Enhancing the light harvesting capability of a photosynthetic reaction center by a tailored molecular fluorophore

Article abstract of DOI:10.1002/anie.201203404

Light machine: The simplest photosynthetic protein able to convert sunlight into other energy forms is covalently functionalized with a tailored organic dye to obtain a fully functional hybrid complex that outperforms the natural system in light harvesting and conversion ability. Copyright

Full text of DOI:10.1002/anie.201203404

Angewandte
Chemie
DOI: 10.1002/anie.201203404
Hybrid photosynthetic complexes
Enhancing the Light Harvesting Capability of a Photosynthetic
Reaction Center by a Tailored Molecular Fluorophore**
Francesco Milano, Rocco Roberto Tangorra, Omar Hassan Omar, Roberta Ragni,
Alessandra Operamolla, Angela Agostiano, Gianluca M. Farinola,* and Massimo Trotta*
In memory of Luigi Lopez
Building artificial photosynthetic molecular machines capa-
ble of exploiting solar energy for photocatalysis and electrical
energy production has attracted considerable interest in
recent years.[1] The studies aim to mimic the main functions
of a natural photosynthetic apparatus: harvesting light,
converting it into a charge-separated state, and using this
state to drive redox reactions such as water splitting.[2]
Photosynthetic organisms share a common functional organ-
ization of the protein complexes that form their photo-
synthetic apparatus. Pigment–protein complexes (the light
harvesting complexes) act as antenna, collecting solar light
and funneling it to a central photochemical core (the reaction
center) where this energy is converted into an electron-hole
pair that is eventually used for fueling the metabolism of the
organism.[3]
The construction of hybrid systems combining a syntheti-
cally tailored antenna with a natural photoconverter appears
to be a suitable approach to associate tunable and effective
light harvesting with an efficient conversion apparatus that
has been optimized by billion years of evolution. Recently,
fluorescent quantum dots (QDs) have been proposed as
artificial antennas[5] in a report on the first example of
efficient transfer of excitation energy from QDs to a photo-
synthetic reaction center (RC). In this case, the non-specific
electrostatic interactions between the two counterparts pre-
vent the controlled positioning of the artificial antenna with
respect to the protein hindering the efficiency of energy
transfer processes. Moreover, the size of the bulky spherical-
shaped QDs, comparable to the RC one, may represent
a drawback for the activity of the hybrid system.
Ideal biomimetic systems must act as antennas, efficiently
harvesting the sunlight and then effectively converting it into
a stable charge-separated state with a sufficiently long
lifetime to allow ancillary chemistry to take place. The
combination of these requirements has not thus far been fully
attained: whereas efficient light harvesting and energy trans-
fer have been obtained in artificial systems, the lifetime of
charge separated states hardly reaches the millisecond range,
thus leading to limited overall energy conversion yields.[4]
Herein we propose the concept of tailored organic
fluorophores as molecular antennas, an approach which
offers considerable advantages: the molecular diversity of
organic compounds enables very fine tuning of spectroscopic
and electronic properties, as well as control of molecular
shape and flexibility. This reduces the impact of the artificial
antenna on the RC structure and function. Moreover, the
chemistry of bioconjugation can affix the fluorophore to
selected amino acid residues. We have designed and synthe-
sized a hybrid system combining a bacterial RC with a tailored
molecular fluorophore, which acts as an antenna to extend the
light harvesting capability of the natural system and enhance
its activity in a wavelength range where the unmodified
biological system does not efficiently absorb.
The chosen reaction center is the well-known model
system isolated from R. sphaeroides R26[7] (Figure 1). It is
a membrane-spanning protein composed of three subunits
named L, M, and H. Nine cofactors are found in the protein
scaffold, two ubiquinone-10 molecules (QA, QB), one iron ion,
two bacteriopheophytins (BF), and four bacteriochlorophylls
(Bchl), two of which form a functional dimer (D). The
purified RC is surrounded by a toroid of detergent mole-
cules,[8] which prevent its precipitation in water. Upon photon
absorption, which occurs with an efficiency close to unity, one
electron sitting on D is excited and shuttled to the electron
acceptor QA and then to QB. (Supporting Information,
[*] Dr. F. Milano, Prof. A. Agostiano, Dr. M. Trotta
Istituto per i Processi Chimico Fisici
Consiglio Nazionale delle Ricerche
Via Orabona, 4, 70126 Bari (Italy)
E-mail: m.trotta@ba.ipcf.cnr.it
Dr. O. Hassan Omar
Istituto di Chimica dei Composti Organometallici
Consiglio Nazionale delle Ricerche
Via Orabona, 4, 70126 Bari (Italy)
Dr. R. R. Tangorra, Dr. R. Ragni, Dr. A. Operamolla,
Prof. A. Agostiano, Prof. G. M. Farinola
Dipartimento di Chimica
Universitꢀ degli Studi di Bari Aldo Moro
Via Orabona, 4, 70126 Bari (Italy)
E-mail: farinola@chimica.uniba.it
[**] Ministero dell’Istruzione, dell’Universitꢀ e della Ricerca (MIUR),
and Universitꢀ degli Studi di Bari Aldo Moro Project PRIN09
PRAM86 “Innovative Materials for Organic and Hybrid Photo-
voltaics” are acknowledged for their financial support. The COST
Action CM0902 “Molecular machineries for ion translocation across
biomembranes” is also acknowledged.
Figure S3.1). In the absence of external reductants, a charge
recombination reaction occurs: D+QBꢀ!DQB (or D+QA
!
ꢀ
DQA, if the QB functionality is inhibited or removed).
Conversely, in the presence of an exogenous electron donor
to the oxidized D, the RC absorbs a second photon and
shuttles a second electron so that the final quinone QB, upon
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201203404.
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Figure 2. Absorption spectra of RC (c) and AE (c). Fluorescence
spectrum of AE (c). RC (1 mm), AE (4 mm) in Tris (20 mm), EDTA
(1 mm), TX-100 (0.03%, pH 8; TTE buffer). AE spectra are multiplied
by a factor of two. The spectrum of the RC shows several peaks, which
arise from the D (860 nm), the monomer BChl (801 nm), and from the
BF (765 nm).
Figure 1. Crystallographic structure of the R. sphaeroides R26 reaction
center (PDB code: 1AIJ[6]). Subunits: H orange, L yellow, and M cyan.
Bchl, BF, and quinones are in pink. The iron ion is a pink van der
Waals sphere. Lysines are van der Waals green spheres. The terminal
NZ Lys targets of bioconjugation are in blue. The detergent toroid
surrounding the hydrophobic portion of the protein is shown as a grey
cylinder, including the detergent molecules (not to scale). The struc-
ture of the activated AE antenna is shown in the upper left corner
(C cyan, N blue, O red, and S yellow). AE and RC structures are to
scale.
presence of the triple bonds, as well as the propyl arm that
distances the fluorophore skeleton from its activated carboxyl
site are believed to alleviate the steric hindrance of AE
molecules on the surface of the RC. All of these features
make the reaction highly selective for the functionalization of
the protein in the most appropriate positions, thus enabling
the RC to be photoactive even at wavelengths where
negligible light conversion would normally take place.
The absorption spectrum of the purified AE–RC complex
(Figure 3A) confirms bioconjugation, with an average of
4.1 ꢁ 0.3 fluorophores per protein, as calculated from the
absorption at 450 nm. Sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) shows that the binding of
AE occurs on all three protein subunits (Figure 3B), showing
that Lys are indeed distributed throughout the entire protein
(Table S7.1). MALDI-TOF mass measurements show the
presence of up to two AEs on the H subunit and up to one on
the L and M subunits (Figure S5.1).
The actual AE ability to transfer energy to the RC without
significantly modifying the energetics and the activity of the
protein was investigated with three different experiments. As
a first experiment, the possible alteration of electrochemical
midpoint potentials of the semiquinones in the protein
(Figure S3.1) were ruled out, as the charge recombination
reaction kinetic measured in RC and AE–RC was found to be
substantially unchanged. (Figure S4.1).
To verify that bioconjugation to the lysines L82, L268, and
M110 close to the binding site of the cytochrome had not
jeopardized the correct protein functioning, RC activity was
studied by measuring the rate of photocycle in the presence of
exogenous cytochrome and quinone. The rate of the photo-
cycle can be obtained by measuring the changes in the
absorption intensity at 551 nm, where the difference in
absorption between reduced and oxidized cytochrome is at
a maximum.[13] The measurements (Figure 4) were carried out
at the diagnostic wavelengths of 600 and 450 nm using
a photon flux of 1.50 ꢀ 10ꢀ2 mEsꢀ1 cmꢀ2 (corresponding to an
double reduction and protonation, is released and substituted
by a new quinone molecule from an exogenous pool.[7] This
photocycle can be reconstituted in solution and driven until
the exogenous pools of external electron donor (cytochrome)
or quinone become exhausted.[9]
Lysines and cysteines are common targets for the biocon-
jugation of proteins and enzymes.[10] In the RC from R.
sphaeroides, five cysteine residues are present, out of which
only H156 is available for bioconjugation.[11] Conversely, 22
lysines (Lys) are found, nine of which are good candidates for
bioconjugation targets because of their positions (see below).
The organic fluorophore AE (Figure 1), which belongs to
the class of aryleneethynylenes, has been designed and
synthesized[12] to fulfill the spectroscopic, chemical, and
steric requirements to act as antenna for the RC. The
conjugated backbone of AE, comprised of a bis(thiophene)-
benzothiadiazole core with two phenylenethynylene arms,
absorbs light at 450 nm (where the RC absorption is at
a minimum) and has a large Stokes shift with an emission
maximum at 602 nm, which corresponds to an RC absorption
peak (Figure 2). The succinimidyl ester group in AE enables
selective covalent binding of the fluorophore to the Lys of the
RC (see the Supporting Information, Section S2). The
surfactant Triton X-100 conveys the apolar AE molecules in
the aqueous reaction medium, driving them to react with the
nine Lys located in proximity to the RC portion surrounded
by the Triton X-100 toroid and close to the chlorin pigments
(D and Bchl) involved in energy photoconversion. The
bioconjugation in this portion of the RC is favored by the
intercalation of the n-hexyl chains attached to the thiophene
rings of AE with the surfactant molecules surrounding the
protein. Moreover, the almost linear shape of AE given by the
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Figure 4. Light driven cytochrome c oxidation at 450 nm (upper) and
600 nm (lower) for RC (c) and AE–RC (c). In both cases, photon
flux=1.50ꢁ10ꢀ2 mEsꢀ1 cmꢀ2. Conditions: RC or AE–RC (0.5 mm), Cyto-
chrome c (5 mm), decyl-quinone (50 mm) in TTE buffer with KCl
(100 mm).
out at 600 and 450 nm with the same photon flux in both cases.
At 600 nm the final concentrations of charge separated RC
and AE–RC are coincident. At 450 nm it was observed that
the concentration of the charge separated state generated in
the AE–RC equals that obtained at 600 nm, whereas in the
non bioconjugated protein it is five times smaller.
Figure 3. a) Absorption spectrum of the AE–RC molecule. Conditions:
RC 4.5 mm in TTE buffer. b) SDS-PAGE of AE–RC and RC. On the left
side, the three subunits are clearly visible in both cases (AgNO3
staining). On the right side, the fluorescence of the bioconjugated
subunits is visible under UV light (lex =302 nm).
Time resolved spectroscopy has been used to further
characterize the hybrid system. The exited state of AE in
buffer solution decays with a single exponential with a lifetime
of 6.01 ꢁ 0.02 ns. The corresponding AE emission spectrum
has a maximum at 602 nm. In AE–RC, the fluorescence
lifetime is faster, with an average lifetime 2.8 ꢁ 0.1, and the
emission spectrum shows the same maximum position, but
with an intensity (after correctign for internal absorption)
almost three times smaller than that of AE (Figure 6).
To gain insight into the mechanism for the energy transfer
between AE and RC, the Fçrster distance was calculated for
AE (R0 = 54 ꢁ; see the Supporting Information, Section S6).
Considering the distance between the nine best candidate
lysines and the chlorin pigments (Table S7.3), 26 out of 27 are
shorter than the Fçrster distance, which suggests that energy
transfer may occur by fluorescence resonance. Such a fluores-
cence resonance energy transfer (FRET) mechanism, also
proposed in the case of the QDs associated to RC,[5] would
involve any AE molecule bound to any of the nine lysines as
irradiance of 3 mWcmꢀ2 at 600 nm) in both cases. When
illuminated at 600 nm, both RC and AE–RC showed the same
rate constant (3.88 ꢁ 0.03 sꢀ1 and 3.77 ꢁ 0.08 sꢀ1 respectively),
as the absorption of the protein alone drives the photocycle.
At 450 nm, the photocycle was found to be almost three times
faster in the AE–RC than in the unmodified protein (0.78 ꢁ
0.01 sꢀ1 in RC and 1.90 ꢁ 0.01 sꢀ1 in AE–RC). This result not
only confirms that the bioconjugation is not detrimental to the
protein activity but, more importantly, that it enhances the
ability of the RC to drive the photocycle.
In a third independent experiment (Figure 5) the antenna
effect of the AE was investigated by studying the efficiency of
generating the charge separated state in native and function-
alized RCs. Under continuous illumination and in the absence
of exogenous electron donors, the RC shuttles electrons from
the donor to the acceptor and accumulates a charge separated
state up to a final concentration that remains constant until
the light is switched off. The measurements were again carried
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Figure 6. Fluorescence measurements. a) Fluorescence decay of AE
(c) and AE–RC (c). b) Steady state fluorescence of AE (c) and
AE–RC (c). Excitation wavelength 450 nm. Conditions: AE (4 mm) in
the steady state experiment, AE (10 mm) in time resolved experiment.
AE–RC (AE/RC=4:1) in TTE buffer.
Figure 5. Concentration of D+QA generated by continuous illumina-
tion at 450 nm (upper) and 600 nm (lower) at photon
ꢀ
flux=1.50ꢁ10ꢀ2 mEsꢀ1 cmꢀ2. RC (c) or AE–RC (c) at a concen-
tration of 1 mm in TTE buffer with KCl (100 mm).
of hybrid materials with functional properties going beyond
those of natural photosynthetic systems.
donors, transferring energy to any of the chlorin pigments
functioning as acceptors.
Received: May 3, 2012
Published online: && &&, &&&&
Altogether the data clearly indicate that the AE cova-
lently binds to the reaction center and correctly functions as
an antenna. In fact, at 450 nm the hybrid system outperforms
the protein by a factor of three according to the photocycle
assay, and by a factor of five according to charge separation
and steady state fluorescence measurements. Thus, light
collected by the fluorophore at 450 nm is efficiently trans-
ferred by a FRET mechanism to the protein, and used for its
enzymatic activity without any loss of functional integrity.
In conclusion, we have shown that a tailored molecular
organic dye can be covalently conjugated with the photo-
synthetic RC of Rhodobacter sphaeroides to synthesize
a hybrid system capable of absorbing light and efficiently
performing photoconversion in a wavelength range where the
non-conjugated protein does not absorb. This method can
selectively functionalize the lysine residues that are best
located for efficient energy transfer, and the molecular
structure of the AE dye allows the enzyme to maintain full
activity. These findings show that it is possible to design
effective organic/biological hybrid photosynthetic machines
for energy conversion, and paves the way to a new generation
Keywords: bioconjugation · fluorophores · hybrid structures ·
.
photosynthesis · protein modifications
[1] a) J. Barber, Chem. Soc. Rev. 2009, 38, 185 – 196; b) P. Marꢂti, M.
Trotta in CRC Handbook of Organic Photochemistry and
Photobiology, Vol. 1, 3rd ed. (Eds.: A. Griesbeck, M. Oelgemçl-
ler, F. Ghetti), CRC, Boca Raton, FL, 2012.
[2] A. Sartorel, M. Carraro, F. M. Toma, M. Prato, M. Bonchio,
Energy Environ. Sci. 2012, 5, 5592 – 5603.
[3] G. D. Scholes, G. R. Fleming, A. Olaya-Castro, R. van Gron-
delle, Nat. Chem. 2011, 3, 763 – 774.
[4] a) V. Balzani, G. Bergamini, P. Ceroni, Nanopart. Nanodevices
Biol. Appl. 2009, 4, 131 – 158; b) V. Balzani, A. Credi, M.
Venturi, ChemSusChem 2008, 1, 26 – 58.
[5] I. Nabiev, A. Rakovich, A. Sukhanova, E. Lukashev, V.
Zagidullin, V. Pachenko, Y. P. Rakovich, J. F. Donegan, A. B.
Rubin, A. O. Govorov, Angew. Chem. 2010, 122, 7375 – 7379;
Angew. Chem. Int. Ed. 2010, 49, 7217 – 7221.
4
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Angew. Chem. Int. Ed. 2012, 51, 1 – 6
These are not the final page numbers!

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Chemie
[6] M. H. Stowell, T. M. McPhillips, D. C. Rees, S. M. Soltis, E. C.
Abresch, G. Feher, Science 1997, 276, 812 – 816.
Le Droumaguet, K. Velonia, Appl. Microbiol. Biotechnol.
2006, 73, 243 – 254.
[7] G. Feher, J. P. Allen, M. Y. Okamura, D. C. Rees, Nature 1989,
339, 111 – 116.
[11] S. Osvꢃth, J. W. Larson, C. A. Wraight, Biochim. Biophys. Acta
Bioenerg. 2001, 1505, 238 – 247.
[8] M. Roth, A. Lewit-Bentley, H. Michel, J. Deisenhofer, R. Huber,
D. Oesterhelt, Nature 1989, 340, 659 – 662.
[9] F. Milano, L. Gerencser, A. Agostiano, L. Nagy, M. Trotta, P.
Maroti, J. Phys. Chem. B 2007, 111, 4261 – 4270.
[10] a) G. T. Hermanson, Bioconjugate Techniques, 2nd Edition ed.,
Academic Press, London, UK, 2008; b) P. Thordarson, B.
[12] a) G. M. Farinola, F. Babudri, A. Cardone, O. Hassan Omar, F.
Naso, Pure Appl. Chem. 2008, 80, 1735 – 1746; b) A. Opera-
molla, R. Ragni, O. Hassan Omar, G. Iacobellis, A. Cardone, F.
Babudri, G. M. Farinola, Curr. Org. Synth. 2012, in press.
[13] L. Gerencsꢄr, G. Laczkꢂ, P. Marꢂti, Biochemistry 1999, 38,
16866 – 16875.
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Communications
Hybrid photosynthetic complexes
Light machine: The simplest photosyn-
thetic protein able to convert sunlight in
other energy forms is covalently func-
tionalized with a tailored organic dye to
obtain a fully functional hybrid complex
that outperforms the natural system in
light harvesting and conversion ability.
F. Milano, R. R. Tangorra,
O. Hassan Omar, R. Ragni,
A. Operamolla, A. Agostiano,
G. M. Farinola,*
M. Trotta*
&&&& — &&&&
Enhancing the Light Harvesting
Capability of a Photosynthetic Reaction
Center by a Tailored Molecular
Fluorophore
6
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ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2012, 51, 1 – 6
These are not the final page numbers!