22 April 2002
Chemical Physics Letters 356 (2002) 347–354
www.elsevier.com/locate/cplett
The primary photoreaction of photoactive yellow
protein (PYP): anisotropy changes and excitation
wavelength dependence
T. Gensch
a,c
, C.C. Gradinaru b,d, I.H.M. van Stokkum b, J. Hendriks c,
K.J. Hellingwerf c, R. van Grondelle b,*
a
Institute for Biological Information Processing 1 (IBI-1), Research Centre J€ulich, D-52425 J€ulich Germany
Department of Physics and Astronomy, Faculty of Sciences, Institute of Molecular Biological Sciences, Vrije Universiteit,
De Boelelaan 1081, NL-1081 HV Amsterdam, Netherlands
c
SILS, Department of Chemistry, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, Netherlands
d
Department of Biophysics, Leiden University, 2333 CA, Leiden, Netherlands
b
Received 1 February 2002; in final form 26 February 2002
Abstract
The absorption and stimulated emission changes in the first 535 ps of the PYP photocycle can be described by four
life times of 0.7, 6.3, and 220 ps and long lived. Two intermediates, I0 and Iz0 , were identified. We did not obtain indications for a significant excitation wavelength dependent primary photochemistry as found in low temperature absorption spectroscopy. The anisotropy of the primary photoproduct I0 and its successor Iz0 amounts to 0.3 – significantly
lower than that of the bleached ground state (0.4). This distinctive change of the transition dipole moment orientation
in the product state (24°) reflects changes of the chromophore geometry and electron density distribution caused by the
photoisomerisation. Ó 2002 Published by Elsevier Science B.V.
1. Introduction
The photoactive yellow protein (PYP) from the
eubacterium Halorhodospira halophila (formerly
Ectothiorhodospira halophila [1]) is the photoreceptor for a negative phototactic (photophobic)
response of this photosynthetic organism induced
by blue light [2]. It belongs to a family of blue light
*
Corresponding author. Fax: +31-20-444-7999.
E-mail address: rienk@nat.vu.nl (R. van Grondelle).
photoreceptors – the xanthopsins – found in several other photosynthetic bacteria [3–5]. After
absorption of a blue photon PYP starts a cyclic
photoreaction with several intermediates and finally forms a long-living state, pB355 . Since the
pB355 state differs substantially from the ground
state (pG446 ) [6–14], it is supposed that this difference is detected by the organism and leads to a
signal for the flagella.
Apart from the blue-shifted pB355 state, the
photocycle of PYP consists of a number of earlier
intermediates. Ultrafast transient absorption
0009-2614/02/$ - see front matter Ó 2002 Published by Elsevier Science B.V.
PII: S 0 0 0 9 - 2 6 1 4 ( 0 2 ) 0 0 3 4 4 - 5
348
T. Gensch et al. / Chemical Physics Letters 356 (2002) 347–354
spectroscopy studies indicated the existence of a
primary intermediate with red-shifted absorption
I0 ðPYPB Þ after a few picoseconds [15–18]. The
existence of yet another intermediate with redshifted absorption spectrum ðIz0 Þ similar to I0 is still
debated. A red-shifted state, pR460 [6,19] is formed
in a few nanoseconds [16,18]. Recently, a branching in the early photochemistry of PYP was suggested with two primary photointermediates
involved – one red ðI0 ð¼ PYPB ÞÞ and one blue
shifted ðPYPH Þ – and both leading to pR460 [18].
A multiexponential decay of the PYP fluorescence has been observed [20–22] with major components of 700 fs and 4 ps, which sets the time
frame for the primary photoreaction event to the
first 10 ps after excitation. Interestingly, in the free
p-coumaric acid chromophore the trans–cis
photoisomerisation occurs monoexponentially and
without an intermediate state involved on a time
scale 10 times slower than the fastest process observed for wt-PYP [22,23].
The ultrafast photoisomerisation – also found
in other photoreceptors (visual rhodopsins, phytochromes, sensory rhodopsins) as the primary
step – may give rise to a change in the direction of
the transition dipole moment of the chromophore
in the photoproduct compared to that of the
ground state. Anisotropy measurements are sensitive to such dipole moment reorientations. Haran
et al. [24] observed in bovine rhodopsin very fast
(<10 fs) anisotropy changes and the formation of
bathorhodopsin with an anisotropy of 0.34, which
is significantly lower than 0.4 (the expected value
for parallel dipole orientations of rhodopsin and
bathorhodopsin). This result was in agreement
with the time of the photoisomerisation (<1 ps for
rhodopsin) obtained in other studies and has
proven the ability of (absorption) anisotropy
measurements to reveal structural rearrangements
during ultrafast photoisomerisation reactions.
2. Experimental
2.1. Sample preparation
PYP was prepared as described [14]. It was used
without removal of its poly-histidine tail. The
concentration of PYP in the different experiments
was 220 lM. A total sample volume of 4–5 ml was
loaded into a flow system and circulated by means
of a peristaltic pump (Watson Marlow 313S) with
a speed of 5–7:5 ml min1 . The cuvette for optical
detection was a flow-through quartz cuvette with 1
mm pathlength (5 mm high). The sample had an
OD of 1, 0.39, and 0.075 at 446, 400, and 485 nm,
respectively. The sample was exchanged after
measurement times of about 12 h. No changes in
the steady state absorption spectrum were observed when comparing spectra obtained before
and after measurements.
2.2. Femtosecond transient absorption spectroscopy
Transient absorption spectra were recorded on
a home-built femtosecond spectrometer described
in [25]. For excitation on the red flank of the
pG446 absorption, the non-collinear optical
parametric amplifier output was tuned to 485 nm
(with a FWHM of 12 nm) and a pulse energy
of ca. 200 nJ. The duration of the pulse was ca.
60 fs FWHM. For 400 nm excitation, we used a
small fraction of the frequency doubled output
from the regenerative amplifier (150 nJ). A
part of the amplifier output is sent through a
variable delay line (minimal steps of 0:1 lm) and
subsequently focused on a sapphire plate in order to generate a white light continuum as probe
light. This light is split into two almost identical
beams (signal and reference). All three beams,
i.e. pump, signal and reference are focused to a
spotsize of 120 lm. For 485 nm the frequency
was set to 1 kHz, while this was lowered to 100
Hz for the 400 nm pump, to prevent excitation
of pB355 produced in the preceding pulse. This
assured that virtually all PYP molecules in the
excitation volume were in the pG446 state before
excitation.
The polarisation of the pump beam was set to
parallel, magic angle and perpendicular relative to
that of the probe beam, by using a Berek prism
compensator (New Focus 5540). The spectral detection window for PYP was set to 430–580 nm.
Typically, 5000 spectra were recorded for each
time delay, and 250 delays were sampled up to 535
ps, which was the largest delay achievable. The
T. Gensch et al. / Chemical Physics Letters 356 (2002) 347–354
maximal change in the transmission of the sample
was 100 mOD, and the noise level was around 1
mOD.
The used excitation area and pump energies
resulted in photon densities of 2.7 1015 ðkexc ¼
400 nmÞ and 4:3 1015 photons cm2 per pulse
ðkexc ¼ 485 nmÞ. However, these relatively high
photon densities lead only to excited state populations of 8% ðkexc ¼ 400 nmÞ and 2 % ðkexc ¼
485 nmÞ, because of the relatively low absorption
coefficient of PYP (7:5 1017 cm2 at 446 nm) and
the choice of pump wavelengths on the blue and
red side of the absorption maximum. The experiments were performed in the linear excitation regime.
2.3. Data analysis
The time-gated spectra were analysed with a
global fitting program described elsewhere [25,26]
from which one obtains life times and decay associated difference spectra (DADS). The instrument response function, fitted with a Gaussian
profile, had a FWHM of ca. 100 fs, and the probe
pulse dispersion was described with a third order
polynomial function of the wavelength. The dispersion parameters agree well with the ones measured in a series of experiments performed under
similar conditions [25,27].
With 400 nm excitation, difference absorption
spectra recorded under parallel and perpendicular
polarisation orientations of the probe relative to
the pump light were fitted by target analysis
[28,29]. Using a kinetic model the (magic angle)
time dependence of the concentration of each state
was calculated. For each state bleach, stimulated
emission and absorption can possess their own
anisotropy r. The anisotropies are assumed to be
time independent on the 500 ps time scale. The
model function for parallel data is the sum of the
magic angle concentrations multiplied by ð1 þ 2rÞ
and by the species associated difference spectrum
(SADS) for each contribution. Likewise, for perpendicular data the anisotropy factor is ð1 rÞ. In
order to estimate all unknown parameters (rate
constants, spectra and anisotropies) spectral constraints are necessary, which are explained in the
text.
349
3. Results and discussion
3.1. Transient absorption with excitation at 400 nm
Immediately after the excitation pulse negative
signals are detected at all observation wavelengths,
reflecting strong ground state bleaching and stimulated emission signal contributions. At later
times, ground state bleaching below 470 nm,
stimulated emission above 460 nm and product
formation around 500 nm are identified. Fig. 1
shows the time-course of these processes at suitable wavelengths. At 452 nm the ground state
bleaching is probed, whereas at 471 nm a fast decay of the stimulated emission is observed. In the
492 and 506 nm traces the stimulated emission
decays more slowly, and product formation occurs
– as seen at times later than 6 ps. A product state
transition is present on the long ps time scale. At
471 nm the initial negative signal approaches zero
at about 4 ps and stays near zero for the next 530
ps. In other words, this wavelength is the isosbestic
point between the ground state and the primary
photoproduct.
Three exponential decays with life times of 0.8,
8 and 900 ps were sufficient to describe the kinetics of the absorption and stimulated emission
changes. It was not possible to reliably estimate a
fourth life time. The longest life time is uncertain
due to an incomplete overlap with the observation time window. The decay associated difference
spectra (DADS) are depicted in Fig. 2a. The first
two components (dotted and dashed) are dominated by bleach recovery and stimulated emission
decay, and by the formation of the primary
product in the region 480–560 nm. The multiexponential stimulated emission decay and the differences in spectral shape of the DADS (extrema
of first and second DADS are at 480 and 500 nm,
respectively) point towards two different excited
states ES1 and ES2. The slowest component
shows absorption decrease and increase in the
product region (centered at 500 nm) and the
bleach region, respectively. The latter could be
product relaxation back to the ground state as
well as formation of a new product with blueshifted absorption relative to the primary product
(see also Section 3.3). The formation of pR460 was
350
T. Gensch et al. / Chemical Physics Letters 356 (2002) 347–354
Fig. 2. DADS estimated from global analysis with three life
times. Key: life times (respectively dotted, dashed, solid): 0.8, 8
and 900 ps (a, 400 nm excitation) and 1.5, 11 and 800 ps (b, 485
nm excitation). The vertical axis unit is mOD.
study on the free deprotonated p-coumaric acid
chromophore [23].
3.2. Transient absorption with excitation at 485 nm
Fig. 1. Magic angle transient absorption data (solid) and model
fits (dashed) observed at 452, 471, 492 and 506 nm after excitation with 400 nm light. The bottom trace was observed at 506
nm after excitation at 485 nm, and shows a small remainder of a
coherent artefact. Note that the time axis is linear from )5 to
5 ps, and logarithmic from 5 to 500 ps. The vertical axis unit is
mOD.
not observable due to the observation time window of 535 ps compared to the formation time of
3 ns [16,18]. The small but significant difference
of the second and third DADS in the bleach region indicates a contribution of excited state absorption around 450–470 nm blue-shifted to the
stimulated emission. A similar excited state absorption has been found in a transient absorption
Several low temperature experiments revealed a
more complicated, branched photocycle of PYP
before the formation of pR460 [30–33]. Imamoto et
al. report a distinct excitation wavelength dependence of the relative efficiency of formation of the
two primary photoproducts, PYPB ð¼ I0 Þ and
PYPH when using cw illumination. At excitation
wavelengths larger than 460 nm significantly less
PYPB ð¼ I0 Þ is formed, while for excitation below
460 nm a nearly constant concentration ratio is
obtained. We tested the possibility that the primary photochemistry is excitation wavelength dependent by comparing the data with excitation at
400 and 485 nm. For the latter, the signal was not
reliable at observation wavelengths below 470 nm
due to the four times lower excited state population compared to that with 400 nm excitation and
the low intensity of the probe light below 470 nm.
As a consequence a crucial part for the analysis of
the absorption difference spectra – namely the
T. Gensch et al. / Chemical Physics Letters 356 (2002) 347–354
ground state bleach region – could not be recorded.
Three exponential decays with life times of 1.5,
11 and 800 ps are sufficient to describe the data. The
existence of two excited states can be concluded
from the biexponential decay of the stimulated
emission. In Fig. 2b the DADS are plotted. The
spectral shapes and the extrema of the DADS are
similar to that for excitation at 400 nm, only the
early stimulated emission (dotted) shows less intensity below 500 and above 540 nm. By comparing
these DADS we infer that the quantum yield of the
formation of the red-shifted intermediate is similar
or somewhat smaller for 485 nm excitation.
The only significant difference in the experiments
at the two excitation wavelengths is the somewhat
slower kinetics of the primary photochemistry. The
first two life times are larger for the 485 nm excitation by factors of, respectively, 1.9 and 1.4. This
can be seen by comparing the traces in the product
absorption range at 506 nm (Fig. 1). The zero
crossing point, which is found at 6 ps for excitation
at 400 nm, shifts to 12 ps for excitation at 485 nm.
On the basis of our experiments, we can exclude
a change in the primary photochemistry for excitation in the red edge of the ground state absorption spectrum. Instead, the excitation wavelength
dependence at low temperatures has to be explained by photobackreactions from PYPB and
PYPH to the ground state and the development of
photoequilibria. Whether the branching in the
early photocycle is also present at room temperature remains to be proven. With the exception of
the data presented in Imamoto et al. at 4 ns [18] no
evidence for such behaviour has been found.
The similar quantum yield of I0 formation for
excitation at 485 and 400 nm is an important result
with respect to a recently published time-resolved
structure analysis by Laue X-ray diffraction on
PYP crystals [34]. In this study, which revealed a
number of important molecular motions during
the PYP photocycle, an excitation wavelength of
495 nm was used. From our study it seems clear
that the photochemistry is most probably similar
in the first 535 ps to that for excitation near the
PYP absorption maximum, although small differences in the reaction rates exist. Most likely, I0 is
also produced with excitation at 495 nm.
351
3.3. Time-resolved anisotropy with excitation at 400
nm
The data set measured with excitation at 400
nm included observation with parallel, perpendicular and magic angle polarisation conditions. It
therefore allows us to determine the anisotropy of
the PYP primary photoreaction in a time-resolved
manner. In Figs. 3a–c the time course of traces
with parallel and perpendicular polarisation in the
bleach and stimulated emission as well as product
region are shown. While the bleach region shows a
constant limiting anisotropy of the expected 0.4,
the product formed within 6 ps is characterised by
a lower anisotropy. This behaviour is exemplified
in Fig. 3d, where the difference absorption spectra
at 80 ps with parallel and perpendicular orientation of probe and pump beam polarisation are
shown. The inset depicts the anisotropy calculated
from the raw data with values around 0.4 and 0.3
in the bleach and product regions, respectively.
A target analysis including two excited states
(ES1, ES2) and two product states ðI0 , Iz0 Þ was
performed assuming the kinetic scheme ES1 ! GS
(ground state, 40%), ES1 ! ES2 (35%),
ES2 ! GS (100%) and ES1 ! I0 ! Iz0 (25% and
52%, respectively). Spectral constraints were imposed, namely that the bleach parts of the difference spectra of I0 and Iz0 are identical, and that
these products do not absorb below 468 nm. The
life times of ES1, ES2, and I0 , were then found to
be 0.7 and 6.3, and 220 ps. The anisotropy of the
ES1 species was fixed at 0.4, as was the anisotropy
of the bleach part of the difference spectra of ES2,
I0 and Iz0 . Then, the anisotropy of the stimulated
emission of ES2 was 0.33, while the anisotropy of
the absorption of I0 and Iz0 was 0.30 and 0.31. The
estimated relative errors in all parameters are 10%,
except for the life time of I0 ð220 50 psÞ. As seen
in Figs. 3a–e with this model the data are well
fitted for all 500 difference spectra in the complete
time window. The anisotropy values of I0 (0.30)
and Iz0 (0.31) are significantly lower than that of
the ground state (0.4) which we attribute to the
isomerisation reaction (see below). We have no
explanation for the low anisotropy of ES2.
The first two life times obtained in the target
analysis compare well with that from a simple
352
T. Gensch et al. / Chemical Physics Letters 356 (2002) 347–354
Fig. 3. Anisotropy data (solid) and model fits (dashed). (a)–(c) Traces at 452 nm (ground state bleach), 477 nm (stimulated emission
and formation of Iz0 ) and 492 nm (stimulated emission and formation and decay of I0 ). Estimated life times 0.7, 6.3, and 220 ps and long
lived. (d) and (e) Difference spectra at 80 (mainly I0 ) and 535 ps (mainly Iz0 ). For all plots, the parallel data ðkÞ has the largest absolute
magnitude and perpendicular the least. Note that the time axis in (a)–(c) is linear from )5 to 5 ps, and logarithmic from 5 to 500 ps. The
inset in (d) and (e) depicts the anisotropy calculated from the raw data (solid) and fit (dashed). (f) SADS resulting from target analysis.
Key: dot–dashed ES1, dotted ES2, dashed I0 , solid Iz0 . The vertical axis unit is mOD.
three exponential fit. The spectral assumptions of
the target analysis enable to resolve the 220 ps
from the 900 ps life time found with global
analysis of the magic angle data. The results obtained from the target analysis – besides delivering quantitative information like SADS and
species anisotropy – are also more reliable, since
the target analysis uses constraints known about
the underlying physical processes. The observation of a near 200 ps component is in agreement
with Ujj et al. [16].
The SADS of the primary photoproduct I0
agrees well with that obtained by Ujj et al. [16].
Their spectrum of Iz0 (lower absorption and slightly
blue-shifted compared to I0 ), however, looks different from the one obtained here, where the absorption of Iz0 is also blue-shifted but the extinction
coefficient is two times larger than I0 . The latter
reflects the low efficiency (52%) of the I0 ! Iz0
transformation, which is visualised by the almost
two times smaller bleach signal at 535 ps (mainly
Iz0 ) compared to that at 80 ps (mainly I0 ) (Figs. 3d
and e). This also is the reason for the bleach recovery observed in the 452 nm trace and the decay
of absorption in the 492 and 506 nm magic angle
traces in Fig. 1 in the long ps time range. The
absorption in the product region decreases, although a product ðIz0 Þ with increased absorption is
formed, but with a smaller concentration. An additional indication for the larger absorption of Iz0 is
the blue shift of the zero crossing point in the
absorption difference spectra at 80 ps (474 nm) and
at 535 ps (472 nm) (Figs. 3d and e).The different Iz0
spectrum of Ujj et al. [16] arises from their assumption that the I0 ! Iz0 occurs with a quantum
yield of 1.
T. Gensch et al. / Chemical Physics Letters 356 (2002) 347–354
The anisotropy values can be related to a
change of the transition dipole moment of the
new state compared to the ground state. The
decreased anisotropy value of I0 (0.30) reflects a
change in the orientation of the transition dipole
moment of b ¼ 24° relative to that of pG446
(calculated from r ¼ 2=5 ð3 cos2 b 1Þ=2). The
most obvious event causing this change is the
photoisomerisation of the PYP chromophore.
Transient absorption studies on mutants [35],
structures of early intermediates from X-ray diffraction crystallography [34] and a recent low
temperature FTIR study [33] all give experimental evidence that the isomerisation is the first step
in the photoreaction. The crystallographic studies
have also identified more details of the isomerisation which is a double isomerisation around the
double bond and an adjacent single bond flipping
of the carbonyl group into a hydrophobic pocket
while leaving the phenol part of the chromophore
rather fixed.
Similarly large and even 10-fold faster anisotropy changes have been observed in a single
wavelength transient absorption anisotropy study
on rhodopsin [24]. In the latter study a similar
decreased anisotropy (0.34 corresponding to a
change of 16°) was measured for bathorhodopsin,
the fully isomerised photoproduct of rhodopsin
which reflects the nuclear motion due to isomerisation. In addition they detected an even lower
anisotropy value at 50 fs (0.25 corresponding to
30 °) which was interpreted as sudden redistributions of charges on the retinal chromophore of
rhodopsin accompanying excitation. Analogous
processes are not detected for PYP.
The low anisotropy of the primary photoproduct ðI0 Þ of PYP offers strong experimental support
for the hypothesis, that the photoisomerisation is
the first event of the photocycle and takes most
likely place in the first picosecond. The corresponding large dipole moment change of 24° represents the superposition of the geometrical
displacement of the chromophore nuclei due to the
isomerisation as well as changed electronic wavefunctions. The different environment the chromophore carbonyl group is surrounded by, after
isomerisation, could have, in fact, a large impact
on the electronic wavefunctions.
353
Acknowledgements
T.G. thanks the Royal Dutch Academy of Sciences and the Nordrhein-Westf€alische Akademie
der Wissenschaften for a Casimir-Ziegler fellowship.
References
[1] J.F. Imhoff, J. Suling, Arch. Microbiol. 165 (1996) 106.
[2] W.W. Sprenger, W.D. Hoff, J.P. Armitage, K.J. Hellingwerf, J. Bacteriol. 175 (1993) 3096.
[3] T.E. Meyer, J.C. Fitch, R.G. Bartsch, G. Tollin, M.A.
Cusanovich, Biochim. Biophys. Acta 1016 (1990) 364.
[4] R. Kort, W.D. Hoff, M. van West, A.R. Kroon, S.M.
Hoffer, K.H. Vlieg, W. Crielaard, J.J. van Beeumen, K.J.
Hellingwerf, EMBO J. 15 (1996) 3209.
[5] Z.Y. Jiang, L.R. Swem, B.G. Rushing, S. Devanathan, G.
Tollin, C.E. Bauer, Science 285 (1999) 406.
[6] T.E. Meyer, E. Yakali, M.A. Cusanovich, G. Tollin,
Biochem. 26 (1987) 418.
[7] M.E. van Brederode, W.D. Hoff, I.H.M. van Stokkum,
M.L. Groot, K.J. Hellingwerf, Biophys. J. 71 (1996)
365.
[8] G. Rubinstenn, G.W. Vuister, F.A.A. Mulder, P.E. Dux,
R. Boelens, K.J. Hellingwerf, R. Kaptein, Natl. Struct.
Biol. 5 (1998) 568.
[9] W.D. Hoff, A. Xie, I.H.M. van Stokkum, X.J. Tang, J.
Gural, A.R. Kroon, K.J. Hellingwerf, Biochem. 38 (1999)
1009.
[10] C.J. Craven, N.M. Derix, J. Hendriks, R. Boelens, K.J.
Hellingwerf, R. Kaptein, Biochem. 39 (2000) 14392.
[11] A. Xie, L. Kelemen, J. Hendriks, B.J. White, K.J.
Hellingwerf, W.D. Hoff, Biochem. 40 (2001) 1510.
[12] R. Brudler, R. Rammelsberg, T.T. Woo, E.D. Getzoff, K.
Gerwert, Natl. Struct. Biol. 8 (2001) 265.
[13] B.-C. Lee, P.A. Croonquist, T.R. Sosnick, W.D. Hoff, J.
Biol. Chem. 276 (2001) 20281.
[14] J. Hendriks, T. Gensch, L. Hviid, M. van der Horst, K.J.
Hellingwerf, J.J. van Thor, Biophys. J. (2002) 1632.
[15] A. Baltuska, I.H.M. van Stokkum, A. Kroon, R. Monshouwer, K.J. Hellingwerf, R. van Grondelle, Chem. Phys.
Lett. 270 (1997) 263.
[16] L. Ujj, S. Devanathan, T.E. Meyer, M.A. Cusanovich, G.
Tollin, G.H. Atkinson, Biophys. J. 75 (1998) 406.
[17] S. Devanathan, A. Pacheco, L. Ujj, M. Cusanovich, G.
Tollin, S. Lin, N. Woodbury, Biophys. J. 77 (1999)
1017.
[18] Y. Imamoto, M. Kataoka, F. Tokunaga, T. Asahi, H.
Masuhara, Biochem. 40 (2001) 6047.
[19] W.D. Hoff, I.H.M. van Stokkum, H.J. van Ramesdonk,
M.E. van Brederode, A.M. Brouwer, J.C. Fitch, T.E.
Meyer, R. van Grondelle, K.J. Hellingwerf, Biophys. J. 67
(1994) 1691.
354
T. Gensch et al. / Chemical Physics Letters 356 (2002) 347–354
[20] H. Chosrowjan, N. Mataga, N. Nakashima, Y. Imamoto,
F. Tokunaga, Chem. Phys. Lett. 270 (1997) 267.
[21] P. Changenet, H. Zhang, M.J. Vandermeer, K.J. Hellingwerf, M. Glasbeek, Chem. Phys. Lett. 282 (1998) 276.
[22] N. Mataga, H. Chosrowjan, Y. Shibata, Y. Imamoto, F.
Tokunaga, J. Phys. Chem. B 104 (2000) 5191.
[23] P. Changenet-Barret, P. Plaza, M.M. Martin, Chem. Phys.
Lett. 336 (2001) 439.
[24] G. Haran, E.A. Morlino, J. Matthes, R.H. Callender,
R.M. Hochstrasser, J. Phys. Chem. A 103 (1999) 2202.
[25] C.C. Gradinaru, I.H.M. van Stokkum, A.A. Pascal, R. van
Grondelle, H. van Amerongen, J. Phys. Chem. B 104
(2000) 9330.
[26] I.H.M. van Stokkum, T. Scherer, A.M. Brouwer, J.W.
Verhoeven, J. Phys. Chem. 98 (1994) 852.
[27] C.C. Gradinaru, J.T.M. Kennis, E. Papagiannakis, I.H.M.
van Stokkum, R.J. Cogdell, G.R. Fleming, R.A. Niedermann, R. van Grondelle, Proc. Natl. Acad. Sci. USA 98
(2001) 2364.
[28] A.R. Holzwarth, in: J. Amesz, A.J. Hoff (Eds.), Biophysical Techniques in Photosynthesis, Kluwer Academic
Publishers, Dordrecht, 1996, p. 75.
[29] B.P. Krueger, S.S. Lampoura, I.H.M. van Stokkum, E.
Papagiannakis, C.C. Gradinaru, D. Rutkauskas, R.G.
Hiller, R. van Grondelle, Biophys. J. 80 (2001) 2843.
[30] W.D. Hoff, S.L.S. Kwa, R. van Grondelle, K.J. Hellingwerf, Photochem. Photobiol. 56 (1992) 529.
[31] Y. Imamoto, M. Kataoka, F. Tokunaga, Biochem. 35
(1996) 14047.
[32] T. Mascianglioli, S. Devanathan, M.A. Cusanovich, G.
Tollin, M.A. ElSayed, Photochem. Photobiol. 72 (2000) 639.
[33] Y. Imamoto, Y. Shiharige, F. Tokunaga, T. Kinoshita, K.
Yoshihara, M. Kataoka, Biochem. 40 (2001) 8997.
[34] Z. Ren, B. Perman, V. Srajer, T.Y. Teng, C. Pradervand,
D. Bourgeois, F. Schotte, T. Ursby, R. Kort, M. Wulff, K.
Moffat, Biochem. 40 (2001) 13788.
[35] S. Devanathan, S. Lin, M.A. Cusanovich, N. Woodbury,
G. Tollin, Biophys. J. 79 (2000) 2132.