The First Picoseconds in Bacterial
Photosynthesis—Ultrafast Electron Transfer for
the Efficient Conversion of Light Energy
Wolfgang Zinth*[a] and Josef Wachtveitl[b]
In this Minireview, we describe the function of the bacterial reaction centre (RC) as the central photosynthetic energy-conversion
unit by ultrafast spectroscopy combined with structural analysis,
site-directed mutagenesis, pigment exchange and theoretical
modelling. We show that primary energy conversion is a stepwise
process in which an electron is transferred via neighbouring chro-
mophores of the RC. A well-defined chromophore arrangement in
a rigid protein matrix, combined with optimised energetics of the
different electron carriers, allows a highly efficient charge-separation process. The individual molecular reactions at room temperature are well described by conventional electron-transfer theory.
1. Introduction
Photosynthesis, the most important process for solar energy
conversion on earth, can be condensed into a chemical reaction scheme in which light drives the synthesis of glucose
from carbon dioxide and water. A closer look reveals a biophysical and biochemical reaction scheme with high complexity where a large variety of specialised biomachines convert
energy from sunlight into chemical energy. Despite the diversity of photosynthetic organisms a few functional principles are
maintained as building blocks: There are always so-called light
reactions, where the biophysical processes of light absorption,
transfer of electronic excitation energy, initial charge separation and vectorial charge transfer take place. Coupling of electron and proton transfer leads to the formation of a proton
gradient across the membrane, which is utilized for the synthesis of adenosine triphosphate (ATP) and other energy-rich compounds. In the subsequent dark reactions the biochemical
energy is converted and stored via the ATP-driven reduction of
carbon dioxide to carbohydrates.
The light-absorbing chromoproteins perform two key functions for the light reactions of photosynthesis: the antenna systems, where pigment–protein complexes with high chromophore contents absorb light, transfer the excitation energy in a
photophysical process towards the other functional units, the
reaction centres. Here, energy conversion and charge transfer
take place as the initial photochemical steps in photosynthesis.
For some photosynthetic bacteria, antenna systems and reaction centres can be separated, and the functions of both have
been studied separately. In most other systems, especially in
higher photosynthetic organisms, a complete separation into
intact units exclusively operating as antenna or reaction centre
cannot be accomplished without loss of main functional features.
Information on the molecular processes of photosynthesis is,
to a large extent, based on the knowledge of the structural arrangement. It was only recently that structure analysis of the
most important photosynthetic units could be completed.[1–8]
These publications demonstrated that primary charge separaChemPhysChem 2005, 6, 871 – 880
DOI: 10.1002/cphc.200400458
tion as the initial energy-conversion process, occurs in the different photosynthetic organisms in pigment–protein complexes with large structural similarity (see Figure 1). Photosyn-
Figure 1. Molecular arrangement of chromophores and other electron carriers
connected with the primary charge separation of PS I (a), PS II (b) and the bacterial RC of B. viridis (c). The structures are drawn using the coordinates from
refs. [1, 3, 6, 8]. For the RC we use the following colour code: Blue for the closely
spaced pair of BChl, which acts as the primary donor. Red is used for the accessory BChl BA and green for the subsequent electron carrier, the BPhe HA. The
two quinones are plotted in yellow. The chromophores of the “inactive” branch
are plotted in grey. Note that the colours used for PS I and PS II are not connected to defined functional properties but should underline the large structural
similarities.
thesis in higher organisms is oxygenic and uses two photosynthetic units, photosystem I (PS I) and photosystem II (PS II).
They act in a concerted way to transfer electrons from water
to NADP + . The arrangement of the chromophores—six chlorophylls (Chl)—in the core of the PS I complex is shown in Fig[a] Prof. Dr. W. Zinth
Department fr Physik, Ludwig-Maximilians-Universitt Mnchen
Oettingenstr. 67, 80538 Mnchen (Germany)
Fax: (+ 49) 89-218-092-02
E-mail: [email protected]
[b] Prof. Dr. J. Wachtveitl
Institut fr Physikalische und Theoretische Chemie
Goethe-Universitt Frankfurt, Marie-Curie-Str. 11
60439 Frankfurt (Germany)
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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W. Zinth and J. Wachtveitl
ure 1 a.[6] The pigments are arranged in two branches which intersect at one end at a pair of Chl (P700) and at the other end
at the electron acceptor A0, an iron/sulfur cluster. The structural arrangement of the chromophores in the core of the PS II
complex exhibits some similarity to PS I.[8] Again the chromophores are arranged in two branches, two Chl are closely
spaced (see Figure 1 b, top). On each branch there is an adjacent Chl and a pheophytin (Phe) prior to a plastoquinone. The
two branches meet at the pair of Chl molecules and at the
nonheme iron.[9–11] In addition there are further Chl chromophores with antenna functions and similar absorption spectra,
which cannot be separated from the core. Thus electronic
energy transfer and electron transfer upon primary charge
separation are hard to disentangle.
The photosynthetic reaction centres (RCs) of purple bacteria
(see Figure 1 c)[1, 3] allow a direct observation of photoreduced
electron transfer (ET) since 1) the functional RC can be separated from the light-collecting antenna systems and 2) the absorption spectra of the bacterial chlorine chromophores show
well-separated absorption bands (see Figure 2). It finally
(BChl) molecules, two bacteriopheophytins (BPhe) and two quinones (Q) are imbedded in this protein and function as photoreceptors and/or electron carriers. Two of the BChl are closely
spaced and excitonically coupled. They are called the special
pair P. The lowest energy absorption of the special pair P is in
the low-energy shoulder of the RC absorption spectrum, thus
P acts as a trap for the electronic energy supplied from the antenna system and acts as the primary electron donor. In the
photosynthetic RC the electron is transferred subsequently via
the accessory BChl BA and the BPhe HA to the quinone QA. This
primary electron transfer, which occurs within 200 ps, has been
subject of many investigations and will be treated in detail
below. It is only on the microsecond timescale that the electron is transferred further to the secondary quinone QB, a process that apparently involves the cooperation of other electron
or proton carriers.[12] After re-reduction of the special pair via a
cytochrome, the transfer of a second electron from P to QB
can be photoinduced. This process is coupled to proton
uptake, thus the quinone QB leaves the RC as a hydroquinone
and promotes the transport of protons across the cytoplasmic
membrane via the cytochrome bc1 complex. The electron
transfer cycle is closed via additional cytochromes. With the rebinding of a quinone at the QB site the photosynthetic function of the reaction centre is restored.
2. Techniques for the Investigation of the
Primary Steps in Photosynthesis
Figure 2. Visible and near-IR absorption spectrum of RC from Rb. sphaeroides,
together with a schematic view of the bacteriochlorines. The colour code of the
chromophores is repeated in the spectrum and relates the absorption bands to
specific chromophores.
became evident that the bacterial RC can also serve as a realistic model for PS II, when the recent structural analysis for PS II
revealed a high similarity between the pigment arrangement
of PS II and that of the bacterial reaction centre (Figure 1 c).
The first photosynthetic system where structural analysis could
be accomplished was the photosynthetic reaction centre of
the purple bacterium Blastochloris (B.) viridis (formerly called
Rhodopseudomonas (Rps.) viridis). Four bacteriochlorophyll
872
Primary reactions in photosynthesis comprise processes occurring on the picosecond and sub-picosecond timescales. The
most direct access to these ultrafast reactions is provided by
time-resolved optical spectroscopy using ultrashort light pulses
at appropriate wavelengths. The interesting spectral region is
illustrated in Figure 2, where the main bacteriochlorine absorption bands of the photosynthetic reaction centre of the purple
bacterium Rhodobacter (Rb.) sphaeroides in the visible and
near-infrared are found. At long wavelengths around 865 nm
there is a broad absorption band due to the Qy transition of
the excitonically coupled special pair P. Around 800 nm (Qy)
and 590 nm (Qx) absorptions due to the accessory BChl molecules are observed. The Qy and Qx transitions of BPhe are
found around 760 nm and 530 nm, respectively. When the
electronic structure of the chromophore is changed, for example, by reduction or oxidation, the absorption spectrum is
modified. Upon reduction of BChl and BPhe the original absorption bands disappear and anion bands appear around
650 nm (BChl , BPhe) and 1020 nm (BChl).[13] These changes
in the absorption spectrum yield the most direct access to the
primary reaction dynamics. In time-resolved experiments, a
first ultrashort light pulse is used to initiate the photosynthetic
reaction. The subsequent changes in optical properties of the
sample are observed as a function of time. In general, the ultrafast experiments monitor molecular properties via transient
absorption. For this purpose a secondary, properly delayed
probing pulse at the wavelength lpr is applied and the absorption change initiated by the excitation pulse is recorded. This
type of experiments has been performed throughout the
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ChemPhysChem 2005, 6, 871 – 880
First Picoseconds in Bacterial Photosynthesis
whole visible, near- and mid-infrared range with sub-picosecond temporal resolution. Supplementary information on excited electronic states could be obtained by time-resolved emission experiments.
Information on the involved molecular processes, especially
on reaction principles, energetics and optimisation, was considerably extended by variation of the sample properties:
Changing the sample temperature allowed to distinguish between activated and nonactivated transfer processes. Details
on the energetics of intermediates were obtained, when specific chromophores were exchanged by modified pigments.[14–16] Site-specific mutagenesis with the replacement of
specific amino acids yielded insight into operational principles
and structural optimisation of the reaction centre.[17–21]
3. The Primary Reaction Steps
The photosynthetic energy conversion processes start with the
excitation of the special pair P by excitation energy transfer
from the antenna pigments, or by direct absorption of light.
The first experiments with temporal resolution in the ten-picosecond domain were based on Nd:glass lasers and could
mainly use excitation wavelengths around 530 nm. Thus the
reaction centre was excited with pronounced excess energy
and energy transfer processes occurred prior to the excitation
of the lowest transition of the special pair. In addition, most of
these experiments used high light intensities, where each reaction centre absorbed more than one photon per excitation
pulse. Nevertheless important information on the reaction intermediates could be obtained.[22–27] It was shown that the reduction of the BPhe occurs on the timescale of several picoseconds and that the electron transfer to the quinone QA takes
about 150 ps. Other authors found absorption transients in the
20 ps time domain, which were assigned to a transient formation of a BChl anion.[26] Later on, a continuous improvement of
the ultrafast techniques led to experiments with excitation directly into the long-wavelength electronic transition of the
special pair P. Fast transients have been observed, however no
convincing assignment of these transients to a specific electron-transfer reaction could be made.[28–32] With the first structure determination of RC from B. viridis, the arrangement of
the chromophores of the RC in two branches (see Figure 1 c)
leading from the special pair to the electron-accepting quinones suggested two different ET pathways. However it
became evident that the reduction of quinone occurred via
the A branch. A first indication of the functional asymmetry
came from structure analysis itself and from optical spectroscopy on crystallised RC.[1, 33–35] In the first published structures
only one of the two quinones was present. Nevertheless lightinduced absorption experiments on these RC crystals showed
that the intermediate P + QA was formed to a large extent.[32, 36]
Finally it was demonstrated that under native conditions more
than 99 % of the ET occurs via the active branch on the A side.
On the microscopic scale, different processes may contribute
to this unidirectionality: The two branches show a slightly
asymmetric arrangement with larger intermolecular distances
on the B side and with different amino acid compositions in
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the vicinity of the chromophores.[1, 3] Local amino acid chromophore interactions are different on the two branches. They
may influence the energetics of the chromophore states and
hence the ET. More polar groups on the A side allow a better
stabilisation for intermediates P + BA and P + HA .[19, 21, 37, 38] Recently, it has been shown that the yield for ET over the B side
can be largely increased by multiple mutations, swapping
amino acids in the two branches.[39–42] These data underline
the importance of the energetic differences for directed electron transfer. In addition, details in the structural arrangement
of the chromophores on the two branches may contribute to
the asymmetry: the temperature factors from structure analysis
indicate that the chromophores on the A branch are structurally better defined and the phytyl chains of the chromophores
of the A branch are arranged in a way to keep BA and HA in
close contact with high electronic coupling.[3, 4, 43, 44]
In the mid-1980s a series of ultrafast experiments were performed by J. L. Martin and J. Breton with strongly improved experimental techniques.[45–48] The authors used a high temporal
resolution on the femtosecond timescale, they performed the
experiments with excitation at the appropriate wavelengths in
the long-wavelength absorption band of the special pair. RC
from two organisms were studied, Rb. sphaeroides contained
type-a bacteriochlorines and B. viridis type-b chromophores.
Both types of RC exhibit very similar reaction dynamics. By recording the time dependence of the stimulated emission on
the long-wavelength side of the P* absorption band, Martin
and Breton were the first to demonstrate that the lifetime of
the electronic excited state P* is 2.8 ps. The authors also
showed that the reduction of the BPhe occurs with the same
time constant. They did not find indications for the existence
of another, shorter-lived intermediate. Therefore they concluded that the ET should occur with one step from the special
pair P to the BPhe HA according to reaction scheme given in
Equation (1):
hn
2:8 ps
200 ps
P !P* !Pþ HA !Pþ QA ð1Þ
Since the BPhe HA and the special pair P are not direct neighbours—the accessory BChl BA is placed in between (see Figure 1 c)—the primary ET had to occur over the large distance
of 1 nm in one step. The authors deduced from their data
that an intermediate state P + BA—if it existed—should have a
lifetime of less than 100 fs.[46] These data were taken as an important indication for a superexchange ET as the primary
charge-separation process in photosynthesis: the electron
should not be transferred step by step between neighbouring
chromophores as was expected from conventional Marcustype ET theory, considering the structural arrangement of the
chromophores. The apparent lack of intermediate P + BA indicated that this state should not be populated and could not
serve as a real intermediate in the ET process. Thus the role of
the accessory BChl BA located between P* and HA became
highly debated: the free energy of state P + BA was assumed
to be much higher than that of P* and no real population of
P + BA was expected. Under these conditions the accessory
BChl BA would work as a virtual electron carrier and promotes
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W. Zinth and J. Wachtveitl
the one-step ET only by increasing the electronic coupling.
Only this assumption allows an ET over the large distance between P and HA with the short time constant of 2.8 ps. This superexchange mechanism for ET was supported by quantum
chemical simulations which yielded an energy of intermediate
P + BA far above P* and excluding any real population of this
state at ambient temperature.[49]
Even if the superexchange model seemed to be convincingly supported by experiments and theory,[30, 50, 51] a number of
unsolved questions or contradictory results had to be considered: Studies on the initial reaction dynamics and intended to
develop a unifying view of the reaction centre, had difficulties
to reconcile a superexchange forward reaction with recombination dynamics.[51–55] Inspecting the data of Martin and
Breton, R. Marcus found that these data would not exclude the
existence of an intermediate P + BA even with a relatively long
lifetime of up to 1 ps.[52] Simulations on the energetics of intermediates that considered the published structure of the reaction centre and polar molecules in the vicinity of the electron
carriers indicated that the energy of intermediate P + BA could
be at or even below the energy of P*, facilitating step-wise ET
via P + BA .[56–59]
In 1989 Kirmaier and Holten presented new experimental
data on the primary reaction. Using a data evaluation procedure with one time constant in the few-picosecond time
domain, they presented a pronounced wavelength dependence of the single ET time with values between 1.3 ps and
4 ps.[60, 61] They interpreted the data by considering a heterogeneity of the RC. At the same time we published a new set of
experimental data recorded under improved experimental conditions.[62–66] These experiments were performed with high
temporal resolution and excitation in the long-wavelength
band of the special pair P. A high sensitivity was established to
record weak components in the transient absorption changes.
A wide set of probing wavelengths, including spectral positions indicative for the reduced chromophores and different
polarisation of the probing pulses were used. These experiments have shown unambiguously that there is an intermediate electron carrier prior to BPhe HA and that the intermediate
state contains a BChl anion. A reaction scheme [see Eqs. (2)
and (3)] with three ground-state intermediates (I2, I3, I4) in the
picosecond time domain could be established and the molecular interpretation with I2 being state P + BA was confirmed:
hn
3 ps
0:9 ps
200 ps
P !I1 !I2 !I3 !I4
hn
3 ps
0:9 ps
ð2Þ
200 ps
P !P* !Pþ BA !Pþ HA !Pþ QA ð3Þ
In detail, the experimental observations can be summarized
as follows:
1) The transient absorption data show a weak but clearly visible kinetic component with a time constant of 0.9 ps (Rb.
sphaeroides) and 0.65 ps (B. viridis).[64, 65]
2) Over wide spectral ranges, this component has a weak amplitude or even vanishes.
874
3) The new, fast component is clearly evident and pronounced only at certain wavelengths, for example, at
665 nm, 790 nm, 1020 nm (see Figures 3 and 4). That is, in
spectral ranges where BChl or its reduced form BChl has
absorption bands, where absorption changes are expected
upon reduction of BChl.[64, 65]
Figure 3. Transient absorption data of RC for Rb. sphaeroides.[63, 64] At 920 nm
(a), in the red wing of the P absorption band, the signal is dominated by the
3 ps decay of the electronically excited special pair P*. At 785 nm (b) and
665 nm (c) one observes the 0.9 ps kinetic component of the P + BA to P + HA
transition as well as the 200 ps ET to the quinone QA. In b) and c) fit curves
with (a) and without (c) the 0.9 ps component are shown. Please note
that the time axis is linear until a delay time of 1 ps and logarithmic for larger
delay times.
4) In Figure 5 we plotted the relative population of the intermediates for the reaction schemes of Equations (2) or (3),
where the intermediate I2 is populated with 3 ps but depopulated faster (0.9 ps). In this model intermediate I2 will
reach only weak occupation, while the other intermediates
show peak populations in the > 90 % range. The weak
peak population of I2 (below 20 %) is the reason for the
small absorption changes found for the 0.9 ps kinetic component. However, when this order of reaction rates is used
for the evaluation of the difference cross-sections, the
weak absorption signal together with the small occupation
leads to reasonable values for the cross-section.
5) The application of a sequential reaction model with the
order of time constants given in Equation (2) for the evaluation of the different absorption cross-sections leads to the
spectra shown in Figure 4. The difference spectra for the
second and third intermediates are well-known from previ-
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ChemPhysChem 2005, 6, 871 – 880
First Picoseconds in Bacterial Photosynthesis
Figure 4. Difference cross-section spectra for the intermediates of the primary
ET in RC of Rb. sphaeroides. The difference spectra are calculated from the experimentally recorded absorption changes connected to the observed decay
times using the sequential reaction scheme of Equation (2). The dotted curve
displays the difference spectrum obtained by Fajer et al.[13] upon reduction of
BChl-a (spectrum shifted in order to consider the different surroundings of the
molecules).
Figure 5. Relative population of the different intermediate states calculated
with the time constants given in Equation (2). For the situation realised in photosynthetic reaction centres where the intermediate I2 is more slowly populated
(3 ps) than depopulated (0.9 ps), the resulting population of this intermediate is
very small and remains below 20 %. This leads to weak absorption changes
connected with the 0.9 ps kinetic component.
ous publications. They are directly related to the intermediates
P + HA and P + QA . The other two spectra need to be discussed
in more detail: The initial state corresponds to the first excited
electronic level P* of the special pair P. It shows the bleaching
of the special pair band around 865 nm and 600 nm, absorption features around 800 nm due to changed excitonic coupling and a spectrally broad absorption increase throughout
most of the visible and near-IR range. The second intermediate
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shows absorption decrease due to oxidation of the special
pair P (similar as in intermediate P + QA) together with an
absorption decrease in the BChl (Qy) band and absorption
increases around 650 nm and 1020 nm.[67] When comparing
these features with absorption difference spectra from neutral versus in vitro reduced BChl-a (see dotted curve in
Figure 4) it is evident that intermediate I2 is identical to P +
BA .
6) Experiments with different polarisations between exciting
and probing light pulses have been performed in the spectral range of lpr = 665 nm and 1020 nm.[64, 67] In these transient dichroism experiments the relative orientation between the photoselected transition dipole moment of the
special pair P and that of the intermediate I2 was determined. From these experiments a value of 268 88 was obtained. A comparison with the angles between the Qy transition of the special pair P and those of the accessory BChl
(298) or BPhe (738) gives another strong argument for I2
being intermediate P + BA .
7) The final argument for the assignment given in Equation (3) comes from the transient experiment at lpr =
1020 nm.[67] In this spectral range neither BPhe nor BPhe
show absorption, but here BChl has a distinct absorption
band (see Figure 4). Also in this uncluttered spectral range
the fast 0.9 ps kinetic component was observed with the
signatures expected from the transient population of the
intermediate P + BA .
In the meantime, a number of other publications appeared
clearly supporting the stepwise nature of the primary ET processes.[68–74] The primary ET reaction consists of a sequence of
individual ET steps transferring an electron between neighbouring chromophores. The ET starts at the excited special pair
P*. Within 3 ps an electron is transferred to the accessory
BChl BA. In a second and faster ET step the electron reaches
the BPhe HA. The final step on the picosecond timescale brings
an electron from HA to QA within 200 ps. Within the picosecond time domain photosynthesis has accomplished a charge
separation over a distance of 25 A. The experimental results
show convincingly that a superexchange mechanism is not required to ascertain efficient forward ET. It will be shown below
that the observed ET reactions can be described well within
the scope of conventional ET theory and that they are optimised for highest quantum efficiency.
4. Conventional Theory and the Primary
Photosynthetic Electron Transfer
Theoretical descriptions of photosynthetic ET in RC were provided by different approaches. An overview can be obtained
from textbooks and recent review articles.[75–77] Classical Marcus
theory is still widely used. A number of more elaborate techniques using spin-boson models, quantum mechanical treatments of nuclear degrees of freedom or molecular dynamics
simulations have been introduced to explain photosynthetic
ET over the wide range of parameters used experimentally.[36, 50, 51, 54, 55, 58, 59, 76, 78–122]
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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W. Zinth and J. Wachtveitl
The photosynthetic ET processes in native RC at room temperature are well described by the nonadiabatic Marcus
theory.[71] Here ET from a donor molecule D to an acceptor
molecule A is treated by three molecular parameters:[75] the
electronic coupling V, the reorganisation energy l and the gain
in free energy DG. V may be deduced from the overlap of the
electronic wavefunctions of D and A. In standard theory V is
assumed to be independent of time. This assumption corresponds to a static arrangement of donor and acceptor molecules. For ET in biological systems the electronic coupling
often depends exponentially on the donor-acceptor distance x
as described in Equation (4)[123]
VðxÞ / expðbxÞ
ð4Þ
where b 7 nm1.
The nuclear motion of donor and acceptor molecules and
their surroundings modulate the energetics of the initial state
DA and the charge-separated state D + A . These modulations
ensure energy conservation during the ET process. According
to the Franck–Condon principle the transfer of the electron
should occur with fixed nuclear coordinates, at the position
where the potential curves of DA and D + A intersect. In conventional nonadiabatic ET theory, the nuclear motions are
treated via the thermally averaged Franck–Condon factor FC(T)
[see Eq. 6)]. In the nonadiabatic limit, where typical ET times
are much longer than the timescale of the nuclear motion, the
ET rate [given in Eq. (5)] becomes:[55, 121, 124]
kET ðTÞ ¼
1
2p 2
¼
V FCðTÞ
tET
h
1
FCðTÞ ¼ pffiffiffiffiffiffiffiffiffiffiffiffiffi exp½ðDGlÞ2 =4lkT
4plkT
1
¼ pffiffiffiffiffiffiffiffiffiffiffiffiffi expðE A =kTÞ
4plkT
ð5Þ
ð6Þ
Equation (5) gives the ET rate as a function of the electronic
coupling V and the Franck–Condon factor FC(T), which depends exponentially on the activation energy EA, which is
given by EA = (DGl)2/4l. In order to obtain an analytical expression for the Franck–Condon factor, assumptions on the
nature of the nuclear motions are required. In a simple version
(see Figure 6) the potential curves of the initial state DA and
the charge-separated state D + A are assumed be harmonic
with the same vibrational frequency w. ET becomes possible at
the intersection of the two potential energy curves with a
probability connected to the electronic coupling V. The motion
on the DA potential curve controls the access to the interaction region. At ambient temperatures, only three molecular parameters, V, DG and l control each ET step. The two quantities,
l and DG may be expressed as the activation energy EA controlling the exponential part of the Franck–Condon factors.
EA = 0 or DG = l, yield nonactivated ET with accelerated transfer rates towards low temperatures. Activated ET, EA ¼
6 0, can be
separated into the normal region with DG < l and the inverted
region DG > l. Within standard nonadiabatic ET theory, the
Franck–Condon factor controls the temperature dependence
876
Figure 6. Schematic of the potential energy surfaces for standard Marcus-type
ET used for the analysis of the different ET steps in the RC. The two quantities,
gain of free energy DG and reorganisation energy l, are indicated. The electronic coupling V determines the probability of ET between the two potential
curves in the intersection region.
of the ET. Towards cryogenic temperatures the quantum mechanical nature of the nuclear motions becomes important
and deviations from Equation (6) have to be considered.
Experiments on the temperature dependence of the electron-transfer reaction have shown that all reaction steps of the
primary ET are accelerated towards lower temperatures, indicating that each individual ET process (i) is nonactivated: DGi =
li.[31, 38, 47, 62, 71] For the initial ET reactions the gain in free energy
DG and the reorganisation energies l could be obtained from
emission experiments on native and modified RC (l1 DG1 =
400–600 cm1, l2 DG2 1200 cm1). Within the limits of the
simplifying nonadiabatic ET theory and nonactivated reactions,
one may obtain an estimate for the electronic coupling. Evaluation of Equations (4–6) for the first two ET steps yielded typical values for the electronic coupling of V1 = 25 cm1 and V2
50 cm1.[70, 71] The experiments on native RC performed at different temperatures allowed only a qualitative picture of primary ET reactions. For a more detailed description additional
information from specifically modified RC was required.
5. Variation of ET Parameters by Protein
Engineering and Chromophore Exchange
Since the late 1980 s selective perturbation of the RC can be
accomplished by site-directed mutagenesis or pigment exchange.[14–21] To understand the molecular principles that underlie the photosynthetic reaction, a fine tuning of the ET parameters DG, l and V for each individual ET step is required.
The exchange of chromophores in RC of Rb. sphaeroides yielded reliable site-specific information by adjusting the redox potential of electron acceptors. The replacement of the accessory
bacteriochlorophyll by a (3-vinyl)-BChl-a caused an energetic
rise of the intermediate state P + BA .[125, 126] As expected from a
stepwise reaction model a reduction in primary ET rate with
the accompanying significant decrease of the transient P + BA
population and less efficient charge separation was found. Important information was obtained when the secondary acceptor BPhe was replaced by different types of modified BPhe and
Phe molecules. It could be shown that the change of the
redox potential upon the incorporation of a Phe-a molecule
drastically altered the reaction dynamics and yielded another
proof for the stepwise ET scheme.[127] The more negative redox
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First Picoseconds in Bacterial Photosynthesis
potential of Phe-a as compared to BPhe-a (by 220 mV) increases the free energy of the electron acceptor HA. Thus the
initial states P* and P + BA get into energetic proximity to P +
Phe-a at room temperature. This modifies reaction dynamics
and reveals information on the energies of states P + Phe-a
and P + BA relative to P*.[74, 127] In Figure 7 data are shown for
one class, where the effects of the mutations are expected to
change only the energetics of the investigated ET step; and
the other class, where side effects have to be considered. It
has also been shown recently that quite often the electronic
coupling is influenced.[69, 70] Presumably, this is due to longrange structural changes upon exchange of individual amino
acids and the highly sensitive dependence of the electronic
coupling on donor–acceptor distance.
6. Stepwise ET Transfer Scheme—A General
Model for the Photosynthetic Energy
Conversion
Figure 7. Transient absorption data for wild-type RC of Rb. sphaeroides (*)
and for the modified RC, where pheophytins replace the bacteriopheophytins
(pheo, *) plotted on a linear (< 1 ps) and logarithmic (> 1 ps) scale. In the
spectral range of 1020 nm, where the absorption is dominated by the (oxidised)
special pair P + and the (reduced) BChl , one observes for the “pheo” sample a
long-lasting population of the intermediate P + BA populated in thermal equilibrium with state P + HA . From the evaluation of the data, the energetic position of the intermediate P + BA could be determined.[74]
the BChl anion band around 1020 nm. Instead of the shortlived population of the BChl anion in wild-type RC, connected
with a 0.9 ps kinetic component, we find in the modified RC,
absorption dynamics related with a long-lasting population of
P + BA . The decay of this absorption change goes in parallel
with the decay of P + Phe-a , indicating that the two intermediates are in thermal equilibrium. From the evaluation of amplitudes and rates, the relative energy difference between P + BA
and P + Phe-a were determined along with an estimate for the
energy of P + BA relative to P*: P + BA is found to be below P*
by 450 cm1. This value finally proves stepwise ET via P + BA
instead of a one-step superexchange process.
A series of studies addressed the ET dynamics in site-directed mutants. A strong reduction in the primary ET rate was obtained when tyrosine M 210 was replaced by phenylalanine or
leucine.[19, 21, 38] The absence of the polar side-chain of tyrosine
M 210 leads to a considerable energetic rise of state P + BA .
The effect on the primary ET is similar to that observed upon
BChl-a to (3-vinyl)-BChl-a exchange. Room temperature ET is
slowed down, but still proceeds via the accessory BChl. However, the reaction is now thermally activated; upon decreasing
temperature, a further reduction of the reaction rate was observed. Systematic variations of the RC properties by site-directed mutagenesis have been performed for Rb. capsulatus,
Rb. sphaeroides and B. viridis. Initially it was a generally accepted working hypothesis, that the mutations should mainly
modify the energetics of the RC.[19] However it became rapidly
evident that mutations influence the RC in a much more complex way. For a consistent discussion of the primary ET reaction, the mutants must be sorted into two different classes:[122]
ChemPhysChem 2005, 6, 871 – 880
www.chemphyschem.org
The experiments presented until now treated the primary ET
reaction in the photosynthetic RC of two purple bacteria, Rb.
sphaeroides and B. viridis. These RC were solubilised by detergents, separated from the naturally embedding membrane
and from the antenna systems. Experiments on membranebound RC from an antenna-deficient mutant of Rb. capsulatus
yielded the same scheme of intermediate states.[128] There was
a fast kinetic component with a time constant of 0.6 ps related
to the transient reduction of BChl. Again the stepwise reaction
model could be applied to this RC. It could thus be excluded
that the solubilisation of the RC was the origin of the transient
population of P + BA . Experiments on RC from Rb. sphaeroides
have shown a certain decrease of the ET rate when membrane
bound preparations were used.[68, 129]
Additional studies have been performed on RC of the thermophilic green bacterium Chloroflexus (Cf.) aurantiacus possessing a different pigment composition (BPhe-a instead of
BChl-a at position BB). Also for this RC ultrafast absorption dynamics indicative of a step-wise ET via the accessory BChl BA
were found.[130]
The recent structural analysis of PS II has shown a large similarity with bacterial RC. However, the lack of an isolated, lowlying excited state of a special pair complicates assignment of
femtosecond and picosecond transients to specific ET processes. On the other hand the large similarity of the structure of
PS II with that of bacterial RC suggests that similar reaction
principles should apply to both types of photoactive centres.
7. Optimisation of Photosynthesis
Since the appearance of the first photosynthetic organisms
some 3 109 years ago, evolution generated a vast variety of
photosynthetic organisms, bacteria, algae and plants: oxygenic
photosynthesis using the two photosystems (PS I and PS II)
and the nonoxygenic photosynthesis where one energy converting unit, the bacterial RC is used. The combination of the
building blocks of photosynthesis, light-collecting antenna and
energy-converting RC allowed life to populate ecological
niches with extreme conditions: habitats in full sunlight, where
each chlorophyll molecule absorbs at least one photon within
1 ms or a location deep in the ocean where at a depth of
about 100 m a chromophore molecule can only absorb one
photon every other minute. While the extreme variations in
available photon densities can be overcome by optimisation of
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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W. Zinth and J. Wachtveitl
antenna size, structure and chromophore composition, basic
chemical requirements have to be fulfilled in order to optimise
the energy conversion process within the RC. The photosynthetic RC is highly efficient in charge separation and ET. The
formation yield of intermediate P + QA can be as high as
97 %.[131] This high quantum efficiency can only be reached by
an optimal design of an RC, which has to meet different environmental, chemical and physical requirements:[70, 71, 73, 132]
1) Many photosynthetic bacteria occupy ecological niches
shaded by photosynthetic organisms using chlorophylltype chromophores. Only the long-wavelength part of the
solar light, l > 700 nm, not absorbed by the higher photosynthetic organisms can be used. Thus photosynthetic bacteria have to use bacteriochlorine pigments absorbing in
the near-IR. Most photosynthetic bacteria capture light between 750 nm and 870 nm. Of special interest is the bacterium B. viridis containing a BChl-b antenna collecting photons beyond 1000 nm and a special pair with a Qy transition at somewhat shorter wavelengths around 960 nm. B.
viridis is one of the few photosynthetic organisms using
light on the long-wavelength side of the 970-nm water absorption band.
2) When solar light energy is absorbed in the antenna
system—often containing more than 100 individual chromophores—this electronic excitation must be guided efficiently to the RC and trapped there, otherwise this energy
would be lost due to internal conversion or radiation from
the antenna chromophores. To ascertain efficient collection
of excitonic energy the absorption band of the special pair
must be tuned to the optimum position, generally in the
long-wavelength absorption band of the antenna. This is
accomplished by using the correct chromophore type (for
example BChl-b in B. viridis), by the strong excitonic coupling of the two BChl molecules in the special pair and by
a suitable shift of the absorption wavelength tuneable via
the interaction of the BChl molecules with the surrounding
amino acids. It has been shown that the specific hydrogenbond pattern between the special pair and the amino
acids modifies both the position of the absorption peak
and the redox potential of the special pair.
3) After the efficient collection of excitonic energy on the 20–
50 ps timescale, the excitation must be trapped irreversibly
to prevent loss either due to back transfer to the antenna
or to internal conversion from P*. Since the special pair is
surrounded by many antenna pigments (in resonance with
the P* transition) back transfer from P* to the antenna is
very efficient. The only way to trap the excitation is by a
fast and almost irreversible reaction step. This is accomplished by the initial charge separation with the formation
of P + BA within 3 ps and by the even more rapid secondary ET with 0.9 ps to the BPhe HA. Charge separation to
P + BA alone, with a weak loss in free energy (DG
450 cm1) is not sufficient to obtain irreversibility since
DG is of the order of (room temperature) kBT. It is only after
the second ET process with a total energy drop of
2000 cm1 that back transfer to the antenna is prevented.
878
4) Another important loss channel is a direct recombination
from an early radical-pair state via electron back transfer to
the ground state P. While a fast forward step with sufficiently large energy difference avoids energy back transfer
to the antenna, recombination is more difficult to prevent.
A fast forward reaction, (for example from P* to P + BA) can
always be accomplished by the combination of large electronic coupling V and an appropriate Franck–Condon
factor. Basically the recombination process from P + BA to
ground state P is assumed to have a similar electronic coupling with a smaller Franck–Condon factor. However, the
different energetics of forward and backward reactions do
not lead to a significant decrease in the recombination,
since high-frequency intramolecular modes may always
allow energy conservation in this inverted regime of ET.
Thus recombination cannot be overcome by a single ET
step. Only the combination of several ET processes permits
transfer of the electron in the forward direction with the required high speed over a distance large enough to prevent
recombination.
5) The energy maintained in the RC after charge transfer must
be stored over a sufficiently long time (ms to ms) to allow
the required coupling to the slower proton-transfer steps.
At this stage the reaction is so slow that thermal equilibrium between all earlier intermediates exists. Under such
conditions thermal population of earlier intermediate
states in the ET chain follow Boltzmann distribution. Only if
the energetic position of the long-lived intermediates is
sufficiently lowered compared to P* may recombination be
prevented. A rough estimate indicates that an energy loss
of around 0.5 eV must be established to allow high quantum efficiency for the microsecond chemical reactions.
Thus a considerable fraction of the photon energy (1.4 eV)
has to be dissipated in order to maintain high quantum efficiency. The relative amount of stored energy versus
photon energy strongly drops for long-wavelength-absorbing bacterial RC. Here nature had to find a compromise between the occupation of new ecological niches and the
loss in reaction efficiency.
6) For the individual parts of the step-wise ET process the RC
has to fulfil the basic requirements of any efficient reaction
chain: for each single reaction step the forward rate must
be much larger than the recombination rate.
7) If one compares the native RC, which has been highly optimised upon evolution, with the different mutated RC,
often decreased transfer rates in the mutated RC are
found. This points to a loss in the charge separation efficiency. However, for one mutation, where the hydrogen
bonding to the special pair P is modified for B. viridis we
find a considerable increase in the initial ET, which should
improve the quantum efficiency of charge separation. However, since the modification also changed the spectral position of the special pair band, the spectral overlap with the
antenna was reduced, leading to an overall reduction of
photon-to-energy efficiency. While many mutations lead to
a considerable reduction of photosynthetic efficiency, a certain amount of charge separation is often maintained. This
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemphyschem.org
ChemPhysChem 2005, 6, 871 – 880
First Picoseconds in Bacterial Photosynthesis
demonstrates that the design of the RC exhibits some robustness: the RC is not only built for optimum use of light
energy but also for some insensitivity with respect to point
mutations. This feature may be a signature of the evolutionary optimisation procedure and allows the bacteria to
adapt to future environmental modifications.
8. Summary and Outlook
The function of the bacterial RC as an important energy conversion unit has been described in detail by ultrafast spectroscopy combined with structural analysis, site-directed mutagenesis, pigment exchange and theoretical modelling. It has been
shown that primary energy conversion is a step-wise process
where an electron is transferred via neighbouring chromophores of the A branch of the RC. A well-defined chromophore
arrangement in a rigid protein matrix combined with optimised energetics of the different electron carriers, allows a
highly efficient charge-separation process. The individual molecular reactions can be well described by conventional ET
theory. It is only the well-designed combination of the different
processes that makes the photosynthetic RC an energy-conversion system optimised for highest quantum efficiency, good
energy use and insensitivity with respect to environmental
changes.
Acknowledgements
The authors acknowledge H. Scheer and D. Oesterhelt for highquality samples and many helpful discussions, and the Deutsche
Forschungsgemeinschaft for financial support.
Keywords: chromophores · electron
chemistry · photochemistry · proteins
transfer
·
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Received: September 22, 2004
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