Journal of Luminescence 48 & 49 (1991) 199-203
North-Holland
199
Energy transfer and dynamics of photosynthetic antenna
complexes and biological model systems
A study by photochemical hole-burning
H. van der Laan, Th. Schmidt and S. Völker
Center for the Study of the Excited States of Molecules, Huygens and Gorlaeus Laboratories, University of Leiden, Leiden,
The Netherlands
Photochemical hole-burning (PHB) experiments have been performed on two types of complex organic systems. Results
on the Q 0-0 transition of bacteriochlorophyll-a (BChI-a) in triethylamine (TEA) between 0.4 and 4.2 K, obtained with a
Thom, follows a Tls*oL dependence. Such a power law has
diode
laser atfor780nm,
show that
the homogeneous
width,
been found
many organic
glassy
systems at lowline
temperatures.
The absolute value of ‘hon~ in a solution of water and
detergent is almost one order of magnitude larger than in TEA, which suggests that energy transfer takes place between
BChI-a molecules within a micelle. Holes burnt in the 800 nm band of the B800-850 antenna pigment-protein complex of
purple bacteria Rhodobacter sphaeroides show no variation in hole width between 1.2 and 30 K. The results yield an energy
transfer time of 2.3 ±0.4 ps between the aggregated BChl 800 and BChI 850 molecules. The same value is found for intact
chromatophores at 1.2 K.
1. Introduction
Complex organic amorphous systems and
photosynthetic biological complexes show broad
inhomogeneous spectral bands with widths, F~nhom,
of the order of a few hundred cm~1,even at liquid
helium temperatures. Many of these systems
absorb in the near infrared region and often show
weak luminescence. In order to study the excitedstate dynamics of such systems, it is necessary to
know the homogeneous line width, Thom, which is
hiddenThom
under
the by
inhomogeneously
broadened
is given
the optical dephasing
time,
band.
T
2:
1
1
1
(1)
where T1 is the population decay time of the
excited state, and T~the pure dephasing time
determined by thermally induced fluctuations of
the optical transition frequency (e.g. phonon scattering). The first term includes direct de-excitation
to the ground state and all energy transfer processes that occur from the excited state.
0022-2313/91/$03.50
©
1991
-
Spectral hole-burning (HB) is a powerful highresolution laser technique which allows to determine the value of Thom in the MHz-regime [1]. We
present here the results of HB-studies of two
related complex organic amorphous systems which
absorb at about 800 nm. The first one is bacteriochlorophyll-a (BCh1-a) as guest in organic glasses.
In this case, T
1 ~ Ti’, and the hole width is mainly
determined by pure dephasing processes. Since the
temperature has a strong influence on the value of
Thom, the second term in eq. (1) is dominant.
Further, weThom•
will show that the environment greatly
influences
The second system is a photosynthetic antenna
pigment-protein complex of purple bacteria. Such
complexes collect and transfer the energy of light
to the reaction center, where the charge separation
occurs that leads to the primary electron transfer
process. The antenna complexes in these bacteria
are mainly built up by aggregates of BChI
molecules embedded in a protein [2,3]. We will
show that for the B800-850 complex of Rhodobacter sphaeroides, T1 ~ T~,and the hole width is
mainly determined by population decay [4]. The
Elsevier Science Publishers By. (North-Holland)
200
H. van der Laan et a!.
/ Dynamics of photosynthetic
widths of the holes yield the energy transfer time
from BCh1800 to BChl85O.
2. Experimental
Samples of bacteriochlorophyll-a (BCh1-a)
were prepared at concentrations of ~°3 X
i05 mol/l in various glassy hosts. We used
triethylamine (TEA), a solution of water:glycerol
(1: 1) with the detergent lauryldimethylamine Noxide (LDAO, 0.5 vol%), and a solution of water
with LDAO at various concentrations (0.1 to
3Ovol%). All samples had an optical density at
780 nm of OD 1.0.
The B800-850 pigment-protein complex was
isolated from chromatophores of Rhodobacter
sphaeroides strain 2.4.1 using the detergent LDAO
(for a detailed description of the preparation, see
ref. [4]). Concentrated solutions of the isolated
complex were first diluted with 20 mM Tris-HCI
buffer (pH8) and 0.1% LDAO. In order to obtain
transparent glassy samples at liquid helium temperatures, the solutions were diluted with ~60%
glycerol. The samples had an optical density at
800nm of 0D~1.2 [4].
To burn and probe spectral holes in the BChI-a
samples we have used a cw single-mode laser diode
at 780 nm (Hitachi, HL 7806-G, 5 mW, bandwidth
Tiaser~’°50 MHz). The laser frequency was tunable
over 200 0Hz by varying the temperature
(30 GHz/°C) and the current (3 0Hz/mA). For
larger frequency scans, which were needed for the
antenna complex, we have used a broad band cw
dye-laser (dye Styryl 8, bandwidth 35 GHz)
pumped
by an were
argon-ion
laser.simultaneously by
The holes
probed
fluorescence excitation (with a cut-off filter of
850 nm) and transmission through the sample. In
some cases we have also used photoacoustic spec-
antenna complexes
placed inside the 4He-bath cryostat. By reducing
the vapour pressure of the 3He, temperatures down
to 0.4 K could be reached [6,7]. The temperature
was measured by a calibrated carbon resistor
placed in contact with the sample and via the
vapour pressure (accuracy ±0.01K).
Above 4.2 K, a gas flow cryostat (LeyboldHeraeus) was used. The temperature was measured
by a calibrated carbon resistor via a Wheatstone
bridge (accuracy ±0.05K) [7].
3. Results and discussion
3.1.
Optical dephasing of bacteriochlorophyll-a in
glasses
Figure 1 shows two holes burnt into the Q 0-0
transition of BCh1-a in TEA (11nhom~°550cm’),
and in a solution of water: glycerol (1: 1) with 0.5%
LDAO ([nhom
800 cmt), at 4.2 K, respectively.
The experiments were performed at ~780 nm with
the cw single-mode laser-diode mentioned above.
Both holes have Lorentzian lineshapes but their
widths, of about 700 MHz and 4.3 0Hz, differ by
almost one order of magnitude. The homogeneous
line widths in TEA are comparable with those
obtained for free-base porphin (H
2P) in organic
glasses [1,6,7]; e.g. at 1.2 K, Thom 115 MHz for
BChl-a and Thom 50MHz for H2P [8].
We have determined the value of Thom by plotting the hole width, Thole, as a function of burning
fluence, Pt (P: burning power, t: burning time),
and extrapolating the curve to Pt 0 at each ternperature, T Under theseT’laser
conditions,
50 MHzwe
andtake
the
Thom
2Thole
~~aser,
where to be Lorentzian. In fig.
laser =lineshape
is assumed
2 Thom is given as a function of T for BChl-a in
TEA, between 0.4 and 4.2 K. The data points follow
Thom = T
0 + aT’
with T0 = 40 ±10 MHz. The
latter value yields a fluorescence decay time T, =
(2i,T0)’
=4.0±1.0ns,
is consistent
values reported
in the which
literature
for BCh1-awith
in
various hosts at room temperature [9].
Our results demonstrate that the T’3 dependence of Thom, which was previously found for a
large number of organic amorphous systems [1,6,7]
is also valid for very complex molecules in glasses,
-+
—
~°‘,
troscopy (PAS) at 1.65 K. This technique, which
is based on4He
resonant
detection
second
soundfor
in
(T<2.17
K), isofvery
sensitive
superfluid
detecting nonradiative processes because it allows
to measure against zero background [5].
For temperatures between 1.2 and 4.2 K, a conventional 4He bath cryostat was used (accuracy
±0.01K). Below 1.2 K, a 3He-glass insert was
H. van der Laan et a!. / Dynamics of photosynthetic antenna complexes
201
BChIa/TEA
500-
C
0
-o
0
(MHzl
300
—3
—2
—1
0
_
1
-
200~
2
3
~vlGI-~l
C
.2
-
BChIa/water:gtycerol (1:1) .0.5% LDAO
.
I
100
,
/
/~
Bthla/TEA
-
+i~1
I’
~~ft’i
0
.0
0
/
3~°’
rwmrro•aT~
0
1
2
3
~.
5
perature, T, for BChI-a in TEA between —~
0.4 and T(K)
4.2 K. Notice
Fig. 2. Homogeneous line width, ‘boo, as a function of temthat ~
=
I~)+aT’3°°’, with I~)= 40±10MHz.
3.2. Energy transfer in the B800-850 antenna pig-20
-10
I
0
men t-protein complex of Rb. sphaeroides [41
10
20
tivlQHz)
Fig. 1. Holes burnt in the Q~0-0 absorption band of BChl-a
in TEA (top), and in a solution of water:glycerol (1:1) with
LDAO (0.5%) (bottom), at 4.2 K. The solid lines are Lorentzian
fits to the experimental data (crosses). Notice the almost one
order of magnitude larger hole width of the latter sample.
at least up to 4.2 K. We are currently investigating
the validity of this power law at higher temperatures.
Since the hole widths of BChl-a in water with
the detergent LDAO are larger than in TEA, we
have started a series of PH B-experiments in which
the concentration of LDAO was varied from 30 to
0.1 vol%. Preliminary results indicate that the hole
width increases with decreasing LDAO concentrations. Since BChI-a does not dissolve in water, we
attribute this effect to energy transfer between
BChI-a molecules within a micelle built up of
clusters of LDAO molecules [8].
Rhodobacter
consists
of two
different
antenna sphaeroides
pigment—protein
complexes
called B800—850 (or LH
2) and B875 (or LH,). The
energy transfer time between these two complexes
in intact chromatophores is about 40 ps [10],
whereas the transfer from B875 complexes to the
reaction center takes about lOOps [10,11]. A schematic representation of these primary steps in the
photosynthetic energy transfer process is given in
fig. 3.
Energy transfer within the isolated B800-850
complex from BChI800 to BCh185O is significantly
faster than among different complexes. From psabsorption [12] and ps-fluorescence [11] experiments (resolution =10 ps) it was inferred that this
process occurs on a time scale of 1—2 ps, whereas
from fs-pump-probe experiments (resolution
molSO fs) it was concluded that the energy transfer
takes <lOOfs [13].
H. van der Laan et a!. / Dynamics of photosynthetic antenna complexes
202
X(r,m)
—
-
—
-40 ps
I
—
hv
-ins
________
BChISW
-inSt
________
BChI85O
LH2
I
_____________________
-loops
a
0
C
—
0
.0
0
-ins
I
800
I
I
820
1 12K
I
I
—
~ChI875
LH1
I
780
BChI
2
RC
Fig. 3. Primary energy transfer processes in bacterial photosynthesis. LI-I2 and LH1 are the antenna complexes B800-850 and
0
795~nm
©
799.6 nm
143 0Hz
®
80~nrn~
138 0Hz
130 0Hz
B875, respectively. BChI2 represents the BChI dimer of the
reaction center (RC).
C
a
These controversial results led us to investigate
this energy transfer process by means of spectral
hole-burning. Permanent holes were burnt in the
800 nm absorption band of the isolated B800-85O
antenna complex. Figure 4 shows three of such
holes burnt and probed at 1.2 K. Their widths, of
about 140 0Hz, were found to be independent of
burning wavelength between 791 and 804 m [4].
In order to determine the homogeneous line
width, we have used the same procedure as for
BChI-a in glasses. The hole width was measured
as a function of burning fluence, Pt, and extrapolated to Pt-sO, at each temperature. Thom was
then obtained by deconvolution of the laser bandwidth (35 0Hz) from the extrapolated hole width.
The values of Thom as a function of temperature
are plotted in fig. 5. They show no temperature
dependence between 1.2 and 30 K. Hole-burning
experiments on the intact chromatophores at 1.2 K
yielded the same value, Thom = 69 ±10 0Hz, as that
obtained for the isolated B800-850 complex [4].
If we assume that the pure dephasing contribution to Thom is of the same order as that of BChI-a
in glasses at 1.2 K, (irTfl~ = 1 0Hz (see fig. 2),
and since we did not observe any temperature
dependence of Thom, we conclude that the second
term in eq. (1) can be neglected. The fluorescence
decay term can also be neglected, because it con-
tributes to Thom with
(‘rrr5)’ =
190 MHz. Further,
C
U-
I
I
-1000
0
.1000
~v IOHzI
Fig. 4. Top: absorption band of BChI800 of the B800-850
antenna
complex at 1.2atK.1.2Bottom:
into
this pigment-protein
band at various wavelengths,
K. Theholes
solidburnt
lines
are Lorentzian fits to the data (Crosses). The hole widths are
independent of wavelength between 791 and 804 nm.
energy transfer within the BChl 800 band does not
seem to occur because the hole widths are independent of wavelength. Thus, the homogeneous line
width of the 800 nm band is entirely determined
by the energy transfer process from BCh1800 to
BChl85O, with T, = (2~TThomY’= 2.3 ±0.4Ps [14].
This value is in excellent agreement with recent
fs-transient absorption experiments at room ternperature, which yielded 2.5 Ps (±10%) for this
transfer time [15]. Our data are also consistent
H. van der Laan et a!.
120
/ Dynamics of photosynthetic
Phom
0Hz)
-
References
80
60
[1] references
S. Völker,
Völker, in:
therein.
Ann.
Relaxation
Rev. Phys.
Processes
Chem.in40Molecular
(1989) 499,
Excited
and
40
-
[2]
[3]
20
0
203
(FOM), Chemical Research (SON) and Biophysics (SVB), with financial aid from the
Netherlands Organization for Scientific Research
(NWO).
.
100
antenna complexes
[4]
-
0
I
10
Fig. 5. Homogeneous line width,
20
T (K)
I
30
F
5om, as a function of temperature of the isolated B800—850 complex, between 1.2 and
30 K (open circles),
Tho,. =
and
69 ±
the
10 intact
GHz, chromatophores
which is independent
at 1.2 of
K
(closed circle).
temperature,
yields an energy transfer time of 2.3 ±0.4 ~ [4].
with the previous estimates of 1—2 ps obtained by
ps-absorption [12] and ps-fluorescence decay
experiments [11], but do not agree with the above
mentioned fs-pump-probe results which were
obtained with too high intensity pulses causing
extensive excitation annihilation [13].
From these results we conclude that holeburning is a reliable technique for determining
dephasing processes as well as energy transfer
times in complex biological systems.
Acknowledgements
The investigations were supported by the
Netherlands Foundation for Physical Research
[5]
[6]
States, ed. J. Funfschilling (Kiuwer, Dordrecht, 1989)
p.113.
H. Zuber, Photochem. Photobiol. 42 (1985) 821.
C.N. Hunter, R. van Grondelle and iD. Olsen, TIBS 14
(1989) 72, and references therein.
H. van der Laan, Th. Schmidt, R.W. Visschers, K.J.
Visscher, R. van Grondelle and S. Völker, Chem. Phys.
Lett. 170 (1990) 231.
H.P.H. Thijssen, R. van den Berg, S. Völker and J.H. van
der Waals, Chem. Phys. Lett. 111 (1984) 121.
H.P.H. Thijssen, R. van den Berg and S. Völker, Chem.
Phys. Lett. 120 (1985) 503.
[7] R. van den Berg, A. Visser and S. Völker, Chem. Phys.
[8] H.
Lett.van
144 der
(1988)
Laan,
105.Th. Schmidt and S. Vblker, to be
published.
[9] J.S. Connolly, E.B. Samuel and A.F. Janzen, Photochem.
Photobiol. 36 (1982) 565.
[10] V. Sundström et al., Biochem. Biophys. Acta 851 (1986)
431.
R.vanGrondelleetal., Biochem.Biophys.Acta894(1987)
313.
[11] A. Freiberg, VI. Godik, T. Pullerits and K. Timpmann,
Biochem. Biophys. Acta 973 (1989) 93, and references
therein.
[12] H. Bergstrom et al., Biochem. Biophys. Acta 852 (1986)
279; 936 (1988) 90.
[13] J.W. Petrich,J. Breton andJ.L. Martin, in: Primary Processes in Photobiology, Springer Proceedings in Physics, Vol.
10, ed. T. Kobayashi (Springer, Berlin, 1987) p. 52.
[14] We have confirmed these results at 4.2 K by burning holes
with higher resolution. The laser used was a Nd:YAGpumped dye laser (dye Styryl 9, bandwidth o~3~5
GHz),
which yielded an energy transfer time of 2.6 ±0.3 ps.
[15] J.K. Trautman, A.P. Shreve, C.A. Violette, HA. Frank,
T.G. Owens and AC. Albrecht, Proc. Nat. Acad. Sci. USA
87 (1990) 215.