Backbone modification of retinal induces protein-like excited state dynamics in solution

Article abstract of DOI:10.1021/ja3007929

The drastically different reactivity of the retinal chromophore in solution compared to the protein environment is poorly understood. Here, we show that the addition of a methyl group to the C=C backbone of all-trans retinal protonated Schiff base acceler

Full text of DOI:10.1021/ja3007929

Communication
pubs.acs.org/JACS
Backbone Modification of Retinal Induces Protein-like Excited State
Dynamics in Solution
Tina Sovdat,† Giovanni Bassolino,‡ Matz Liebel,‡ Christoph Schnedermann,‡ Stephen P. Fletcher,*,†
and Philipp Kukura*,‡
†Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford OX1 3TA, U.K.
‡Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1
3QZ, U.K.
S
* Supporting Information
ABSTRACT: The drastically different reactivity of the
retinal chromophore in solution compared to the protein
environment is poorly understood. Here, we show that the
addition of a methyl group to the CC backbone of all-
trans retinal protonated Schiff base accelerates the
electronic decay in solution making it comparable to the
proton pump bacteriorhodopsin. Contrary to the notion
that reaction speed and efficiency are linked, we observe a
concomitant 50% reduction in the isomerization yield. Our
results demonstrate that minimal synthetic engineering of
potential energy surfaces based on theoretical predictions
can induce drastic changes in electronic dynamics toward
those observed in an evolution-optimized protein pocket.
Figure 1. Native and methylated all-trans retinal protonated Schiff
bases.
backbone: the addition of a methyl group to the C10 position
as depicted in Figure 1. Such a modification is expected to
reduce the excited to ground electronic state energy gap after
relaxation out of the Franck−Condon region due to inductive
stabilization of the partial positive charge along the retinal
backbone. While such a modification will have a minor effect on
the ground state, the bond order inversion expected upon
population of the first excited electronic state25 places a partial
positive charge at the 10-position, which is effectively stabilized
by the presence of the methyl group. At the same time, this
modification has the advantage of leaving the absorption
spectrum unchanged (see Supporting Information), thus,
leading to an overall minimal perturbation of the native system.
The addition and removal of methyl groups to the retinal
backbone has been an active field of study to investigate steric
effects within the bR and rhodopsin protein pockets.26−28
Direct comparison with the results presented herein is difficult
due to the different environment, use of other isomers, and the
lack of any information on reaction dynamics in the previous
works. Here, we focus solely on the reactive properties of native
and modified all-trans protonated Schiff bases in solution both
from a photochemical and photophysical perspective.
We performed ultrafast pump−probe measurements on
native and all-trans-10-methyl RPSB in methanol to investigate
the consequences of our structural modification on the excited-
state dynamics. Pump pulses of 25 fs duration centered at 500
nm ensured population of the first excited state, S1. A
differential absorption map for unmodified all-trans RPSB is
shown in Figure 2A. Here, we focus on the probe wavelength
region between 600 and 900 nm, which is free of any ground-
state signatures. Immediately following time zero, we observed
a dual-peaked stimulated emission (SE) band at 690 and 800
nm that decayed biexponentially at 810 nm with 2 and 7 ps
time constants in agreement with previous results.29 The overall
ouble bond cis−trans isomerizations are found ubiqui-
D
tously in nature as the first step in a variety of light-
induced processes.1 A prominent example is the proton pump
bacteriorhodopsin (bR) found in the archaeon Halobacterium
halobium, which converts light energy into chemical energy.2
This photosynthetic system is triggered by the fast, efficient,
and selective all-trans to 13-cis isomerization of a protein-bound
retinal protonated Schiff base (RPSB) chromophore. Isomer-
ization of the chromophore in solution is almost an order of
magnitude slower (∼4 vs 0.5 ps),3,4 exhibits a lower quantum
yield (0.16 vs 0.64),5,6 and prefers the 11-cis over the 13-cis
product. The correlation between ultrafast dynamics and high
reaction efficiency is not exclusive to retinal but is a common
feature of highly efficient light-induced biological processes.7,8
Understanding and eventually controlling the reactivity and
dynamics of RPSBs has thus become an intense field of study
both from an experimental and theoretical perspective.9−17
Attempts to modify the reactivity of RPSBs in solution,
including changes to the solvent,18−20 backbone structure21−23
and introduction of the opsin shift,24 have failed to show any
appreciable effects on the excited-state dynamics or quantum
yield (QY) compared to the reactivity in the protein
environment. Here, we demonstrate that addition of a methyl
group to the retinal backbone results in protein-like photo-
physics for all-trans RPSB in solution while reducing the
photoisomerization yield.
Rather than attempting to mimic the effect of the protein
pocket by changing the environment of the chromophore, we
opted for a straightforward modification of the retinal
Received: January 24, 2012
Published: April 26, 2012
© 2012 American Chemical Society
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Journal of the American Chemical Society
Communication
Figure 2. Ultrafast dynamics of native and all-trans-10-methyl retinal protonated Schiff base (RPSB). (A) Differential transient absorption map of
native RPSB in MeOH following excitation by a 25 fs pulse centered at 500 nm. (B) Corresponding map for all-trans-10-methyl RPSB. (C)
Temporal dynamics taken from fluorescence upconversion measurements on bacteriorhodopsin (bR)31 and (A) and (B), respectively.
appearance of the SE band is attributed to an excited state
absorption band at 760 nm.30
The dynamics for all-trans-10-methyl RPSB under identical
conditions differ drastically as can be seen in Figure 2B. The
excited state lifetime is reduced by almost an order of
magnitude to 0.7 ps. The SE peaks are shifted from 700 to
710 nm and 810 to 860 nm. A direct comparison of the excited-
state dynamics is shown in Figure 2C. We have also included
the excited state decay associated with the all-trans to 13-cis
isomerization in bacteriorhodopsin measured by fluorescence
upconversion for comparison.31
Close inspection of the results depicted in Figure 2 reveals
several differences between the native and modified RPSB
dynamics beyond the reduction of the excited state lifetime. For
native RPSB, a clear relaxation that is complete within 500 fs is
observed that results in a 170 fs rise time of the SE signature at
Figure 3. Schematic of the potential energy surfaces involved in all-
trans RPSB photochemical dynamics. FC, Franck−Condon point; SP,
stationary point; SE, stimulated emission; CI, conical intersection.
810 nm as shown in Figure 2C. This behavior is in contrast to
the near-instantaneous rise of the SE band in the modified
decay. The surface crossing occurs largely on the reactant side,
RPSB. In addition, native RPSB exhibits clear oscillations with a
leading to a low overall reaction yield. Without detailed
100 fs period during the initial relaxation which appear absent
theoretical investigations, however, we cannot exclude alter-
for the modified system.
native mechanisms based on a modification of the hydrogen-
To assess the effect of the accelerated excited state decay on
out-of-plane frequencies in the isomerizing region that have
the photochemistry, we determined the photoisomerization
been shown to be instrumental in determining the isomer-
1
yields for the all-trans to 11-cis reaction using H NMR to be
ization efficiency for rhodopsin.34
0.16 0.03 and 0.09 0.01 for native and all-trans-10-methyl
Addition of the methyl-group to the retinal backbone causes
RPSB, respectively (see Supporting Information).32 No other
several changes to the observed spectra and dynamics. The
isomers, including the 13-cis product generated by bacterio-
competing S1−SX excited state absorption band is shifted to
rhodopsin, were detectable within our experimental error at the
lower energy resulting in a slightly altered peak appearance of
low photoconversion used in our experiments. Thus, in
the SE band. Nevertheless, the width and position of the SE
contrast to the notion that reaction speed and efficiency are
band remains very similar to native all-trans RPSB suggesting
correlated, our modified RPSB exhibited a reversed effect.
comparable stationary points for both species. Such behavior is
Our results can be rationalized using the simple model
depicted in Figure 3. Absorption of a photon is followed by
rapid relaxation along high-frequency coordinates including C−
C and CC stretching modes with minimal displacement
along the isomerization coordinate.25,33 This relaxation results
in a fast and considerable lowering of the S0−S1 energy gap as
evidenced by the SE band in the near-infrared. The lack of
changes to the SE spectrum throughout excited state decay
points toward a constant molecular structure at a local
minimum.33 A barrier on the excited state prevents the system
from rapidly reaching the ground electronic state via a conical
intersection (CI) which results in the observed picosecond-
expected if the major initial structural changes involve backbone
stretching coordinates.35 Subsequent accelerated excited state
decay as observed in bacteriorhodopsin demands a lower
barrier toward the CI.
The observed lower reaction yield in combination with the
inductive effect of the methyl substituent points toward an even
earlier surface crossing resulting in a reduced probability of
generating the cis-product. The lower barrier and QY may also
be attributed to a lowered backbone vibrational frequency near
the 11,12 CC bond, although further experimental work and
detailed theoretical investigations will be necessary to reveal the
exact nature of the effect.
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Journal of the American Chemical Society
Communication
̈
́
G.; Voıtchovsky, K.; Kindermann, M.; Haacke, S.;
(19) Zgrablic,
Chergui, M. Biophys. J. 2005, 88, 2779.
(20) Zgrablic, G.; Haacke, S.; Chergui, M. J. Phys. Chem. B 2009, 113,
4384.
Our work clearly demonstrates that RPSBs in solution can
indeed exhibit vibronic dynamics of the same speed as in the
protein environment. It also strongly suggests that reaction
yield and speed are not correlated in retinal photochemistry, in
agreement with recent studies of an artificial photochemical
switch.36 Nevertheless, it reemphasizes the importance of the
bR protein pocket in activating a reactive channel toward the
13-cis product which appears completely inactive in solution.
́
(21) Mukai, Y.; Imahori, T.; Koyama, Y. Photochem. Photobiol. 1992,
56, 965.
(22) Kandori, H.; Katsute, Y.; Ito, M.; Sasabe, H. J. Am. Chem. Soc.
1995, 117, 2669.
(23) Hou, B.; Friedman, N.; Ruhman, S.; Sheves, M.; Ottolenghi, M.
J. Phys. Chem. B 2001, 105, 7042.
(24) Bismuth, O.; Friedman, N.; Sheves, M.; Ruhman, S. J. Phys.
Chem. B 2007, 111, 2327.
ASSOCIATED CONTENT
■
S
* Supporting Information
(25) Gonzalez-Luque, R.; Garavelli, M.; Bernardi, F.; Merchan, M.;
Robb, M. A.; Olivucci, M. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 9379.
(26) DeLange, F.; Bovee-Geurts, P. H. M.; VanOostrum, J.; Portier,
M. D.; Verdegem, P. J. E.; Lugtenburg, J.; DeGrip, W. J. Biochemistry
1998, 37, 1411.
Experimental procedures and spectroscopic data for all
compounds. UV/VIS absorption spectra and details of QY
calculation. Details of ultrafast pump−probe experiments. This
material is available free of charge via the Internet at http://
pubs.acs.org.
́
(27) de Lera, A. R.; Iglesias, B.; Rodrıguez, J.; Alvarez, R.; Lop
Villanueva, X.; Padros, E. J. Am. Chem. Soc. 1995, 117, 8220.
(28) Koch, D.; Gartner, W. Photochem. Photobiol. 1997, 65, 181.
́
ez, S.;
́
̈
AUTHOR INFORMATION
■
(29) Hamm, P.; Zurek, M.; Roschinger, T.; Patzelt, H.; Oesterhelt,
D.; Zinth, W. Chem. Phys. Lett. 1996, 263, 613.
̈
Corresponding Author
*philipp.kukura@chem.ox.ac.uk; stephen.fletcher@chem.ox.ac.
(30) Bismuth, O.; Friedman, N.; Sheves, M.; Ruhman, S. Chem. Phys.
2007, 341, 267.
uk
(31) Schmidt, B.; Sobotta, C.; Heinz, B.; Laimgruber, S.; Braun, M.;
Gilch, P. Biochim. Biophys. Acta, Bioenerg 2005, 1706, 165.
(32) Childs, R.; Shaw, G. J. Am. Chem. Soc. 1988, 110, 3013.
(33) Ruhman, S.; Hou, B. X.; Friedman, N.; Ottolenghi, M.; Sheves,
M. J. Am. Chem. Soc. 2002, 124, 8854.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(34) Weingart, O.; Altoe,
Garavelli, M. Phys. Chem. Chem. Phys. 2011, 13, 3645.
̀
P.; Stenta, M.; Bottoni, A.; Orlandi, G.;
P.K. and S.P.F. are supported by Career Acceleration
Fellowships awarded by the EPSRC (EP/H003541/1 & EP/
H003711/1). We are immensely grateful to Giulio Cerullo,
Eberhard Riedle and Nicolaus Ernsting and their research
groups for assistance in the construction of the ultrafast
spectrometer, Peter Gilch for sharing experimental data on
bacteriorhodopsin and Barbara Odell for assistance with the
NMR experiments.
́
(35) Schapiro, I.; Ryazantsev, M. N.; Frutos, L. M.; Ferre, N.; Lindh,
R.; Olivucci, M. J. Am. Chem. Soc. 2011, 133, 3354.
́
(36) Briand, J.; Bram, O.; Rehault, J.; Leonard, J.; Cannizzo, A.;
̈
Chergui, M.; Zanirato, V.; Olivucci, M.; Helbing, J.; Haacke, S. Phys.
Chem. Chem. Phys. 2010, 12, 3178.
REFERENCES
■
(1) Dugave, C. Cis-trans Isomerization in Biochemistry; Wiley-VCH:
Weinheim, 2006.
(2) Lozier, R. H.; Bogomolni, R. A.; Stoeckenius, W. Biophys. J. 1975,
15, 955.
(3) Mathies, R. A.; Cruz, C. H. B.; Pollard, W. T.; Shank, C. V.
Science 1988, 240, 777.
(4) Kandori, H.; Sasabe, H. Chem. Phys. Lett. 1993, 216, 126.
(5) Freedman, K.; Becker, R. J. Am. Chem. Soc. 1986, 108, 1245.
(6) Tittor, J.; Oesterhelt, D. FEBS Lett. 1990, 263, 269.
(7) Schoenlein, R. W.; Peteanu, L. A.; Mathies, R. A.; Shank, C. V.
Science 1991, 254, 412.
(8) Sundstrom, V. Annu. Rev. Phys. Chem. 2008, 59, 53.
̈
(9) Becker, R.; Freedman, K.; Hutchinson, J.; Noe, L. J. Am. Chem.
Soc. 1985, 107, 3942.
(10) Huppert, D.; Rentzepis, P. J. Phys. Chem. 1986, 90, 2813.
(11) Walther, M.; Fischer, B.; Schall, M.; Helm, H.; Jepsen, P. Chem.
Phys. Lett. 2000, 332, 389.
(12) Kloppmann, E.; Becker, T.; Ullmann, G. Proteins 2005, 61, 953.
(13) Olivucci, M.; Lami, A.; Santoro, F. Angew. Chem., Int. Ed. 2005,
44, 5118.
(14) Rostov, I.; Amos, R.; Kobayashi, R.; Scalmani, G.; Frisch, M. J.
Phys. Chem. B 2010, 114, 5547.
(15) Valsson, O.; Filippi, C. J. Chem. Theory. Comput. 2010, 6, 1275.
(16) Malhado, J.; Spezia, R.; Hynes, J. J. Phys. Chem. A 2011, 115,
3720.
(17) Zgrablic,
2012, 134, 955.
́
G.; Novello, A. M.; Parmigiani, F. J. Am. Chem. Soc.
(18) Logunov, S.; Song, L.; El-Sayed, M. J. Phys. Chem. 1996, 100,
18586.
8320
dx.doi.org/10.1021/ja3007929 | J. Am. Chem. Soc. 2012, 134, 8318−8320