation of the transition dipoles in the ring almost all oscillator strength is concentrated in the low-energy k ˆ e1 degenerate pair. The mutual orientation of the
transition moments of these two states is orthogonal in the plane of the ring, as
6.2.2.2
199
6.2 Fluorescence-excitation Spectroscopy
2
1
2
k ± 1k = 0
k±2
1
V1~-250 cm
-1
k±3
1
energy (units V)
200
k±4
2
0
-1
2V1
k±4
V2~-230 cm-1
k±3
V1~-250 cm-1
k±2
-2
~9 Å
k±1
k=0
Figure 6.9. Schematic representation of the
energy level scheme of the excited state manifold of a dimer (manifold 1) and of the B850
ring of LH2 (manifold 2). The mutual orthogonal orientation of the transition moments of the
low-energy k ˆ e1 degenerate pair is indicated
y
x
by the red and blue arrows. The transitiondipole moments of the pigments are given by
the small, black arrows. The interaction within
the dimer is V1 (intradimer), whereas the interaction between the dimers is V2 (interdimer).
For a detailed description see Ref. [20,21].
indicated by the red and blue arrow. A more extensive description of the exciton
manifold for the B850 ring can be found elsewhere [20, 21, 42, 43].
B800 band
The fluorescence-excitation spectra of single LH2 complexes show a wide distribution of narrow absorption bands in the B800 region of the spectrum. These bands
are spread over the whole inhomogeneously broadened ensemble spectrum. To understand the origin of these bands we should compare the dipolar coupling between the pigments and their spread in site-energies (diagonal disorder). From
the crystal structure the dipolar coupling between neighboring pigment is estimated to be about 24 cm 1 (Fig. 6.10a) [42]. The variation in site-energies as
estimated from the inhomogeneously broadened ensemble spectrum is around
180 cm 1. This yields a V/DE ratio of about 0.l which is clearly in the weak coupling
regime (Fig. 6.8a) and a strong localization of excitation on the individual BChl a is
expected [43,44]. We therefore attribute the pattern of spectral bands to absorptions
of more or less individual pigments in the B800 band that are separated in their
6.2.2.3
6 Spectroscopy of Individual Photosynthetic Pigment-Protein Complexes
a
V~-24 cm
-1
21 Å
y
x
fluorescence (cps)
b
Figure 6.10. (a) Schematic
representation of the transition
dipole moments of the B800
pigments of LH2.
(b) Dependence of the B800
fluorescence-excitation spectrum of a single LH2 complex
from Rps. acidophila on the
polarization of the incident radiation. The polarization vector
has been changed in steps of
30h from one spectrum to the
next. The vertical scale is valid 100
for the lowest trace, all others
are displaced for clarity. Figure
780
reproduced with permission
from Ref. [18].
790
800
810
820
wavelength (nm)
spectral positions due to differences in their local environment (inhomogeneous
broadening). Based on the kinetic properties of the excited triplet and singlet states
of the BChl a pigments in the LH2 complex we estimate the maximum emission
rate to be about 2x105 photons s 1, similar to the value obtained by Bopp et al. [45].
The collection efficiency of our microscope is approximately 0.05 % which yields a
fluorescence count-rate of roughly 50 100 counts s 1 for the emission of a single
BChl a pigment, in agreement with our observations.
201
202
6.2 Fluorescence-excitation Spectroscopy
Polarization The interpretation of localized excitations in the B800 band is corroborated by the strong dependence of the relative intensities of these lines on
the polarization of the incident radiation. In Fig. 6.10b seven fluorescence-excitation spectra of the B800 band of an individual LH2 complex are shown, each
taken with a different orientation of the polarization vector of the exciting laser
light. It is seen that the intensities of the absorption lines vary appreciably upon
changing the polarization. This is what one would expect if the excitations are
largely localized on the individual BChl a pigments, because their transition dipole
moments are arranged in a circular manner and, as a result of this, all have different orientations. When analyzing the polarization-dependent spectra of a single
LH2 complex, one would expect to observe nine absorption lines, corresponding
to localized excitations of the nine individual BChl a pigments. However, in
these combined spectra typically only six to seven absorption lines were found,
supporting the conjecture that a slight delocalization of the excited state occurs
over two or three neighboring BChl a pigments in the B800 manifold with a
concomitant redistribution of oscillator strength. This arises from occasional
(near)-degeneracy of the site-energies of adjacent B800 pigments.
Intra- and intercomplex disorder The B800 spectra of Fig. 6.7 and Fig. 6.10b reveal
significant variations in the spectral distribution of the individual resonances between different LH2 complexes as well as for the average position of the whole
line pattern. Due to the many conformational substates of proteins, one expects
strong variations of the electrostatic protein pigment interaction, resulting in a
distribution of absorption frequencies of the individual pigments. In general we
have to consider two independent contributions to the spectral distribution. First
the variation of site energies of BChl a pigments within the same LH2 complex
which is referred to as intracomplex heterogeneity or diagonal disorder. Secondly,
for different complexes, the changes in the spectral position of the weighted average (spectral mean) of the whole spectrum which is called intercomplex heterogeneity or sample inhomogeneity. Obviously, the study of individual LH2 complexes
allows one to discriminate between these two contributions and to investigate them
separately.
In order to find a measure for the intercomplex heterogeneity we have defined
the spectral mean value, n, of the fluorescence-excitation spectrum of a single
LH2 complex by
P
nˆ
i
I(i) n(i)
P
,
I(i)
…2†
i
where I(i) denotes the fluorescence intensity at datapoint i, n(i) the spectral position
corresponding to datapoint i, and the sum runs over all datapoints of the spectrum.
The histogram for n, obtained from the spectra of 46 complexes, is depicted in
Fig. 6.11a and has a width of about Z120 cm 1.
6 Spectroscopy of Individual Photosynthetic Pigment-Protein Complexes
intercomplex heterogeneity
intracomplex heterogeneity
n (nm)
790 795 800 805 810
b
a
8
occurrence
8
6
6
4
4
2
2
0
12600 12500 12400 12300
n (cm-1)
Figure 6.11. (a) Distribution of the spectral
mean for 46 LH2 complexes in the B800 region
of the spectrum featuring the amount of intercomplex heterogeneity.
0
0 20 40 60 80 100120
sn (cm-1)
(b) Distribution of standard deviations for the
spread of absorption lines in the individual
fluorescence-excitation spectra for the same 46
LH2 complexes. Figure reproduced with permission from Ref. [19].
The intracomplex heterogeneity or diagonal disorder is extracted from the data
by calculating the standard deviations s n of the intensity distributions in the individual spectra
n2 ]1/2 ,
s n ˆ [n2
…3†
where n2 is given by
P
n2 ˆ
i
I(i) ‰n(i)Š2
P
.
I(i)
…4†
i
The result is shown in Fig. 6.11b. The distribution for s n is centered at a value of
about 55 cm 1. The full width at half maximum (FWHM) of the distribution of
site-energies is obtained by multiplying this value by a factor of 2.36. This yields,
for the FWHM of the diagonal disorder, a value of 130 cm 1. Clearly, an ensemble
spectrum reflects the convolution of both contributions to the heterogeneity. From
our data we expect for the B800 band a total inhomogeneous linewidth of about
180 cm 1, in good agreement with the results from bulk spectra of LH2 of Rps.
acidophila taken at 1.2 K.
203
6.2 Fluorescence-excitation Spectroscopy
B850 band
In all spectra of the B850 band two broad absorption lines around 860 nm are observed. For 40 % of the complexes these two lines are accompanied by a weaker
transition at higher energy (Fig. 6.7). By exciting the complexes with linearly polarized light at different polarization angles, the relative orientation of the transitiondipole moments associated with the two prominent absorptions around 860 nm
was determined. For all the complexes studied we found that these transitiondipole moments were mutually orthogonal (Fig. 6.12a). The high-energy absorption band (Fig. 6.12a, complex 3) appears to be homogeneous, and shows only a
slight dependence of its total intensity on the polarization of the exciting laser
radiation.
In Fig. 6.12b the distributions of the spectral positions of the three observed transitions are depicted. For the red absorption the distribution is centered at 864 nm.
The transition next highest in energy, featuring a transition-dipole moment oriented perpendicular to the former one, shows a distribution centered around
6.2.2.4
285 cm-1
110 cm
b
a
10
1
200
100
5
0
0
10
2
100
50
occurrence
fluorescence (cps)
204
0
3
-1
864
856
5
0
5
841
600
300
0
820
840
860
880
0
820
wavelength (nm)
Figure 6.12. (a) Fluorescence-excitation spectra of the B850 spectral region of three individual LH2 complexes. For each complex two
spectra with mutually orthogonal polarization
(arrows) of the excitation are shown.
(b) Distribution of the spectral positions for
nineteen complexes of the three observed
840
860
880
wavelength (nm)
bands. The average separation between the 864
and 856 bands (dEe1) is 110 cm 1 and the
energy separation between the spectral mean of
these two bands and the higher energy 841
band is 285 cm 1. The experimental conditions
are the same as in Fig. 6.7. Figure reproduced
with permission from Ref. [20].
6 Spectroscopy of Individual Photosynthetic Pigment-Protein Complexes
856 nm. The high-energy absorption present in some of the spectra is distributed
around 841 nm as can be seen in the lower panel of Fig. 6.12b The corresponding
bands in the single molecule spectra will be referred to as the 864, 856 and 841 nm
bands, respectively. The average energy separation between the 841 band and the
spectral mean of the two mutually perpendicularly polarized transitions is
285 e 35 cm 1. The linewidths of the 864 and 856 nm absorption bands in the
individual spectra range from 50 to 250 cm 1 with an average value of 120 cm 1.
This corresponds to a value for the total dephasing time between 100 and 20 fs, in
agreement with the ensemble averaged values reported for the B850 band at low
temperature [15,41].
The two prominent absorptions around 864 and 856 nm (Fig. 6.12b) are assigned
to the low energy k ˆ e1 exciton states, which agrees with their mutual orthogonal
transition-dipole moments lying in the plane of the ring. The degeneracy of these
states is lifted. The absorption around 841 nm (Fig. 6.12b) is attributed to higher
exciton states. The lifting of degeneracy of the k ˆ e1 states as well as the appreciable oscillator strength of the higher exciton states is ascribed to static energetic
disorder in the individual LH2 complexes.
For the energy separation between the k ˆ e1 states, dEe1, we obtain a distribution centered at 110 cm 1. In order to ascertain that this splitting is not a matrixinduced effect we performed additional experiments with LH2 dissolved in glycerol. For this matrix we observed a similar separation in energy for the two mutually orthogonal absorptions. Therefore we conclude that the degeneracy of the
k ˆ e1 exciton states is lifted by an intrinsic effect of the BChl a assembly.
For all the complexes we have studied so far, it was possible to find a polarization
of the incident excitation light such that either the 864 or the 856 band could be
exclusively observed. Consequently, we conclude that the LH2 complexes are oriented within the PVA matrix with their C9 axis perpendicular to the plane of the
substrate at which the sample is deposited. This might result from the spin-coating
procedure, which introduces a shear force in the film, in combination with the
thickness of the film (less than 1 mm) after evaporation of most of the solvent.
PVA layers that were prepared by simply spreading a drop of the LH2/PVA solution
onto the substrate did not show such a high degree of orientation.
In the following subsections we will go into detail on both the higher exciton
states and the origin of the static disorder.
Spectral diffusion and the k ˆ 0 state A crucial check for the applicability of the
exciton model to the B850 system would be the observation of the k ˆ 0 exciton
state. The fluorescence lifetime of LH2 from Rps. acidophila is about 1 ns and corresponds to the decay time of the k ˆ 0 level. Consequently a relatively narrow absorption line is expected for this state. For 3 out of 19 complexes we have indeed
been able to clearly observe such a line on the low-energy side of the k ˆ e1 transitions. Rapid spectral diffusion smears out this line and hinders its experimental
observation (Fig. 6.13, bottom panel). Therefore we modified the data acquisition
in a similar way as reported earlier [19]. Repetitive scans of the B850 spectral region
were recorded at a high scan rate (3 nm s 1) and stored separately in computer
205
6.2 Fluorescence-excitation Spectroscopy
fluorescence (cps)
wavelength (nm)
855
857
859
k=0
100
60
600
time (sec)
300
fluorescence (cps)
206
0
100
60
855 857
859
wavelength (nm)
Figure 6.13. Fluorescence-excitation
spectrum of the red wing of the longwavelength absorption in the B850
band. In the middle panel a stack of
200 consecutively recorded spectra
(3 s per scan) is shown where the
fluorescence intensity is given by the
color code (yellow corresponds to
high intensities). The spectrum in the
bottom panel corresponds to an
average of all scans, whereas the top
panel corresponds to an average of
only those scans that are covered by
the box. Figure taken in part from
Ref. [17].
memory. In this way the spectral features could be followed in time. By selecting a
time interval where the spectral diffusion is limited and taking the average of only
this interval, the presence of the lowest k ˆ 0 exciton state could be ascertained
(Fig. 6.13, top panel).
The k ˆ 0 transition observed for the three complexes carries an oscillator
strength of 2 9 % of the total oscillator strength in the B850 band, in good agreement with theoretical calculations [46]. Although the statistics is poor, the k ˆ 0
transition in the three complexes suggests that the transition dipole moment of
the k ˆ 0 state becomes stronger when the energy separation between the k ˆ 0
and k ˆ e1 states increases. This is in qualitative agreement with model calculations on this system [43,47,48,51].
C2 modulation To analyze our observations on the B850 band in more detail we
first consider the effects of random disorder in the site-energies of the B850 pig-
6 Spectroscopy of Individual Photosynthetic Pigment-Protein Complexes
ments on the properties of the exciton manifold of the unperturbed ring of BChl a
pigments [20,43,47 51] . Such diagonal disorder modifies the spacing between the
exciton levels and removes the pair-wise degeneracy. Secondly, it leads to a redistribution of oscillator strength from the k ˆ e1 states preferentially to the adjacent
exciton states, i. e. k ˆ 0 and k ˆ e2. Consequently we might assign the absorption
around 841 nm to the k ˆ e2 exciton states. From the energy separation of
285 cm 1 between the k ˆ e1 and the k ˆ e2 states we then derive for the average
interaction strength between the pigments a value of 585 e35 cm 1 (Vavg ˆ
[V1‡V2]/2). This value is much larger than the interactions calculated from the
structure, which are found to be in the range 200 400 cm 1 [41 43,48,52]. It
appears that these values are relatively insensitive to the level of sophistication
of the calculations. Although there is some spread in the calculated interaction
energies, we conclude that assignment of the 841 nm band to the k ˆ e2 exciton
states leads to a discrepancy between the experimentally determined interaction
energy and the calculated values. In addition to this discrepancy, several other observations cannot be explained on the basis of purely random diagonal disorder.
Firstly, with the value for the interaction strength as derived above, a random disorder with a FWHM of about 750 cm 1 is needed to explain an average splitting of
110 cm 1 for the k ˆ e1 states. Such a large disorder will lead to a strong broadening of the exciton manifold and, as a result, the spectral width of the simulated
ensemble spectrum would be significantly larger than that of the experimentally
observed absorption spectrum. Secondly, random diagonal disorder affects all degenerate pairs of the exciton manifold in the same way. In other words, a diagonal
disorder sufficient to explain the splitting of the k ˆ e1 states causes also a splitting of 110 cm 1 for the k ˆ e2 which is clearly not observed. Therefore we come
to the conclusion that the 841 nm band cannot be assigned to the k ˆ e2 exciton
states, and that an alternative explanation for the observed features of the electronic
structure of LH2 has to be found.
In order to explain the magnitude of the splitting of the k ˆ e1 exciton states
we propose a C2 -type modulation in terms of symmetry of the interaction between
adjacent B850 pigments in LH2. Such a two-fold symmetric modulation couples
specifically exciton states that differ by Dk ˆ e2 [20,21]. In addition to lifting
the degeneracy of the k ˆ e1 states, it leads to a transfer of oscillator strength
from the k ˆ e1 to the k ˆ e3 states. Based on this conjecture, we assign the
841 nm absorption band to the k ˆ e3 exciton states. From this assignment we
find for the average interaction strength a value of Vavg ˆ 240 e 35 cm 1 which
gives V1 ˆ 254 e 35 cm 1 for the intradimer and V2 ˆ 225 e 35 cm 1 for the
interdimer interaction if the ratio V2 / V1 is calculated using the point-dipole
approximation. These values are within the range of what is currently accepted
[41].
The C2 -type modulation of the interaction lifts the degeneracy of the k ˆ e3
states only in higher order. In view of the width of the lines and the signal-tonoise ratio of our spectra we are not able to resolve the resulting, small splitting
of the k ˆ e3 band. However, as shown in Fig. 6.12a, we do observe a slight
polarization-dependence of the intensity of this band.
207
208
6.2 Fluorescence-excitation Spectroscopy
Another indication for the presence of a C2 modulation of the interaction is provided by the intensity ratio of the 856 and 864 nm bands. With purely random
disorder the average of this intensity ratio would be one. We observe for most of
the complexes that the intensity of the 864 nm band is larger than the intensity of
the 856 nm band (Fig. 6.12a). This result can be reproduced by assuming a structural
deformation, which leads to a C2 -type modulation of the interactions [21].
A model of elliptical deformation The origin of the C2 -type modulation of the interactions may be attributed to a geometrical deformation of the overall LH2 ring
structure. Since the leading term of the deformation of a circle into an ellipse
has the same angular dependence as the C2 -type modulation, we restrict ourselves
to an elliptical deformation in the plane of the ring. Different arrangements of the
18 pigments on an ellipse are possible [21]. It turns out that only one model can
consistently describe the experimental observations. In this model (Model C of
Ref. [21]) the inter-pigment distance on the ellipse is modulated in such a way
that it is the longest at the long axis of the ellipse. This arrangement is generated
by displacing each pigment along the line connecting the center of the ring and the
position of the pigment in the unperturbed circle, retaining its angular coordinate.
To accommodate the deformation of the ring, the pigments were re-oriented in
such a way that the transition moments preserved their angle with the local tangent of the ellipse. In Fig. 6.14a the new arrangement of the pigments after the
elliptical deformation of the ring is illustrated.
The effect of the deformation on the low energy exciton states is shown in
Fig. 6.14b together with a typical single LH2 spectrum at two mutually orthogonal
polarizations. The most pronounced effect of the deformation is the energy splitting of the k ˆ e1 states which exceeds that of the k ˆ e2 and e3 states. To explain a kˆ e1 splitting of 110 cm 1 a deformation amplitude for the ring structure
of dr/r0 z 7 % is required, where r0 is the radius of the unperturbed ring and the
long and short axes of the ellipse deviate from r0 by dr. A value of dr/r0 z 8 % is
needed to explain an intensity ratio of 0.7 between the 856 and 864 band [21].
The mutual orthogonality of the transition-dipole moments of the k ˆ e1 states
is not significantly affected by this deformation, consistent with the experimental
results. This is illustrated in Fig. 6.14c, where the wavefunctions of the two components of the k ˆ e1 states for a deformation of dr/r0 ˆ 8 % are shown. The lower
and the higher component of the k ˆ e1 states are denoted by k ˆ 1low and k ˆ
1high, respectively. The amplitude of the wavefunction of the k ˆ 1low state is largest
at the short axis of the ellipse, resulting from the stronger interaction between the
pigments which yields a decrease in the energy of the state. The wavefunction of
the k ˆ 1high state has the largest amplitude where the interaction is the weakest.
The bi-directional arrow in the figure indicates the absorption intensity and its polarization. Due to the local curvature of the ellipse, the major Qy transition dipole
vectors in the k ˆ 1low (1high) state make a larger (smaller) angle with one other.
Consequently, the sum of the dipolar vectors in the k ˆ 1high state is smaller
than that in the k ˆ 1low state. Because the node of the k ˆ 1high state is orthogonal
to that of the k ˆ 1low state, the two transition dipoles are mutually orthogonal.
6 Spectroscopy of Individual Photosynthetic Pigment-Protein Complexes
a
Figure 6.14. (a) Schematic
representation of the transition
dipole moments of the B850
pigments of LH2 in the perfect
b
ring configuration (gray) and
with a large elliptical deforma12200
tion of dr/r0 ˆ 20 % (yellow).
(b) Schematic representation
of the lowest excited states of
the exciton manifold of the
12000
B850 ring of LH2 in the perfect
k=±3
circular arrangement (manifold
1) and including an elliptical
deformation (manifold 2). The
low energy k ˆ e1 state (k ˆ
11800 k = ± 2
1low) is marked by (*) and the
high energy (k ˆ 1high) by (#).
On the right two experimental
k=±1
spectra are depicted at mutual
k=0
orthogonal orientation. The ex11600
perimental conditions are the
same as in Fig. 6.7.
(c) Pictorial representation of
the k ˆ 1low and 1high states.
The length of each arrow is
proportional to the amplitude c
k = 1 low
of the exciton wavefunction at
the particular pigment position. The bi-directional arrow in
the ellipse represents the absorption intensity and its polarization. The ellipse and the
wavefunctions are drawn for
dr/r0 ˆ 8 %. See also Ref. [21].
1
2
k=±3
#
*
dE±1
k=±1
200 cps
k = 1 high
Thus an elliptical deformation of dr/r0 ˆ 7 % 8 % in a simple point-dipole approximation explains the major features of the single molecule spectra, i. e., dEe1
ˆ 110 cm 1 for the average k ˆ e1 splitting, the average intensity ratio of 0.7 between the 856 and 864 bands, and their mutually orthogonal polarization.
Random disorder It is seen from the histograms of Fig. 6.12a and b that the splitting (dEe1) and the intensity ratio of the 856 and 864 nm bands vary from one complex to the other. Since the C2 -type modulation of the interactions alone would
result in the same splitting and intensity ratio of the k ˆ e1 states for all the complexes, these variations clearly indicate heterogeneity among the B850 pigments.
Such heterogeneity is caused by random disorder that can exist in both the site-
209
210
6.2 Fluorescence-excitation Spectroscopy
energies of the pigments (random diagonal disorder) and/or their interactions
(random off-diagonal disorder). Because dEe1 and the intensity ratio of the k ˆ
e1 exciton states are determined by the disorder within one complex, these disorders should be considered as intracomplex disorder. Similarly, it can be expected
that the amplitude of the elliptical deformation will be subjected to a distribution,
which should be considered as intercomplex disorder. Since it is difficult to separate all the different types of heterogeneity we approximate the description of the
heterogeneity in the B850 band by a two-step procedure. Firstly, we consider the
character of the excited states of the individual complexes, which we assume to
be determined by intracomplex disorder. In the second step we investigate the consequences that result for the ensemble of complexes when intercomplex disorder is
taken into account.
To model the individual complexes we fixed the amplitude of the elliptical deformation to a particular value and only random diagonal intracomplex disorder was
included in the simulations. The result of these simulations are presented in
Fig. 6.15 and compared with the experimental data. The solid black squares in
Fig. 6.15a represent the result of a Monte Carlo simulation of the distribution of
dEe1. The deformation amplitude is dr/r0 ˆ 8.5 % and the intracomplex diagonal
disorder (G intra) has a FWHM of 250 cm 1. It appears that the simulation fits the
experimental distribution very well. The center of the distribution is largely determined by the amplitude of the elliptical deformation, but it is also influenced by
the random disorder. The random disorder mixes the k ˆ 0 state with the lower
component of the k ˆ e1 states, by which oscillator strength is transferred to
the k ˆ 0 state. At the same time, the lower component of the k ˆ e1 states shifts
to higher energy, making the separation between the k ˆ e1 states smaller. Thus,
the center of the dEe1 distribution will be shifted to a smaller value than in the
absence of random disorder. Accordingly, the deformation amplitude derived
from the simulation, which includes random disorder, is slightly larger than the
previously mentioned value of 7 %.
The width of the dEe1 distribution in Fig. 6.15a is assumed to be mainly determined by the random diagonal disorder. The value of 250 cm 1 sets an upper value
for the random diagonal disorder, because the experimentally observed distribution
includes contributions from other types of heterogeneity, which are neglected in
the simulation. The results of the simulation of the distribution of the intensity
ratio between the 856 and 864 nm bands is shown by the solid black squares in
Fig. 6.15b. In the simulation the intensity of the k ˆ 0 state is included in that
of the 864 nm band, since in most of the complexes the narrow k ˆ 0 band is hidden under the broad 864 nm band, and is not distinguishable due to its rapid spectral diffusion. The simulation gives reasonable agreement with the experimental
distribution. Fig. 6.15c shows the simulated distribution of the angle between
the two transition-dipoles of the k ˆ e1 states. These results of the numerical calculations on the single LH2 spectra are confirmed by an analytical analysis of the
data by Mostovoy and Knoester [46].
6 Spectroscopy of Individual Photosynthetic Pigment-Protein Complexes
intracomplex heterogeneity
a
6
2000
2
0
0
0
40
8
80
120
-1
DE±1 (cm )
160
200
b
3000
4
0
0.0
0.4
0.8
1.2
1.6
Intensity (856/864)
occurrence (simulation)
occurrence (experimental)
4
0
2.0
4000
c
2000
0
30
60
Figure 6.15. Comparison between the experimental distributions (histograms) and numerical simulations (solid squares) of the energy
separation, the intensity ratio and the mutual
polarization angle of the k ˆ e1 transitions in
the B850 band. The experimental data refer to
the left vertical scale and have been obtained at
the same experimental conditions as given in
Fig. 6.7. The numerical simulations refer to the
right vertical scale and are based on an elliptical
90
120
f (degrees)
150
deformation dr/r0 ˆ 8.5 %. For more details on
the simulations see Ref. [20].
(a) Energy separation of the two k ˆ e1 transitions.
(b) Intensity ratio 856/864 of the two k ˆ e1
transitions.
(c) Mutual angle between the k ˆ e1 transition-dipole moments. Figure reproduced with
permission from Ref. [20]
Intra- and intercomplex disorder The value of 250 cm 1 FWHM for the random
diagonal disorder in the B850 band of LH2 in a PVA film is smaller than the values
reported by other authors [41]. With this value for the diagonal disorder some structure remains in the simulation of the experimentally observed B850 band of a bulk
sample. In Fig. 6.16 the calculated and the observed spectra are shown, and it is
seen that the splitting between the two components of the k ˆ e1 states, induced
by the elliptical deformation and random disorder, is still visible in the simulated
curve (dashed line), in contrast to the observed spectrum. The reason for this
apparent discrepancy is that the value of 250 cm 1 for the heterogeneity obtained
from the k ˆ e1 splitting reflects only the heterogeneity within individual com-
211
6.2 Fluorescence-excitation Spectroscopy
1
intensity (a.u.)
212
0
820
840
860
880
wavelength (nm)
Comparison between the experimental, low-temperature ensemble spectrum
of LH2 (solid line) and two simulated spectra.
The experimental ensemble spectrum lacks the
long-wavelength tail, because of overlap with
the fluorescence-detection window. For both
simulated spectra the deformation amplitude is
dr/r0 ˆ 8.5 %. Dashed line: only intracomplex
Figure 6.16.
heterogeneity is taken into account. Dotted
line: both, intra- and intercomplex heterogeneity, are included in the simulation, with G intra ˆ
250 cm 1 and G inter ˆ 120 cm 1. The individual
transitions have homogeneous Lorentzian line
shapes with a width (FWHM) of 100 cm 1
(k 0 0) and 10 cm 1 (k ˆ 0). For more details
on the simulations see Ref. [20].
plexes (intracomplex disorder). In order to describe the ensemble spectrum the
sample heterogeneity or intercomplex disorder should also be included [19,48].
Such heterogeneity originates from the slightly different environments of the different LH2 complexes in the host matrix.
From the distribution of the spectral lines observed in the B850 band (Fig. 6.12b)
it is not possible to find an accurate value for the intercomplex diagonal disorder,
since the spectral positions depend not only on the site-energies of the pigments,
but also on their interactions. For the B800 band this is not the case, since the interactions are relatively weak compared to the diagonal disorder [16]. Therefore, a
good estimate for the intercomplex diagonal disorder of the B850 pigments is obtained from the variation in spectral mean in the B800 band of each LH2 complex.
For the B800 band a value of 120 cm 1 FWHM was found [19]. Taking the same value
for the intercomplex diagonal disorder of the B850 band, we performed a Monte
Carlo simulation of the ensemble spectrum. The result is shown in Fig. 6.16 (dotted
line). The simulation nicely reproduces the experimentally measured ensemble
spectrum, which indicates that both inter- and intracomplex disorder are relevant.
The direct insight into both the inter- and intracomplex disorder of an ensemble
of single LH2 complex has important implications for the discussion on the delocalization length in these type of systems. By delocalization length we mean the
number of pigments that contribute to a particular exciton state, which for the
6 Spectroscopy of Individual Photosynthetic Pigment-Protein Complexes
B850 band is obviously a number between 1 and 18. This length is widely perceived
as a key characteristic of the microscopic exciton state. Bakalis and Knoester [53]
discussed the determination of the delocalization length from the width of the
ensemble-averaged absorption spectrum. Since the intercomplex disorder does
not affect the delocalization length, whereas it does contribute to the width of
the ensemble averaged spectrum, this will lead to an underestimation of the delocalization length through this method. A similar reasoning applies to the value for
the delocalization length as determined by transient absorption spectroscopy [53].
On the basis of the single LH2 spectra it can be shown that in the B850 ring of
LH2 the excitation is largely delocalized over the ring [46].
6.2.3
Conclusions
The results presented in Section 6.2 show that single molecule spectroscopic techniques at cryogenic temperatures can be applied successfully to elucidate the electronic structure of photosynthetic pigment protein complexes. The advantage of
the method is that effects of ensemble averaging, which are typical for conventional forms of spectroscopy, are eliminated. The excitations in the B800 band
are localized on a limited number (2 3) of BChl a pigments. The intra- and intercomplex disorder are determinded to be 120 and 130 cm 1 FWHM respectively.
The B850 assembly of BChl a pigments in LH2 represents a strongly coupled
system, where the electronic properties are appropriately described as Frenkel excitons. The observations are consistent with alternating nearest-neighbor interaction energies of 254 e 35 and 225 e 35 cm 1, respectively, with a distribution
of site energies (diagonal disorder) given by a FWHM with an upper limit of
250 cm 1. These parameters imply that the optical excitations are largely delocalized
over the ring of BChl a pigments [46]. In addition to the random diagonal disorder, a
C2 -type modulation of the intermolecular interaction has to be assumed to account
for the relative intensities and the spectral positions of the exciton transitions. This
modulation is presumably caused by a structural deformation of the LH2 complex,
which transforms the B850 ring from a circular to an elliptical geometry.
It is not surprising that our experimental results cannot be interpreted on the
basis of an exciton model for a perfect circular aggregate of nine dimers. The
ensemble [41] and single molecule spectroscopic experiments [17,45,54] on LH2
already indicated the presence of disorder in this system. It is generally believed
that the origin of such disorder is the random variation in the site-energies of
the pigments involved [41]. However, a random distribution of the coupling between the pigments has also been suggested [55]. Such random disorder has
very specific implications for the structure of the exciton manifold but this alone
is not sufficient to describe our experimental observations consistently. Based on
the magnitude of the splitting between the k ˆ e1 states, which can only be caused
by a C2 -type modulation of the interactions, we suggest that the B850 ring is transformed from a perfect in-plane isotropic absorber and emitter into an elliptic one.
Other groups have also found evidence for anisotropy in the fluorescence emission
213
214
6.3 Single Molecule Spectroscopy on the Light-Harvesting Complex II of Higher Plants
of single LH2 complexes at room temperature and low temperature using either
mica plates or polymer films to immobilize the complexes [54,56]. The modulation
of the interaction between the pigments most probably is related to a geometrical
distortion of the LH2 ring. Bopp et al. [56] showed that the anisotropic behavior of
single LH2 complexes at room temperature could be very well explained by an
elliptical deformation of the LH2 ring, which varies in time. Such a temporal
variation of the ellipticity cannot be observed at 1.2 K where the complexes presumably are trapped in local minima of the potential energy surface, each corresponding to a distinct orientation of the elliptical deformation.
The reduced symmetry of the isolated LH2 complexes at low temperature and at
room temperature is at odds with the nine-fold symmetry of LH2 in crystalline
form. It might be that the dense packing of LH2 complexes in the crystal provides
a stabilizing force which is absent in the isolated complexes as already suggested
by van Oijen et al. [17]. Whether or not the static deformation is representative
of the structure of LH2 in detergent solution or in membranes remains to be
answered. Experiments are in progress to study this aspect in more detail. Nevertheless, this does not significantly affect our conclusions about (static) disorder, the
strength of the intermolecular interactions and the delocalization of optical excitations over the ring. Static disorder is a result of the variation of site energies, and
this variation depends on very local conformation parameters. The interaction
strength is determined by intermolecular distances and the average of the modulated interactions in our model correspond to that of a fully symmetric LH2
ring. Delocalization at low temperature (1.2 K) is determined by the degree of
static disorder with respect to the interaction strength. At higher temperatures
thermally induced effects will dominate, and dynamic electron phonon coupling
will lead to a fast hopping-type motion of localized excitations. From the present
experiments we have derived a basic framework for the electronic structure of
LH2, and the parameters that we have obtained should be considered in describing the properties of LH2 at room temperature in detergent solution, as well as
in vivo.
6.3
Single Molecule Spectroscopy on the Light-Harvesting Complex II of Higher Plants
In contrast to LHC-II there are no high resolution structure data available for LHC-II.
In addition presently available data do not suggest a high symmetry of the complex. Nevertheless LHC-II is a particularly interesting complex for single molecule
spectroscopy, since the currently available structure data leave open a number of
questions regarding for example the orientation of the chlorophyll molecules
with respect to the proteins. Single molecule spectroscopy is ideally suited to
answer these questions. The following paragraph describes first steps towards
that goal.
6 Spectroscopy of Individual Photosynthetic Pigment-Protein Complexes
6.3.1
Experimental
LHC-II was prepared from pea leaves (Pisum sativum L.) according to the procedure
of Krupa et al. [57]. The complexes were stored in buffer (10 mM Tricine, pH 7.8) at
77 K until used. For sample preparation the stock solution was diluted with buffer
containing 1 % detergent (n-octyl b-d-glucopyranoside, Sigma). One drop (100 ml)
of the diluted solution was mixed with 1 ml of a 1 % PVA1) solution and immediately spin-coated onto a clean cover glass to obtain thin LHC-II doped films. In the
case of room temperature measurements such a sample shows stable fluorescence
for several hours. Cooled to a temperature less than 77 K the complexes are stable
for weeks. The low final LHC-II concentration allows the spatial selection of individual LHC-II complexes. The complete procedure of final sample preparation and
mounting was done in the dark.
Monomers were prepared by incubation of 1 mg ml 1of the trimeric complex
with 0.1 U ml 1 phospholipase A2 (Sigma, #P-6534) for 2 h at room temperature.
The monomers were separated from the trimers using sucrose gradient centrifugation. (For a detailed description see Nussberger et al. [58]) Sample preparation of
the monomers was exactly the same as for the trimers.
For all optical investigations a home-built beam scanning confocal microscope
was used operating in the temperature range of 300 1.8 K. The objective (63x,
0.85NA, Melles Griot) was mounted inside the cryostat enabling polarization studies as well as fluorescence emission spectroscopy. Excitation via the 647 nm line of
a Kr‡-ion laser was used. Excitation spectra were recorded using a narrow band dye
laser (Coherent 699-21, line width 1 MHz) operating with DCM dye. Fluorescence
light was separated from stray light using holographic notch filters (Kaiser) and
detected by an avalanche photo diode (EG&G) or focused on a spectrograph
(0.25 m, Acton Research) equipped with a back-illuminated CCD camera (Princeton Instruments).
6.3.2
Polarization and Spectral Distribution of the Fluorescence Emission
of Single LHCII Monomers and Trimers
Investigations on the polarization properties of the light emitted by single LHC-II
trimers or monomers have been performed to obtain information on the number
of emitting states. For better separation between excitation and fluorescence
complexes were excited in the Chl b-region at 647 nm. Polarization of the fluorescence was determined using a permanently turning polarizer (Glan Thompsonprism) in front of the detector. The degree of polarization (p) is defined as
Imax Imin
pˆ
, where Imax and Imin are maximum and minimum fluorescence inImax
tensities of the cos2 -fitted intensity traces. For non-polarized emission pˆ0 whereas
1) PVA ± Polyvinyl alcohol
215
6.3 Single Molecule Spectroscopy on the Light-Harvesting Complex II of Higher Plants
fluorescence / a.u.
Figure 6.17. (a) Polarization modulation trace
of a single LHC-II monomer at room temperature. Complexes are immobilized in a thin PVA
matrix and excited via the Chl b absorption
band at 647 nm.
(b) Histogram of the degree of polarization.
Most monomers show linearly polarized emission. The solid line shows a simulation of the
emission polarization assuming a single emitting randomly oriented dipole.
A
2000
1000
0
0
5
40
50
time / s
20
18
16
14
12
10
8
6
4
2
0
0,0
B
# molecules
216
monomeric LHC-II
0,2
0,4
0,6
0,8
1,0
degree of polarisation
for linearly polarized emission pˆ1. At room temperature the investigation of
single light-harvesting complexes is complicated by photobleaching. Nevertheless,
in degassed buffer observation times up to minutes allow one to determine the degree of polarization with sufficient accuracy. Figure 6.17a shows a typical polarization modulation trace of a LHC-II monomer. The fluorescence is linearly polarized
until the complex is irreversibly photobleached in a single step after 50 s. The histogram in Fig. 6.17b shows that most of the monomers emit linearly polarized
light. A few monomers show two-step photobleaching however most complexes
shoe one-step bleaching, typical for single quantum systems. As expected, traces
of trimers are more complicated. This is why the investigation of the fluorescence
polarization of trimers has been carried out at low and at room temperature.
Figure 6.18 contains a histogram of p from 70 single LHC-II trimers at Tˆ1.8 K.
20
15
10
5
0
degree of polarisation
Figure 6.18. Histogram of the degree of polarization of trimers at Tˆ1.8 K. Most complexes
show p values around 0.4.
6 Spectroscopy of Individual Photosynthetic Pigment-Protein Complexes
10
8
# molecules
Figure 6.19. Bleaching behavior of trimers at room temperature. The histograms show the distribution of the degree of polarization before the third last
step (A), the second last step (B), and
the final step (C).
A
trimeric LHC-II at T=300K
third last step
B
second last step
6
4
2
10
# molecules
8
6
4
2
0
16
14
12
10
8
6
4
2
0
0,0
# molecules
C
last step
0,2
0,4
0,6
0,8
1,0
degree of polarisation
From the histogram it is seen that most of the LHC-II complexes emit light with
pˆ0.4. In the temperature range 1.8 I T / KI100 there is no significant effect of
temperature on p. Photobleaching in trimers never commences in a single step.
Most of the trimers exhibit a two or three jump behavior. In a couple of cases we
also found trimers showing more than three jumps, which may be due to trimer
aggregation or intensity fluctuations of monomeric subunits. Most importantly,
at room temperature there are visible intensity jumps in the traces which also
change the degree of polarization. The histograms in Fig. 6.19 summarize the
evolution of p for three-step photobleaching. It also seems worth mentioning that
most of the trimeric complexes show linearly polarized fluorescence before final
bleaching, comparable to the behavior of monomers.
One typical emission spectrum of a single LHC-II trimer at low temperature
(Tˆ1.8 K) is shown in Fig. 6.20A, recorded with an acquisition time of 200 s.
The spectrum represents a convolution of a number of hardly resolved emission
217
218
6.3 Single Molecule Spectroscopy on the Light-Harvesting Complex II of Higher Plants
300
Figure 6.20. Fluorescence emission
spectra of trimeric LHC-II at 1.8 K.
(A) Acquisition time of 200 s.
(B) Example of an emission spectrum
with an acquisition time of 5 s.
A
200
100
0
20
675
680
685
680
685
B
10
0
675
wavelength / nm
lines. The spectral width is roughly 100 cm 1. Different trimers show differences
in spectral width and shape. The complex spectral shape is due to spectral diffusion which is easily seen when reducing the acquisition time (see Fig. 6.20B).
For a closer investigation of this spectral diffusion we recorded a series of 500 spectra shown in Fig. 6.20B using an acquisition time of 5 s per spectrum. Typical
fluorescence emission spectra of LHC-II monomers and trimers with short acquisition times are compared in Fig. 6.21. In general, monomeric LHC-II complexes
show less lines than trimers. Often we find a single line, while trimers frequently
show three lines. The lines are separated by 20 100 cm 1.
LHC-II monomers build a strongly coupled system of 12 Chl with fast energy
transfer towards the red-most Chl a molecule in the complex [24,27]. It is generally
accepted that there is a large density of states in the red wing of the absorption
spectrum. Currently it is believed that 5 Chl molecules absorb light within an interval of 250 cm 1 from the energetically lowest Chl a molecule. Assuming Boltzman equilibrium, at room temperature all of these molecules should be populated
and hence contribute to the total fluorescence. The finding that LHC monomers
emit linear polarized fluorescence is thus rather surprising. The most trivial ex-
6 Spectroscopy of Individual Photosynthetic Pigment-Protein Complexes
8
Fluorescence Intensity / a.u.
6
4
2
4
2
676
678
680
682
684
Wavelength / nm
Figure 6.21. Typical emission spectra of a trimeric (a) and monomeric (b) LHC-II at low
temperature (Tˆ1.8 K). Acquisition time in both cases was 10 s.
planation would be that the transition dipole moments of all Chl molecules contributing to the fluorescence are parallel. Structural data reveal the binding sites of
Chl molecules and also determine the orientation of the plane of the Chls but
do not uncover the orientation of the transition dipole moments within this
plane. Thus it cannot be excluded that the transition dipole moments of the five
molecules are parallel though from structure data [59] this seems to be highly unlikely. Of course the assumption that there is thermal equilibrium between the redmost Chl could also be wrong, possibly because of a large energy barrier between
the individual molecules. There will be thermal equilibrium among the five red
most molecules only if there is a sizeable coupling between them. The coupling
among Chl a molecules is known to be low [58]. However time-resolved optical
219
220
6.3 Single Molecule Spectroscopy on the Light-Harvesting Complex II of Higher Plants
spectroscopy shows that the slowest time constant in the energy transfer among
Chl a is of the order of 10 ps [23]. This is faster than the lifetime of the first excited
state of the Chls (around 5 ns) such that the lowest Chl states should be in thermal
equilibrium.
A possible explanation of our experimental finding is that the five molecules
have highly different photophysical parameters, such that only one molecule has
a high triplet quenching efficiency.
The fluorescence of a single Chl molecule can only be detected if the triplet state
of this Chl is quenched by a carotenoid. The saturated fluorescence intensity is
given by
Isat ˆ
…k12 ‡ k32 †FF
…2 ‡ k32 /k13 †
…5†
where FF is the fluorescence quantum yield, k12 is the inverse lifetime of the first
excited singlet state, k32 the population rate of the triplet state and k13 is the decay
rate of this state. Realistic values for Chl yield a maximum fluorescence intensity
Isatˆ500 photons s 1. With a detection efficiency less than 1 %, the resulting number of photocounts s 1 will be below the dark count rate of the detector. Efficient
triplet quenching by carotenoids may reduce k13 by two orders of magnitude and
hence Isat will increase by the same factor. The fluorescence signal of one Chl possessing an efficiently quenched triplet state can thus be much higher than that of
the other Chls. Triplet quenching of the red most Chl also makes sense from a physiological point of view since this molecule will be the one with the highest probability for intersystem crossing. Efficient quenching of only a single Chl would also
explain the discrepancy between ensemble absorption and single molecule emission data. Due to the low excitation intensities in ensemble experiments quenching
of the triplet state often does not influence the extinction coefficient. In single molecule experiments one usually works in the nonlinear regime i. e. close to saturation of the singlet transition.
The p values for trimers at low temperatures are distributed between 0 and 1
with a marked peak around 0.4. This is strong indication that there is more
than one emitting state per trimer. From previous ensemble measurements [27]
and our fluorescence emission spectra (see Fig. 6.21) it seems to be evident that
the red-most Chl a molecules are only weakly coupled to Chl a in neighboring
monomers. It is thus reasonable to assume that there are three emitting states
per trimer. By further assuming that the trimers are randomly oriented in the polymer film one can try to simulate the histogram (Fig. 6.22). If one assumes that the
transition dipole moments of the red-most Chl a are oriented in the symmetry
plane of the LHC II trimer (membrane plane) a broad distribution of p-values between 0 and 1 is obtained, which does not fit the data (Fig. 6.22, dashed line). To
achieve a distribution obtaining a pronounced peak around pˆ0.4 it is necessary to
tilt the dipoles out of the symmetry plane. In the case of an isotropic distribution of
orientations within the PVA matrix the best fit is obtained by assuming an angle of
either 25h or 47h between the plane of symmetry and the transition dipole mo-
6 Spectroscopy of Individual Photosynthetic Pigment-Protein Complexes
6
5
4
3
2
Simulation of the histogram
assuming a tilt angle of 0h (dashed line) and
47h (solid line) between the symmetry plane
(membrane plane) and the orientation of the
dipole moments of the trimer.
Figure 6.22.
1
0
0,0
0,2
0,4
0,6
0,8
1,0
degree of polarisation
ments of the Chls as shown in Fig. 6.22, solid line. The discrepancy between the
simulation and the measured histogram may be due to a distribution of orientations which is not completely isotropic in thin polymer films. Such behavior was
found in spin-coated samples of light-harvesting complexes of purple bacteria [17].
In contrast to monomers the trimers photobleach in multiple steps. This is
further indication that monomeric subunits in trimers are only weakly coupled,
otherwise we would expect to find single step photobleaching. In previous studies
the coupling among Chl molecules in different monomeric subunits has been
estimated to be roughly 5 cm 1 [27]. From our fluorescence emission spectra we
know that the energetic disorder among monomers is around 100 cm 1. From
our experiments we thus conclude that the excitation energy is localized on individual Chl a molecules in the monomeric subunits and not delocalized over the trimer. Fluorescence emission spectra of monomers and trimers show a number
of well resolved lines. The linewidth of the emission spectra in Fig. 6.20 and
6.21 is limited by the spectrometer resolution. Fluorescence excitation spectra
(see Fig. 6.23) show a line width of the red-most Chl a states of around
G= 0,65 GHz
75
50
25
681,6
681,8
682,0
682,2
Excitation Wavelength, nm
682,4
Fluorescence excitation spectrum of the red transitions in a LHC-II trimer. The inset
show the red-most line on an enlarged scale. The experiments have been carried out at Tˆ2 K.
Figure 6.23.
221
6.3 Single Molecule Spectroscopy on the Light-Harvesting Complex II of Higher Plants
0.02 cm 1, in good agreement with previous hole-burning experiments [27]. At
Tˆ4.2 K the line width of these molecules should be determined by the exited
state lifetime (tˆ5.6 ns) which corresponds to a width of around 0.001 cm 1. Possibly the measured value is larger than this number because of spectral diffusion.
Fig. 6.24 shows a larger scan range which not only includes the red-most trap
states but also transitions of energetically higher lying Chl a molecules. We find
two prominent bands around 677.5 and 678.5 nm which may be attributed to
Chl a molecules. The linewidth of the most intense peak is around 17 GHz.
This linewidth is most probably determined by energy transfer to the red-most
Chl a molecules. The corresponding energy transfer time is around 30 ps in
good agreement with previous ensemble measurements which find 10 ps as the
slowest energy transfer time in the complex. In contrast to the light harvesting
(LH2) complex of purple bacteria, [54] LHC-II shows narrow fluorescence emission
lines. However although we used short acquisition times for LHC-II we were never
able to measure faster than the spectral diffusion, in contrast to LH2 where it is
possible to record excitation spectra with acquisition times faster than the typical
time constant for spectral diffusion [19]. In contrast to glasses, proteins are
know to have a certain ªcut-offº time t min for spectral diffusion below which no diffusion is measurable [60]. The value for t min depends on the immediate environment of the chromophore one looks at. Chromophores buried within the protein
have less internal degrees of freedom in its environment and hence a larger t min
[50]. The fact that t min is larger for LH2 than it is for LHC-II may be due to the
fact that the Chl a molecule which is emitting light in LHC-II is exposed at the
outside of the protein in contrast to LH2 where it is located in a double walled cylinder formed by the a-helices of the proteins [7]. Spectral jumps in LHC-II trimers
G= 17 GHz
300
Ifl, Cts/s
222
200
100
677
678
679
680
681
682
Excitation Wavelength, nm
Figure 6.24. Fluorescence excitation spectrum of a single LHC-II trimer with a large scan range.
The inset shows the resonance line at 678.5 nm on an enlarged scale together with the linewidth
G . Experiments were carried out at Tˆ2 K.
6 Spectroscopy of Individual Photosynthetic Pigment-Protein Complexes
occur in an uncorrelated fashion. This indicates that, for LHC-II protein chromophore interaction is more important than chromophore chromophore interaction.
In conclusion, the experimental data so far provide various lines of evidence that
the monomer subunits within the LHC-II trimer are rather uncoupled and that
there are three emitting states per trimer and one per monomer with an emission
wavelength of 681.3e0.1 nm. The width of those lines measured by fluorescence
excitation spectroscopy is around 0.02 cm 1. Those spectra show a couple of lines
of energetically higher lying Chl a molecules. It is appealing to combine polarization spectroscopy with fluorescence excitation spectroscopy which would one allow
to determine the orientation of the transition dipole moment of these Chl molecules providing so far missing information about the orientation of Chl molecules
in the LHC-II complex.
6.3.3
Single Molecule Spectroscopy on Photosystem I Pigment-Protein Complexes
In the preceding paragraphs single molecule spectroscopy has been applied to investigate antenna complexes. In this section a complex containing a reaction center
is discussed.
PSI from the cyanobacterium Synechococcus elongatus was isolated as described
previously [61]. Purified PSI trimers were diluted in buffer containing 20 mM Tricine, pH 7.5, 25 mM MgCl2 and 0.02 % (w/w) of detergent (b-DM, Sigma) to reach
a final concentration of ca.10 6 M Chl. The detergent concentration was slightly
above that which induces micelle formation to avoid PSI aggregation. For measurements, PSI containing buffer was diluted again (1/100) in a solution containing
1 % (w/w) PVA, 25 mM MgCl2 and 5 mM Na-ascorbate and spin-coated on a
cover glass to obtain thin PSI-doped films. The low final PSI concentration allows
the spatial selection of individual PSI trimers. Sample preparation and mounting
was done in the dark. The experiments were carried out using a home-built beam
scanning confocal microscope able to operate from 1.8 to 300 K.
Figure 6.25 shows fluorescence images of single PSI complexes recorded at 17
and 236 K. Every bright spot corresponds to the fluorescence emission of a single
PSI trimer. A drastic decrease in the fluorescence intensity with increasing temperature is visible. This strong temperature dependence of the fluorescence intensity was observed in ensemble experiments [32,62]. It is a signature of thermally
activated uphill energy transfer from the low-energy Chl pool to the reaction center.
At physiological temperature energy trapped by the red pool of Chl can be transferred to the reaction center part via thermally populated vibrational levels. At
low temperature this channel is blocked, leading to a more intense fluorescence
emission. The quenching behavior therefore contains information concerning
the energy barrier between red pool pigments and P700. Analysis of temperature
activated fluorescence quenching was performed for 15 aggregates. In Fig. 6.25 we
also show results for two typical complexes. The fluorescence intensity decreases
by an order of magnitude as the temperature is increased from 17 to 280 K. A simple Arhenius law has been used to fit the experimental data [62]. The activation
223
224
6.3 Single Molecule Spectroscopy on the Light-Harvesting Complex II of Higher Plants
17 K
236 K
158 K
Complex 1
Complex 2
60
120
180
T, K
Figure 6.25. Temperature dependence of single PSI fluorescence intensity. Upper part: confocal fluorescence images of single PSI isolated
in PVA film. Images have been recorded at temperatures as indicated.
Fluorescence intensity is decoded in
gray scale. The excitation wavelength
was 680 nm. Lower part: Fluorescence intensity of two single PSI trimers as a function of temperature.
Experimental data have been fitted
with an Arrhenius equation: (A‡B
exp(-DE/kBT)) 1. The activation energies for the two complexes were 550
and 490 cm 1.
energy derived from data on single PSI complexes is 500 cm 1. This value is
slightly higher than that obtained using ensemble measurements (363 cm 1).
The difference may be due to the method chosen. When dealing with single molecules we select those having sufficient fluorescence quantum yield to be capable
of detection. These are aggregates, which may be characterized by a low rate of uphill energy transfer to the RC and hence a high energy gap between the red Chl
pool and the RC. Classical measurements are the results of averaging over the
whole molecular ensemble, also containing low-energy gap aggregates. The slight
difference in activation energy for the two aggregates presented in Fig. 6.25 can be
explained in terms of differences in P700 or/and pigment energy levels among the
complexes. From hole burning data [63] it is known that the P700 absorption band
is distributed over roughly 100 cm 1. This is of the order of the observed differences in DE in our single molecules. It is noticeable that single PSI fluorescence
emission can be detected at room temperature regardless of the low fluorescence
quantum yield.
Photobleaching is one of the most important obstacles in single molecule spectroscopy under ambient conditions. The detection of single pigment protein complexes requires their strong illumination, resulting in a high population of the Chl
triplet state. Fortunately quenching of Chl triplet states by carotenoids allows the
detection of single photosynthetic units. In order to investigate bleaching dynamics
6 Spectroscopy of Individual Photosynthetic Pigment-Protein Complexes
Figure 6.26. Evolution of the
fluorescence spectrum from
single PSI during photobleaching. Three consecutive roomtemperature spectra (1, 2 and
3) are vertically shifted for
clarity. The acquisition time
was 2 s. Note the step-wise
changes in red-most fluorescence between spectra 2 and 3.
The lowest panel shows the
difference between spectra 2
and 3. The arrow indicates the
filter cut-off.
at room temperature we recorded a continuous series of spectra with short acquisition time. The three consecutive spectra presented in Fig. 6.26 show that photobleaching commences from the low-energy Chl pool. Note the marked difference
between the red-pool intensity in spectra 2 and 3. Discrete jumps of red-pool fluorescence emission were observed. Bleaching is a photoinduced process and its efficiency characterizes the excited state population of the pigment pool. The higher
photostability of bulk antennas (C680) as compared to the red pool may thus be
caused by rapid depopulation of C680 excited states. The more rapid photodegradation of red Chls may be due to the existence of an efficient energy transfer
from C680 to low-energy antennas and thus a high population probability of the
red pool Chls. The high photostability of PSI at low temperature make them a
good system for detailed spectroscopic study. In order to probe the spectroscopic
properties of the low-energy antenna we recorded fluorescence excitation spectra
of single PSI complexes. Therefore the excitation laser was scanned in the low-energy antenna region (705 720 nm). Fluorescence was detected at wavelengths
longer than 725 nm. The spectra show narrow excitation lines (Fig. 6.27). The linewidth of the spectra (Z1 cm 1) recorded using a single fast laser sweep across the
resonance was limited by our experimental resolution. Those lines may correspond
to the zero-phonon transitions of individual Chl molecules. Significant line broadening was observed during multiple scan due to spectral diffusion. The number of
the narrow Chl lines observed in the excitation spectra varies from aggregate to aggregate (typically we observed 1 2 lines in the region 705 715 nm, however for
some aggregates up to 4 lines were visible). The quantitative analysis of the spectral
pool composition is difficult because of the intense spectral diffusion. Spectral
jumps of single chlorophylls may be caused by the rearrangement of the local pro-
225
Fluorescence Intensity, Cts/s
6.3 Single Molecule Spectroscopy on the Light-Harvesting Complex II of Higher Plants
60
30
712
713
Excitation Wavelength, nm
Figure 6.27. Low-temperature fluorescence
excitation spectra of the red-most antenna pigments. Main figure: A fluorescence excitation
spectrum of a single PSI aggregate at 2 K.
The spectrum was obtained summing 30 consecutive scans and detecting the fluorescence at
wavelength larger than 725 nm. The background
is limited by the dark count rate of the detector.
Inset: Spectral distribution of single Chl zerophonon lines (spectral region between 705 and
717 nm has been studied).
tein environment. Such processes may be a result of the optical excitation, because
the emission of Stokes-shifted photons by Chl molecule is accompanied by heat
dissipation in their local environment. The absence of a visible phonon structure
in the excitation spectra is remarkable. This effect is related to the difference in
absorption cross section in the zero-phonon line and phonon wing. Even when
the major part of the total spectral intensity is concentrated in a phonon wing,
the absorption cross-section of a pure electronic transition can be higher because
its oscillator strength is concentrated in a narrow spectral interval.
Upon tuning the laser to the maximum of the bulk Chl absorption band
(680 nm), only emission from the red-most Chl is detected in low-temperature
fluorescence emission spectra (see Fig. 6.28). Individual Chl lines resolved in
fluorescence excitation spectra are also visible in fluorescence emission spectra
(fine structure in the spectral region between 710 and 715 nm). The spectral
positions of the observed lines vary from one aggregate to another (this effect
Fluorescence Intensity, a. u.
226
T = 280 K
700
750
800
T = 1.8 K
700
720
740 760 780
Wavelength, nm
Figure 6.28. A fluorescence emission spectrum of single PSI complexes at 2 K. The fine
structure in the 712 nm region corresponds to
800
zero-phonon transitions of individual Chl
molecules.
6 Spectroscopy of Individual Photosynthetic Pigment-Protein Complexes
is related to differences in the local environment of pigments leading to an inhomogeneous broadening in ensemble experiments). Note that the single molecule
approach provides the possibility of unraveling spectral fine structure using nonselective excitation. Close to the narrow emission lines of individual Chl molecules we observe a broad spectral band peaking at 730 nm (see Fig. 6.28).
Since there are no intense Chl vibrations in the low-frequency domain we believe that the broad structure is related to a pigment protein system with strong
electron phonon coupling. Two possible origins of the 730 nm band can be
considered. First, the broad spectral band may be a phonon wing of the zerophonon line found in the 710 715 nm region of Fig. 6.27. Due to strong electron phonon coupling, a large Stokes shift may be associated with the low frequency phonon modes of the protein environment. Second, the 730 nm band
corresponds to a different pigment site. Based on hole-burning studies of pigment protein complexes [37] we assume that the main phonon frequency
responsible for the formation of a phonon wing is 16 cm 1. A large Stokes
shift (300 cm 1) would require a Huang Rhys factor of the 712 nm state of
about 20. Under these conditions the zero-phonon lines should not be observable because of an extremely low Franck Condon factor (exp (-20)).
Based on the ensemble data [35] we can tentatively assign the two observed
bands to the earlier reported C708 and C719 groups of pigments. We expect a
ratio of the oscillator strength of about 5/6 for C708 and C719 Chl sites respectively. The high total intensity of the 730 nm band may be related to the difference
in the triplet quenching efficiency between two pigment pools. Similar to LHCII,
efficient triplet quenching is particularly important because of the high excitation
rate in single molecule experiments. The strong quenching of the red-most state
once again seems to be physiologically motivated because of the efficient energy
transfer to the red-most state at room temperature.
Hence two different Chls pools probably play a role in the formation of the
long-wavelength antenna part of PSI. The absence of narrow zero-phonon lines
in the red-most pool may be explained in terms of an intense spectral diffusion or/and strong coupling leading to low zero-phonon lines intensity. The
efficiency of electron phonon coupling in pigment protein complexes depends
on the change in permanent dipole moment (Dm) [64]. Recently available holeburning data on red pool Chls of PSI from Synechocystis show that Dm increases when the burn wavelength increase from 700 to 715 nm, leading to
significant broadening of the hole spectra and disappearance of zero-phonon
structure on the red edge of the PSI absorption band [37]. A charge-transfer
character of excited antenna state can be associated with a strong electronexchange coupling between closely spaced Chl [65]. Unfortunately Stark-spectroscopy data are not available for Synechococcus elongatus and we can only speculate about the nature of the strong coupling in this system. Nevertheless our
data suggest that the discussion concerning the excitonic nature of the lowenergy C714 state of Synechocystis (see ref. [37,66]) can be expanded to the
red-most state of Synechococcus elongatus.
227
228
References
Summarizing, the spectral analysis suggests that two antenna Chl pools are responsible for the red-most absorption of PSI from Synechococcus elongatus. The
electronic transition of the 712 nm state is not strongly coupled to the phonon
bath of the protein environment whereas electron phonon coupling of the redmost pool leads to a complete disappearance of the zero-phonon structure. Strong
coupling may be associated with excitonic interaction between chlorophylls in the
red-most pool.
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