11488
J. Phys. Chem. 1996, 100, 11488-11495
Energy Transfer and Trapping in Isolated Photosystem II Reaction Centers of Green Plants
at Low Temperature. A Study by Spectral Hole Burning
M. L. Groot,†,‡ J. P. Dekker,† R. van Grondelle,† F. T. H. den Hartog,‡ and S. Vo1 lker*,†,‡
Department of Biophysics, Faculty of Physics and Astronomy, Free UniVersity,
1081 HV Amsterdam, The Netherlands, and Center for the Study of Excited States of Molecules,
Huygens and Gorlaeus Laboratories, UniVersity of Leiden, 2300 RA Leiden, The Netherlands
ReceiVed: January 31, 1996; In Final Form: April 26, 1996X
Spectral hole burning has been performed on the Qy-region of the isolated reaction center of photosystem II,
the D1-D2-cytochrome b559 complex (PSII RC), between 665 and 688 nm, at liquid He temperatures.
The “effective” homogeneous line width Γ′hom at 682 nm, in the red wing of the Qy-band, follows a T1.3(0.1
power law between 1.2 and 4.2 K characteristic of glasses and extrapolates to Γ′0 ) (2πT1)-1 for T f 0 with
T1 ) (4 ( 1) ns, the fluorescence lifetime of the pigments. At these low temperatures, the red-absorbing
“trap” pigments are unable to transfer energy to other pigments. The spectral distribution of the traps has
been determined from hole depth vs λexc experiments. Their linear electron-phonon coupling strength was
found to be rather weak, S ) 0.73 ( 0.05. For λexc< 678 nm, “downhill” energy transfer takes place. Spectral
distributions of pigments characterized by decay times of 200 and 12 ps have further been identified in this
spectral region. The data have been used to reconstruct the fluorescence excitation and absorption spectra.
1. Introduction
The primary process in photosynthesis occurs within a lipid
membrane. It involves the absorption of light by antenna
complexes and the transfer of excitation energy to a primary
electron donor within a reaction center where the energy is
trapped by a sequence of electron-transfer reactions.1,2 In
photosystem II (PSII) reaction centers (RC) of higher plants,
these electron-transfer reactions produce a very high oxidizing
potential (∼1 V) which is used for water oxidation accompanied
by oxygen evolution. This is a major difference between
bacterial and plant photosynthesis.3
An important step forward in the research of the structure
and dynamics of PSII has been the isolation of the D1-D2cytochrome b559 complex (PSII RC), of which the photoactivity
is limited to the primary charge separation.4,5 This PSII RC
consists of the D1 and D2 subunits, cytochrome b559 polypeptides, and the psbI gene product. It contains about six
chlorophylls a (Chl a), two pheophytins a (Pheo a), one or two
β-carotenes, and no plastoquinones because they are lost during
isolation.6-9
After excitation, which may happen directly by light or
indirectly by energy transfer through one of the accessory Chl a
or Pheo a pigments, an electron is transferred from the primary
donor, called P680*, to the first electron acceptor (a Pheo a
molecule) in less than 30 ps.10-15 The primary radical pair
P680+Pheo- decays then by charge recombination in about 40100 ns, depending on temperature.16,17 In the absence of
quinones, electron transfer beyond Pheo a is blocked.
A thermodynamic model for the PSII RC kinetics was
recently proposed18 on the basis of primary exciton equilibration
and charge separation reactions. It describes the experimental
triplet and fluorescence quantum yields obtained as a function
of temperature, from which a distribution of free energy
differences (∆G ≈ 20-80 meV) between the singlet-excited
P680* and the radical-pair state P680+Pheo- results.18 A
* To whom correspondence should be addressed.
† Free University.
‡ University of Leiden.
X Abstract published in AdVance ACS Abstracts, June 1, 1996.
S0022-3654(96)00326-7 CCC: $12.00
consequence of this model is that the radical-pair recombination
reaction to P680* may only take place at temperatures T > 50
K. The increase of the fluorescence quantum yield observed
when lowering the temperature below 50 K was explained by
assuming the presence of accessory pigments (those which do
not constitute P680) energetically degenerate with P680.
Because of inhomogeneous broadening, at very low temperatures, the accessory pigments lying energetically below P680
will act as “traps” for the excitation energy and decay in a time
determined by their fluorescence lifetime of about 4 ns. This
is in agreement with fluorescence lifetimes of ∼4-6 ns observed
at T ∼ 20 K by Roelofs et al.,19 but these authors suggested
that the nanosecond lifetimes reflect charge recombination
fluorescence. If this would be the case, ∆G should be much
smaller than 20 meV, in contradiction to ref 18. The steadystate fluorescence at 4 K observed at λ > 683 nm by Kwa et
al.,20 on the other hand, was attributed to a long-wavelengthemitting Chl a molecule.
By contrast, from persistent spectral hole-burning experiments
at 1.6 K it was concluded that all accessory pigments, when
excited, transfer their energy to P680.21 Holes burnt at ∼682
nm yielded decay times of ∼50 ps, which were interpreted as
energy transfer from Pheo a to P680 implying the absence of
accessory trap pigments in PSII RC.
A lack of consensus in the literature concerning PSII RC has
not only been reported for energy-transfer processes within the
Qy-region, but also for the charge separation rate. The reason
for these controversies has its origin, probably, in the large
overlap of strongly inhomogeneously broadened absorption
bands between 660 and 690 nm. As a consequence, (sub-)
picosecond time-resolved experiments are difficult to interpret.
Several groups have claimed that “slow” (∼20 ps) energy
transfer from 670 to 680 nm absorbing pigments13,19,22 is
followed by “fast” charge separation (∼2 ps).10,13,22 Such times
were supported by transient and permanent hole burning at 1.6
K.11,21 The group at Imperial College,14,15 however, has reported
a “very fast” (∼100 fs) equilibration process between pigments
at 670 and 680 nm, in addition to slow (∼20 ps) charge
separation at room temperature.23 Very recently, (sub-) pico© 1996 American Chemical Society
A Study by Spectral Hole Burning
second transient-absorption experiments at ∼680 nm, at 77 K
have yielded decay times that were interpreted as fast (1-2 ps)
“intrinsic” charge separation and slow (80-100 ps) “energy
transfer-limited” charge separation,24 the latter arising from a
trap state energetically degenerate with P680.18 “Downhill”
energy transfer times of 400-500 fs and ∼14 ps were additionally observed when exciting at ∼670 nm.24
In order to verify whether low-lying trap pigments exist with
a lifetime of about 4 ns which are not involved in any kind of
energy transfer or charge separation at low temperature and to
solve the contradictions related to energy transfer in PSII RC,
we have performed persistent spectral hole-burning (HB)
experiments between 1.2 and 4.2 K over the whole Qy-region
from 665 to 688 nm. This technique, because of its high spectral
resolution and wavelength- and burning-fluence selectivity, is
an attractive tool for studying the excited-state dynamics of
complexes with strongly overlapping spectral bands. HB yields
not only T1- and T2-dephasing times as a function of wavelength
and temperature but also spectral distributions of pigments
characterized by their dynamic properties. Since we have
probed the holes by means of fluorescence excitation spectroscopy, an electronicaly excited pigment may only be detected if
it fluoresces or if it transfers its excitation energy to another
pigment which in turn fluoresces. Because P680* undergoes
very fast charge separation in a few picoseconds, it practically
does not fluoresce. Thus, only the accessory pigments are
sensitive to hole burning detected in this way.
While performing these HB experiments, we have indeed
found holes in the red wing of the Qy-region for which the width
extrapolated to temperature T f 0 corresponds to a fluorescence
lifetime of ∼4 ns. Thus, “4 ns” trap pigments are present in
PSII RC, at least at very low temperatures up to 4.2 K, their
dynamics being controlled by “pure” dephasing processes. We
have also obtained the spectral distribution of these 4 ns traps
(section 3.1).
Further toward the blue of the Qy-region, we have identified
two types of pigments characterized by energy transfer times
of 200 and 12 ps (section 3.2). From the spectral distributions
of the three identified pigments and from two additional
distributions indirectly obtained, one for “P680” and another
one for pigments of unidentified decay time, we were able to
reconstruct the fluorescence excitation and absorption spectra
(section 3.3).
2. Experimental Section
The reaction center of photosystem II, called D1-D2cytochrome b559 (PSII RC), was isolated from the CP47-RC
complex by means of a short Triton X-100 treatment as
described in refs 9 and 25. The latter was obtained from spinach
using the nonionic detergent n-dodecyl β,D-maltoside.26
Prior to the low-temperature measurements, the samples were
diluted in a buffer containing 20 mM BisTris (pH 6.5), 20 mM
NaCl, 0.03% (w/v) n-dodecyl β,D-maltoside, and 85% (w/v)
glycerol. They were stored at 77 K when not used. The PSII
RC complexes contained 6.4 Chl a and 1.6 β-Car per 2 Pheo a.9
To obtain glasses of good optical quality at liquid helium
temperature, the samples were slowly cooled (in about half an
hour) from room temperature to 77 K by keeping the cuvette
(thickness 1 mm) in an empty 4He bath cryostat of which the
outer mantle was filled with liquid nitrogen. Cooling from 77
to 4.2 K was achieved in a few minutes by filling the cryostat
with liquid helium. The temperature was varied between 4.2
and 1.2 K and controlled by the vapor pressure of the 4He. The
temperature was measured using a calibrated carbon resistor in
contact with the sample with an accuracy better than 0.01 K.
J. Phys. Chem., Vol. 100, No. 27, 1996 11489
Figure 1. Qy-region of the absorption spectrum A(λ) (see section 2)
of the isolated reaction center of photosystem II (PSII RC) at 1.2 K.
The curve traced through the data has been used for the reconstruction
of Figure 9b.
Broad-band absorption and fluorescence excitation spectra
at 1.2 K were taken in Leiden with a tunable CW dye laser
(Coherent 599-21, DCM dye, bandwidth ∼30 GHz ) 1 cm-1
without intracavity assembly, amplitude stabilized to <0.5%
by an electro-optic modulator) pumped by an Ar+-laser (Spectra
Physics 2030-15). The wavelength was calibrated with a
Michelson-type interferometric wavemeter (accuracy ∼50 MHz).
Transmission and fluorescence signals were measured simultaneously at 90° with respect to each other with two cooled
photomultipliers (EMI 9658 R). To separate the fluorescence
signal from scattered light, two long-wave pass filters were used
(Schott, RG715 and RG780) such that λdet g 780 nm. Absorption spectra were obtained in two ways. First, by dividing the
transmission signal, It ) I0 × 10-OD, at every wavelength by a
reference spectrum I0(λ) of an empty cuvette, and taking the
logarithm of the ratio, A ) log(I0/I) ) OD, also called
absorbance. The absorption expressed as OD is shown in Figure
1 between 650 and 690 nm. Second, the absorption Ia )
I0(1 - 10-OD) as a function of λ was directly plotted (see section
3.3). Since samples of low optical density (OD ∼ 0.14) were
used, the absorption Ia(λ) and the absorbance A(λ) are proportional to each other, and the shape of the two spectra is the
same. The fluorescence excitation spectrum, which scales with
the absorption spectrum Ia(λ), is then also proportional to the
absorbance at low OD values (see section 3.3).
Spectral hole-burning experiments were performed with the
same CW dye laser using an intracavity assembly by which
the bandwidth of the laser was reduced to its jitter Γlaser ≈ 2
MHz. The laser beam was focused on the sample to an area A
∼3 mm2. Burning-power densities were used in the range from
P/A ∼ 10 µW/cm2 to 10 mW/cm2, with burning times varying
from tb ) 10 s to 30 min. Thus, burning-fluence densities varied
between Pt/A ∼ 5 × 10-5 and 5 J/cm2. The holes were probed
in fluorescence excitation (λdet g 780 nm) with the same laser,
but its power was attenuated by a factor 102-103. The duration
of a probe-burn-probe cycle varied from 5 to 30 min.
The “effective” homogeneous line width Γ′hom was determined by extrapolating half the hole width 1/2Γ′hole to zero
burning-fluence density, Pt/A f 0. The value of Γ′hom at T >
0 may include a contribution from spectral diffusion because
the holes were probed with a delay after burning of a few
minutes.27,28 Since the hole widths of a few hundred megahertz
to a few gigahertz were much larger than the laser bandwidth
(Γlaser ≈ 2 MHz), we took Γ′hom ) 1/2Γhole,Pt/Af0. The hole
profiles were well fitted with Lorentzian curves.
11490 J. Phys. Chem., Vol. 100, No. 27, 1996
Groot et al.
Figure 2. (a) Half the hole width 1/2Γhole as a function of burningfluence density Pt/A at 682.0 nm and 1.2 K. The data extrapolate to
the effective homogeneous line width Γ′hom for Pt/A f 0. Inset: Hole
burnt at Pt/A ) 0.54 mJ/cm2 . (b) Temperature dependence of Γ′hom
between 1.2 and 4.2 K at 682.0 nm. Γ′hom follows a T1.3(0.1 power law
which extrapolates to Γ′0 ) (2πT1)-1 ) 45 ( 10 MHz for T f 0. T1
corresponds to the fluorescence lifetime τfl ) (4 ( 1) ns of chlorophylllike molecules.
3. Results and Discussion
3.1. Trap Pigments Absorbing Furthest to the Red (λ g
680 nm). In order to determine the dynamic behavior of the
accessory pigments absorbing furthest to the red within the Qyregion of the PSII RC complex, we have performed HB
experiments with megahertz resolution as a function of temperature between 1.2 and 4.2 K. The value of the effective
homogeneous line width Γ′hom was obtained, as described at
the end of section 2, by extrapolating half the hole width 1/2Γhole
for burning-fluence densities Pt/A f 0. Such a plot is shown
in Figure 2a for holes burnt at 682 nm, at 1.2 K. Between 678
and 688 nm we have found narrow holes, of the order of a few
hundred megahertz, with a wavelength-independent width at a
given burning-fluence density. A typical hole is shown in the
inset of Figure 2a. It was obtained at a low value of Pt/A )
0.54 mJ/cm2 for which 1/2Γhole 280 MHz is close to Γ′hom.
The temperature dependence of Γ′hom for pigments absorbing
at 682 nm is given in Figure 2b. The data follow
Γ′hom ) Γ′0 + aT1.3(0.1
(1)
between 1.2 K and 4.2 K. A T1.3 power law, as obtained here,
is characteristic for dephasing in doped organic glasses29 and
indicates that for PSII RC the pigment-protein interaction is
similar to that in amorphous systems. The extrapolated value
of Γ′hom for T f 0 is given by Γ′0 ) (2πT1)-1, in which T1 )
Figure 3. (a) Relative hole depth D (%) as a function of excitation
wavelength λ (dashed line) together with the profile of the fluorescence
excitation spectrum (solid line). The holes were burnt with Pt/A ) 0.54
mJ/cm2 at 1.2 K. (b) Spectral distribution of the 4 ns trap pigments
(dashed line) reconstructed in the fluorescence excitation spectrum. The
distribution was obtained after multiplying the two curves in Figure
3a by each other and normalizing the height to the fluorescence
excitation spectrum (solid line).
(4 ( 1) ns proves equal to the fluorescence lifetime τfl of Chl a
and Pheo a molecules. Because T1 ) τfl, these HB results
demonstrate that there are pigments absorbing furthest to the
red within the Qy-region which indeed are traps. These 4 ns
living pigments undergo pure dephasing and decay, but do not
transfer energy to other pigments like P680, at least at low
temperatures up to 4 K. Such traps were predicted for T < 50
K from the kinetic model of ref 18.
To determine the spectral distribution of these 4 ns trap
pigments we have measured not only the hole width but also
the relative depth of the holes D (%) as a function of excitation
wavelength between 677 and 687 nm at a constant, low burningfluence density (Pt/A ) 0.54 mJ/cm2, see Figure 2a) at 1.2 K.
In this wavelength region the holes change their depth but not
their width, indicating that this method selects those pigments
that are involved in a specific dynamic process, in this case
characterized by a 4 ns decay time. The values of D (%) vs λ
are reproduced in Figure 3a together with the profile of the
fluorescence excitation spectrum between 675 and 690 nm. The
dashed curve traced through the hole depth data is a guide to
the eye. By multiplying this curve by the profile of the
fluorescence excitation spectrum, one obtains an approximate
Gaussian profile30 with a maximum at (681.8 ( 0.3) nm and a
fwhm ) (143 ( 5) cm-1, which represents the spectral
distribution of the 4 ns fluorescing pigments.
This distribution is shown in Figure 3b where its height has
been normalized to the fluorescence excitation spectrum.30 By
A Study by Spectral Hole Burning
J. Phys. Chem., Vol. 100, No. 27, 1996 11491
comparing the position and the width of this 4 ns trap
distribution with the broad (∼120 cm-1 width) permanent
satellite hole at 681.6 nm reported by Tang et al.,21 we conclude
that our distribution and their satellite hole most probably are
related to the same pigments. The authors of ref 21 attributed
the pigments absorbing at 681.6 nm to Pheo a, because a hole
burnt at 663 nm caused simultaneously two satellite holes, the
very broad one at 681.6 nm and a second one at 545.7 nm, the
wavelength of the Qx-band of Pheo a. As mentioned in the
introduction, and in contradiction to our results, holes burnt at
∼682 nm were reported to yield decay times of ∼50 ps,21 much
shorter than the 4 ns determined by us. The discrepancy may
be due to too high burning-fluence densities and temperatures
used: the holes of ref 21, which were measured at a single
temperature of 1.6 K, correspond to a value of Γ′hom ) 3.2 GHz.
This is a factor of 10 larger than our value of Γ′hom at the same
temperature and wavelength. Although no burning-fluence
density is mentioned in ref 21, the same group reported in ref
11 a hole of a similar width burnt with a Pt/A value of the order
of 2000 times larger than that in our experiments. As may be
seen from our Figures 2a,b, it is necessary to extrapolate the
hole widths for Pt/A f 0 and T f 0 to get a reliable value of
T1.
We have also determined the linear electron-phonon coupling strength S for the 4 ns trap pigments. The value of S was
obtained from HB experiments carried out under saturating
conditions, i.e. at very high burning-fluence densities by which,
besides the zero-phonon holes, side holes become visible (see
Figure 4a). To burn such deep holes we had to use the CW
laser without the intracavity assembly (bandwidth of ∼1 cm-1,
see section 2). The hole widths (∼4 cm-1) are then not only
limited by twice the laser bandwidth, but additionally, they are
power broadened. The area of the zero-phonon hole Azph was
divided by the sum of Azph and the area of the pseudo-phonon
side hole Apsh, and the ratio was extrapolated to zero burningfluence density (see Figure 4b). This yields the so-called
Debye-Waller factor R which, under these conditions, is related
to S by
R ) (Azph/(Azph + Apsh))Pt/Af0 ) 2e-2S/(1 + e-2S)
Figure 4. (a) Deep holes burnt under saturating conditions, at two
burning-fluence densities Pt/A, at 682.0 nm and 1.2 K. (b) Burningfluence density dependence of the ratio Azph/(Azph + Apsh), where Azph
is the area of the zero-phonon hole and Apsh is the area of the pseudophonon side hole, at 682 nm and 1.2 K. The extrapolated value for
Pt/A f 0 yields the Debye-Waller factor R which is related to S, the
electron-phonon coupling strength. S ) 0.73 ( 0.05 (see text).
(2)
Examples of deep holes and side holes burnt at 682 nm are
shown in Figure 4a for two different Pt/A values. We attribute
the side hole appearing at ∆ν ∼ (16 ( 1) cm-1 from the zerophonon hole to a low-frequency mode of the protein.31 From
the ratio of the hole areas as a function of Pt/A (see Figure 4b),
we have obtained S ) 0.73 ( 0.05, a value which agrees with
that reported for the Pheo a Qy-state.21 It represents a rather
weak electron-phonon coupling strength when compared to S
∼ 2, the value reported for P680 of PSII in ref 11 and for P870/
P960 of purple bacteria.32
Finally, we remark that by burning at 682 nm we also found
a satellite hole at ∼545 nm, the wavelength of the Pheo a Qxstate. Thus the 4 ns trap pigments consist, at least, of Pheo a.
On the other hand, from fluorescence spectra at 4 K it was
concluded that the most red emission arises from Chl a.20
Probably both pigments are present at λ > 680 nm.
3.2. Pigments Absorbing at Higher Energies within the
Qy-Region (λ < 680 nm). By burning at wavelengths shorter
than 678 nm, at Pt/A ∼ 0.5 × 10-3 J/cm2, the narrow holes
representing the 4 ns trap pigments (see Figure 2a) become very
shallow (D < 2%) and disappear (see Figure 3a). However, it
is still possible to detect holes at these shorter wavelengths if
103 times higher burning-fluence densities are used. This is
illustrated in Figure 5, where 1/2Γhole has been plotted as a
function of Pt/A for holes burnt at 676 nm, at 1.2 and 4.2 K.
Figure 5. Half the hole width 1/2Γhole as a function of burning-fluence
density Pt/A, at 676 nm, for 1.2 and 4.2 K. The extrapolated value for
Pt/A f 0 yields Γ′hom at each temperature.
Notice that, while the values of Pt/A ∼ 0.5-1 J/cm2 are indeed
3 orders of magnitude higher than in Figure 2a (holes at 682
nm), the holes are about four times broader. As in Figure 2a,
Γ′hom at a given temperature is obtained from the extrapolation
of 1/2Γhole for Pt/A f 0.
In order to select pigments by their dynamic properties, i.e.
by their hole widths and depths at a given Pt/A value, we have
measured 1/2Γhole as a function of Pt/A at various wavelengths.
Such a plot is shown in Figure 6a. Notice that the widths of
holes burnt at 676 and 678 nm follow the same curve (open
11492 J. Phys. Chem., Vol. 100, No. 27, 1996
Groot et al.
Figure 7. Temperature dependence of Γ′hom of pigments absorbing at
676 nm compared to that of the 4 ns trap pigments at 682 nm (Figure
2b). At 676 nm, Γ′0 ) (2πT1)-1 > (2πτfl)-1 with T1 ) (200 ( 10) ps
(see text).
Figure 6. (a) Burning-fluence density dependence of 1/2Γhole under
saturating conditions (Pt/A values up to 5 J/cm2), for three excitation
wavelengths at 1.2 K. (Upper curve) 676 nm (open circles) and 678
nm (closed circles). (Lower curve) 685 nm (diamonds). Notice that
pigments absorbing at 676 and 678 nm, having the same hole widths,
belong to the same spectral distribution (see text). (b) 1/2Γhole vs burningfluence density (for Pt/A < 0.15 J/cm2), at 678 nm (closed circles) and
685 nm (diamonds), at 1.2 K. Notice that pigments absorbing at 678
and 685 nm, having the same hole widths, belong to the same spectral
distribution. Thus, pigments absorbing at 678 nm may belong to either
of the two distributions depending on their decay time (see text).
and closed circles) and saturate to a width of ∼4 GHz, which
is much larger than that of holes burnt at 685 nm with 1/2Γhole
∼ 2 GHz for Pt/A f ∞ (diamonds). Thus, the pigments
absorbing at 676 and 678 nm belong to the same spectral
distribution characterized by a faster dynamics than the pigments
absorbing at 685 nm which decay in 4 ns.
If, on the other hand, we follow the values of 1/2Γhole in the
region of Pt/A < 0.15 J/cm2 (see Figure 6b), we notice that the
widths of holes burnt at 678 nm have crossed over from the
676 nm curve to the 685 nm curve. We have to conclude that
pigments absorbing at 678 nm also belong to the 4 ns trap
distribution (see section 3.1). This is understandable if we look
at the spectrum in Figure 3b, where at 678 nm not only pigments
belonging to the 4 ns distribution absorb, but also others that
can only be burnt at much larger Pt/A values and undergo a
faster dynamics.
In Figure 7 the dephasing between 1.2 and 4.2 K of pigments
absorbing at 676 nm has been plotted and compared to that of
the 4 ns trap pigments absorbing at 682 nm (from Figure 2b).
We observe that for both types of pigments Γ′hom follows a T1.3
dependence, i.e. a glasslike behavior, but the extrapolated value
Γ′0 for T f 0 is different at the two wavelengths. At 676 nm,
Γ′0 ) (2πT1)-1 > (2πτfl)-1 with T1 ) (200 ( 10) ps.
Apparently, the decay of pigments absorbing at 676 nm is 20
times faster than that of pigments in the red wing. The direct
fluorescence to the ground state will, therefore, be very weak.
Because holes burnt between 672 and 678 nm at a constant
Pt/A value yielded the same hole widths, we call the spectral
distribution of these pigments the “200 ps” distribution. Its
maximum is at ∼675 nm and has a width of ∼140 cm-1 (see
section 3.3 and Table 1). We attribute the 200 ps decay to
downhill energy transfer to the lower-lying P680 and 4ns
pigments.
When burning at 672 nm, two types of holes are observed,
as illustrated in Figure 8a. The first type belongs to pigments
of the 200 ps distribution. It has a width 1/2Γhole ∼ 2 GHz at
Pt/A ≈ 1 J/cm2 at 1.2 K. When burning at about five times
higher burning-fluence density this type of hole saturates (see
also Figure 6a) and its base line shifts downward suggesting
that a much broader hole has been formed. This second type
of hole is very broad (lowest one in Figure 8a). It has a width
1/ Γ
2 hole ≈ 13 GHz, about 15 times larger than Γ′hom of the 200
ps distribution. Between ∼5 and 10 J/cm2 the holes do not
broaden significantly. Since pure dephasing up to 1.2 K
contributes only little to the hole width (at most 0.2 GHz),
T1 ≈ (2π 13 GHz)-1 ) (12 ( 2) ps. The latter type of holes
is observed between ∼665 and ∼673 nm, from which we
have estimated that the “12 ps” distribution is centered at ∼669
nm and has a width of about 200 cm-1 (see section 3.3 and
Table 1).
Similar holewidths corresponding to a decay time of 12 ps
at 1.6 K were reported in the literature when burning at ∼665
nm.21 Also picosecond-time-resolved experiments at higher
temperatures (from 15 K to room temperature) yielded decay
times of 10-20 ps in this wavelength region.13,19,24,33 We
attribute the fast decay at 1.2 K to downhill energy transfer
from the 12 ps pigment distribution to the 200 ps, 4 ns, and
P680 distributions.
In Figure 8b we have summarized our hole-burning results
by plotting the values of Γ′hom as a function of excitation
wavelength, for given values of Pt/A, together with the
fluorescence excitation spectrum. The regions of constant
holewidths represent the spectral distributions of pigments which
have been labeled with the values of their corresponding decay
times: 4 ns, 200 ps, and 12 ps.
3.3. Reconstruction of Spectra from the Spectral Distributions of Pigments Characterized by Their Decay Times.
To reconstruct the fluorescence excitation spectrum at 1.2 K
from the spectral distributions of pigments obtained from our
hole-burning experiments, we have to make assumptions about
A Study by Spectral Hole Burning
Figure 8. (a) Holes burnt at various burning-fluence densities at 672
nm and 1.2 K. (From top to bottom) Pt/A ∼ 1 J/cm2, 5 J/cm2, and 10
J/cm2. The hole at Pt/A ∼ 10 J/cm2 has a width 1/2Γhole ) 13 ( 2 GHz
which corresponds to a transfer time T1 ) (2πΓ′0)-1 ) (12 ( 2) ps.
(b) Values of Γ′hom at different spectral positions within the fluorescence
excitation spectrum. Burning-fluence densities are given together with
the name of their spectral distribution.
the shape of these distributions, their positions, heights, and
widths. With respect to the shape, we have assumed that at
liquid He temperature it is Gaussian because the spectral
distributions are inhomogeneously broadened (holes can be burnt
into them). The position and width of the 4 ns trap pigments
distribution have been accurately determined as described in
section 3.1 (see Figures 3a,b) with a maximum at (681.8 ( 0.3)
nm and a fwhm ) (143 ( 5) cm-1.
For the positions and widths of the 200 ps and 12 ps pigment
distributions, we have used an iterative procedure. In a first
approximation, we have taken for the value of fwhm the
wavelength region over which the holes have equal width when
burnt at constant Pt/A. By the choice of the burning-fluence
density Pt/A, we select pigments that decay with a specific time
constant, cf. the discussion of Figures 6a,b above. For the
maximum of each distribution we have taken the center of the
region over which the holes have an equal width. Although by
adjustment of their relative heights the 4 ns, 200 ps, and 12 ps
distributions fill in the fluorescence excitation spectrum reasonably well over a large wavelength region, they leave a gap
around 679 nm. This gap can only be filled in if we assume at
least one distribution in the 679 nm region with a total width
of ∼100 cm-1. We have not attempted to interpret this gap
because we have not identified further decay times by hole
burning in this region. From time-resolved pump-probe
experiments at room temperature in the same wavelength region,
it was concluded that very fast equilibration of the order of 100
fs takes place between pigments absorbing in the red and in
the blue of the Qy-region.14,15 If such a fast equilibration would
also occur at liquid He temperatures, we would expect extremely
J. Phys. Chem., Vol. 100, No. 27, 1996 11493
Figure 9. (a) Reconstruction of the fluorescence excitation spectrum
at 1.2 K by a superposition of inhomogeneously broadened bands. The
latter were taken as Gaussians; they represent the spectral distributions
of pigments characterized by a specific decay time. Those labeled with
4 ns, 200 ps, and 12 ps were obtained from the widths and depths of
holes at different wavelengths. The distribution represented by dots
was added in order to fill in the fluorescence excitation spectrum without
gaps (see text). (b) Reconstruction of the absorption spectrum Ia(λ) at
1.2 K (see section 2) by superposition of the four inhomogeneously
broadened (Gaussian) distributions of Figure 9a, but with different
heights, and an indirectly obtained distribution which we attribute to
P680 (see text, section 3.3). The area of the fluorescence excitation
spectrum has been scaled to 0.25 of that of the absorption spectrum.18
The latter Ia(λ) is proportional to the absorbance A(λ) ) OD only at
low OD. Since OD ∼ 0.14, the proportionality is valid (see sections 2
and 3.3).
TABLE 1: Position of the Maxima and Widths of the
Spectral Distributions of Pigments Characterized by Their
Decay Time, at 1.2 Ka
distribution
λmax (nm)
fwhm (cm-1)
4 ns trap (from HB)
P680
679 (unidentified decay)
200 ps (from HB)
12 ps (from HB)
681.8 ( 0.3
680.8 ( 0.5
678.8 ( 0.3
675.1 ( 0.4
669.1 ( 0.5
143 ( 5
150 ( 5
101 ( 3
142 ( 5
196 ( 6
a The fluorescence excitation and absorption spectra in the Q -region
y
of PSII RC were reconstructed by superposition of the spectral
distributions discussed in section 3.3 (see Figure 9a,b).
broad holes of ≈50-100 cm-1 width. Such holes would be
very shallow and, therefore, probably not detectable within the
noise.
The four distributions of pigments are given in Figure 9a
together with the fluorescence excitation spectrum. The three
dashed curves represent the spectral distributions estimated from
hole burning, whereas the curve with thin points is that of
11494 J. Phys. Chem., Vol. 100, No. 27, 1996
hypothetical pigments absorbing at ∼679 nm with one or more
unidentified decay time(s). The superposition of all four
distributions is given by the curve with thick points. It
reproduces the fluorescence excitation spectrum well, with the
exception of the far blue wing which should probably be
accounted for by additional distributions or vibronic bands.
Next, we made an attempt to reconstruct the absorption
spectrum Ia(λ), which at low concentration (OD ) 0.14) is
proportional to the absorbance A(λ) (see section 2), starting from
the distributions obtained in the fluorescence excitation spectrum. The reconstruction was carried out in three steps. First,
the fluorescence excitation spectrum was scaled in such a way
that its area corresponds to 25% of that of the absorption
spectrum Ia(λ) (see Figure 9b). The reason for this is based on
the results of ref 18 where it was calculated that after
nonselective excitation at ∼610 nm at 4 K, about 75% of the
excitation is used for charge separation. Thus, the remaining
25% will decay through the accessory pigments to the lowestlying 4 ns trap and should be detectable in fluorescence
excitation. As a consequence, the height of the 4 ns trap
distribution is fixed to that of the fluorescence excitation
spectrum.
Second, we have assumed that the red wing of the absorption
spectrum at 1.2 K consists of 4 ns trap pigments and P680
pigments only, at least for λ > 683 nm. This assumption is
based on various types of experiments: (1) our HB experiments
at 1.2 K which have shown that the red wing of the fluorescence
excitation spectrum only consists of 4 ns trap-pigments, (2)
triplet-minus-singlet absorption difference spectra34 which show
a broad bleaching at 680.6 nm (width 6.5 nm) and were
attributed to depletion of the P680 ground state, (3) magnetophotoselection experiments at 6 K35 which have proven the
presence of P680 pigments in the red edge of the absorption
spectrum, and (4) transient hole-burning experiments at 4.2 K
in the 681 nm region in which it was reported that the absorption
maximum of P680 lies at 680.3 nm,11 or 681.7 nm,21 with an
inhomogeneously broadened width of about 140 cm-1.11,21 Since
the 4 ns distribution is fixed in position, width, and height, the
P680 distribution, under the assumptions made here, follows
automatically because it has to fill in the red wing of the
absorption spectrum. The maximum of P680 then appears at
(680.8 ( 0.5) nm with a width of (150 ( 5) cm-1. Its height
was matched to that of the absorption spectrum (see Figure 9b).
In this procedure, the spectral location of P680 is not measured
by us but follows from a comparison of the band shapes of the
red wings of the fluorescence and absorption spectra. It is
gratifying that the spectral distribution of P680 thus derived is
so similar to that reported in the literature by other techniques.11,21,34,36 The very small shoulder at 667 nm in the wing
of the distribution, reported in ref 34, does not appear in the
reconstructed spectrum of P680. Our results do not give any
indication on the nature of P680, i.e. whether it is a multimer
of several weakly coupled pigments37 or a dimer,35 nor on the
charge separation time.
Third, to reconstruct the rest of the absorption spectrum we
have used the positions and widths of the 200 ps, 12 ps, and
the 679 nm-distribution of pigments from the fluorescence
excitation spectrum and only varied their relative heights until
they filled in the absorption spectrum. This means that, for
simplification, we have assumed that no other distributions are
present in this part of the absorption spectrum at 1.2 K, for
which we do not have any proof. If at 1.2 K, like at room
temperature,14,15,37 very fast equilibration would occur over a
large part of the spectrum, we would expect very broad and
shallow holes to be difficult to detect. The results of the
Groot et al.
Figure 10. Energy-level scheme of PSII RC corresponding to the data
obtained in this work at 1.2 K. The arrows denote the possible decay
channels from the spectral distributions of pigments identified by hole
burning and from the indirectly obtained P680 (see section 3.3). No
distributions of pigments have been included which are not directly or
indirectly determined here.
reconstruction under the given assumptions are shown in Figure
9b. The absorption spectrum is well reproduced by only five
pigments distributions, of which four were taken from the
fluorescence excitation spectrum, while the P680 pigment
distribution arose from a need to fill in a gap left over in the
red part of the absorption spectrum as compared to the
fluorescence excitation spectrum. The P680 pigments are not
detected by hole burning because they do not fluoresce owing
to the fast charge separation within a few picoseconds.
Nevertheless it is surprising that, under the conditions assumed,
the five distributions reproduce a large part of the absorption
spectrum rather well without the need for additional distributions.
As in the fluorescence excitation spectrum, the extreme blue
wing of the Qy-region of the absorption spectrum is not
accounted for by the five distributions mentioned. In this
spectral region there probably are vibronic bands or other
pigments which could not be identified by hole burning.
In Table 1 the positions and widths are given for (1) the three
spectral distributions deduced from our HB experiments at 1.2
K, (2) the distribution which we have attributed to P680, and
(3) another distribution of hypothetical pigments with an
unidentified decay time with a maximum at ∼679 nm and a
width of ∼100 cm-1.
We have summarized the results obtained in this work in the
energy-level diagram of Figure 10. Each spectral distribution
of pigments within the Qy-region of PSII RC is characterized
by a specific decay time. These distributions were obtained
by persistent hole burning probed by fluorescence excitation at
1.2 K. The possible decay pathways are also given in the figure.
We think that the “12 ps”-pigments decay via energy transfer
to the 200 ps, the 4 ns, and the P680 pigments, whereas the
200 ps pigments decay via energy transfer to the 4 ns and the
P680 pigments. The 4 ns pigments decay only to the ground
state via emission of fluorescence and nonradiative decay. From
the experiments we cannot conclude anything about the internal
dynamics of P680. Within the framework of the multimer
model,37 P680 reflects the core pigments of the PSII RC
including the pheophytins and may show dynamics on a
subpicosecond time scale before charge separation occurs.
A Study by Spectral Hole Burning
Because of the low temperatures of our experiments, we have
not considered any energy transfer processes back from P680
to the various pigments absorbing at higher energy which
become important at room temperature.
4. Conclusions
We have proved by means of megahertz-resolution holeburning spectroscopy that trap pigments, absorbing in the red
wing of the isolated PSII RC complex and with a fluorescence
lifetime of 4 ns, exist at liquid He temperatures. From the hole
depth as a function of λexc we have obtained the spectral
distribution of these trap pigments. This set of molecules has
an approximate Gaussian distribution centered at 681.8 nm with
a width of ∼140 cm-1. From HB experiments performed under
near-saturating conditions, and by taking the ratio of Azph to
the sum of Azph plus Apsh, extrapolated for zero burning-fluence
density Pt/A f 0, we have determined the linear electronphonon coupling strength, S ) 0.73 ( 0.5. These trap pigments
consist, at least, of pheophytin a molecules because a hole burnt
at 682 nm yields a satellite hole at ∼545 nm, the spectral
position of their Qx-band. According to ref 20, also Chl a should
be present at 682 nm, which we have not identified here.
By measuring holewidths and hole depths as a function of
excitation wavelength for λexc < 678 nm, we have verified that
downhill energy transfer takes place. Between 672 and 678
nm it competes with pure dephasing and occurs in 200 ps,
whereas between 665 and 672 nm it is much faster and occurs
in 12 ps.
Because hole burning is selective as a function of wavelength
and burning-fluence density, we were able to determine the
spectral distributions of pigments characterized by their decay
times. After determining the positions and widths of these
distributions, the fluorescence excitation and absorption spectra at 1.2 K were reconstructed. Although not identified
here by hole burning, we inferred that the P680 spectral
distribution should be centered at 680.8 nm and have a width
of ∼150 cm-1. It is satisfying to see that the results for P680
are consistent with results from the literature obtained by other
techniques.11,21,34,36 In addition, we obtained a distribution
centered at 678.8 nm with a width of ∼100 cm-1 which
corresponds to hypothetical pigments of one or more unidentified decay times.
The results presented demonstrate that hole burning, because
of its high spectral resolution and wavelength- and burningfluence selectivity, represents a powerful tool for disentangling
the low-temperature excited-state dynamics of photosynthetic
complexes with strongly overlapping spectral bands.
Acknowledgment. We thank C. Eijckelhoff and H. van
Roon for providing us with the PSII RC samples and M. P.
Bakker for assistance in some of the hole-burning experiments.
Further, we acknowledge J. H. van der Waals for valuable
remarks regarding the manuscript. The investigations were
supported by the Netherlands Foundation for Physical Research
(FOM) and Chemical Research (SON) with financial aid from
the Netherlands Organization for Scientific Research (NWO).
References and Notes
(1) van Grondelle, R.; Dekker, J. P.; Gillbro, T.; Sundstrom, V.
Biochim. Biophys. Acta 1994, 1187, 1.
(2) Fleming, G. R.; van Grondelle, R. Phys. Today 1994, 47, 48.
J. Phys. Chem., Vol. 100, No. 27, 1996 11495
(3) Govindjee; van Rensen, J. J. S. In The Photosynthetic Reaction
Center; Deisenhofer, J., Norris, J. R., Eds.; Academic Press, Inc.: San Diego,
CA, 1993; Vol. I, p 357.
(4) Nanba, O.; Satoh, K. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 109.
(5) Chapman, D. J.; Gounaris, K.; Barber, J. Biochim. Biophys. Acta
1988, 933, 423.
(6) Kobayashi, M.; Maeda, H.; Watanabe, T.; Nakane, H.; Satoh, K.
FEBS Lett. 1990, 260, 138.
(7) Gounaris, K.; Chapman, D. J.; Booth, P.; Crystall, B.; Giorgi, L.
B.; Klug, D. R.; Porter, G.; Barber, J. FEBS Lett. 1990, 265, 88.
(8) van Leeuwen, P. J.; Nieveen, M. C.; Van de Meent, E. J.; Dekker,
J. P.; van Gorkom, H. J. Photosynth. Res. 1991, 28, 149.
(9) Eijckelhoff, C.; Dekker, J. P. Biochim. Biophys. Acta 1995, 1231,
21.
(10) Wasielewski, M. R.; Johnson, D. G.; Seibert, M.; Govindjee Proc.
Natl. Acad. Sci. U.S.A. 1989, 86, 524. Wiederrecht, G. P.; Seibert, M.;
Govindjee, Wasielewski, M. R. Proc. Natl. Acad. Sci. U.S.A. 1994, 91,
8999.
(11) Jankowiak, R.; Tang, D.; Small, G. J.; Seibert, M. J. Phys. Chem.
1989, 93, 1649.
(12) Durrant, J. R.; Hastings, G; Hong, Q.; Barber, J.; Porter, G.; Klug,
D. R. Chem. Phys. Lett. 1992, 188, 54.
(13) Schelvis, J. P. M.; van Noort, P. I.; Aartsma, T. J.; van Gorkom,
H. J. Biochim. Biophys. Acta 1994, 1184, 242.
(14) Rech, Th.; Durrant, J. R.; Joseph, D. M.; Barber, J.; Porter G.;
Klug, D. R. Biochemistry 1994, 33, 14768.
(15) Klug, D. R.; Rech, Th.; Joseph, D. M.; Barber, J.; Durrant, J. R.;
Porter, G. Chem. Phys. 1995, 194, 433.
(16) Danelius, R. V.; Satoh, K.; van Kan, P. J. M.; Plijter, J. J.; Nuijs,
A. M.; van Gorkom, H. J. FEBS Lett. 1987, 213, 241.
(17) van Kan, P. J. M.; Otte, S. C. M.; Kleinherenbrink, F. A. M.;
Nieveen, M. C.; Aartsma, T. J.; van Gorkom, H. J. Biochim. Biophys. Acta
1990, 1020, 146.
(18) Groot, M.-L.; Peterman, E. J. G.; van Kan, P. J. M.; van Stokkum,
I. H. M.; Dekker, J.P.; van Grondelle, R. Biophys. J. 1994, 67, 318.
(19) Roelofs, T. A.; Kwa, S. L. S.; van Grondelle, R.; Dekker, J. P.;
Holzwarth, A. R. Biochim. Biophys. Acta 1993, 1143, 147.
(20) Kwa, S. L. S.; Tilly, N. T.; Eijckelhoff, C.; van Grondelle, R.;
Dekker, J. P. J. Phys. Chem. 1994, 98, 7712.
(21) Tang, D.; Jankowiak, R.; Seibert, M.; Yocum, C. F.; Small, G. J.
J. Phys. Chem. 1990, 94, 6519.
(22) Roelofs, T. A.; Gilbert, M.; Shuvalov, V. A.; Holzwarth, A. R.
Biochim. Biophys. Acta 1991, 1060, 237.
(23) Hastings, G.; Durrant, J. R.; Hong, Q.; Barber, J.; Porter, G.; Klug,
D. R. Biochemistry 1992, 31, 7638.
(24) Visser, H. M.; Groot, M. L.; van Mourik, F; van Stokkum, I. H.
M.; Dekker, J. P.; van Grondelle, R. J. Phys. Chem. 1995, 99, 15304.
(25) Kwa, S. L. S.; Newell, W. R.; van Grondelle, R.; Dekker, J. P.
Biochim. Biophys. Acta 1992, 1099, 193.
(26) Dekker, J. P.; Bowlby, N. R.; Yocum, C. F. FEBS Lett. 1989, 254,
150.
(27) Wannemacher, R.; Koedijk, J. M. A.; Völker, S. Chem. Phys. Lett.
1993, 206, 1.
(28) Koedijk, J. M. A.; Creemers, T. M. H.; den Hartog, F. T. H.; Bakker,
M. P.; Völker, S. J. Lumin. 1995, 64, 55.
(29) Völker, S. In Relaxation Processes in Molecular Excited States;
Fünfschilling, J., Ed.; Kluwer: Dordrecht, 1989; p 113 and references
therein. Annu. ReV. Phys. Chem. 1989, 40, 499 and references therein.
(30) De Caro, C.; Visschers, R. W.; van Grondelle, R.; Völker, S. J.
Phys. Chem. 1994, 98, 10584.
(31) van der Laan, H.; Smorenburg, H. E.; Schmidt, Th.; Völker, S. J.
Opt. Soc. Am. B 1992, 9, 931.
(32) Tang, D.; Johnson, S. G.; Jankowiak, R.; Hayes, J. M.; Small, G.
J.; Tiede, D. M. In Twenty-Second Jerusalem Symposium: PerspectiVes in
Photosynthesis; Jortner, J., Pullman, B., Eds..; Kluwer Academic Publishers: Boston, MA, 1990; p 99.
(33) Holzwarth, A. R.; Muller, M. G.; Gatzen, G.; Hucke, M.; Griebenow, K. J. Lumin. 1994, 60, 497.
(34) Kwa, S. L. S.; Eijckelhoff, C.; van Grondelle, R; Dekker, J. P. J.
Phys. Chem. 1994, 98, 7702.
(35) Bosch, M. K.; Proskuryakov, I. I.; Gast, P.; Hoff, A. J. J. Phys.
Chem. 1995, 99, 15310.
(36) van der Vos, R.; van Leeuwen, P. J.; Braun, P; Hoff, A. J. Biochim.
Biophys. Acta 1992, 1140, 184.
(37) Durrant, J. R.; Klug, D. R.; Kwa, S. L. S.; van Grondelle, R.; Porter,
G.; Dekker, J. P. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 4798.
JP960326N