Phytochrome as molecular machine: Revealing chromophore action during the Pfr → Pr photoconversion by magic-angle spinning NMR spectroscopy

Article abstract of DOI:10.1021/ja9108616

The cyanobacterial phytochrome Cph1 can be photoconverted between two thermally stable states, Pr and Pfr. The photochemically induced Pfr → Pr back-reaction has been followed at low temperature by magic-angle spinning (MAS) NMR spectroscopy, allowing two

Full text of DOI:10.1021/ja9108616

Published on Web 03/05/2010
Phytochrome as Molecular Machine: Revealing Chromophore
Action during the Pfr f Pr Photoconversion by Magic-Angle
Spinning NMR Spectroscopy
Thierry Rohmer,† Christina Lang,‡ Christian Bongards,§
Karthick Babu Sai Sankar Gupta,† Johannes Neugebauer,† Jon Hughes,‡
Wolfgang Ga¨rtner,§ and Jo¨rg Matysik†,*
Leiden Institute of Chemistry, Leiden UniVersity, P.O. Box 9502, 2300 RA Leiden,
The Netherlands, Pflanzenphysiologie, Justus-Liebig-UniVersita¨t, Senckenbergstrasse 3,
D-35390 Giessen, Germany, and Max-Planck-Institut fu¨r Bioanorganische Chemie, Stiftstrasse 34-36,
D-45470 Mu¨lheim an der Ruhr, Germany
Received January 2, 2010; E-mail: j.matysik@chem.leidenuniv.nl
Abstract: The cyanobacterial phytochrome Cph1 can be photoconverted between two thermally stable
states, Pr and Pfr. The photochemically induced Pfr f Pr back-reaction has been followed at low temperature
by magic-angle spinning (MAS) NMR spectroscopy, allowing two intermediates, Lumi-F and Meta-F, to be
trapped. Employing uniformly 13C- and 15N-labeled open-chain tetrapyrrole chromophores, all four statess
Pfr, Lumi-F, Meta-F, and Prshave been structurally characterized. In the first step, the double bond
photoisomerization forming Lumi-F occurs. The second step, the transformation to Meta-F, is driven by
the release of the mechanical tension. This process leads to the break of the hydrogen bond of the ring D
nitrogen to Asp-207 and triggers signaling. The third step is protonically driven allowing the hydrogen-
bonding interaction of the ring D nitrogen to be restored. Compared to the forward reaction, the order of
events is changed, probably caused by the different properties of the hydrogen bonding partners of N24,
leading to the directionality of the photocycle.
Introduction
Phytochromes comprise a well-characterized family of red-/
far-red light sensitive photoreceptors found in plants, cyano-
bacteria, bacteria, and fungi.1,2 Their photochemically active
chromophore is an open-chain tetrapyrrole covalently bound to
the protein via a thioether linkage (Figure 1). The chromophore
triggers the conversion between two thermally stable states, Pr
and Pfr. In the Pr ground state, the chromophore absorbs red
light (λmax ∼ 658 nm in the case of Cph1) triggering a Z-to-E
photoisomerization of the C15dC16 double bond (for number-
ing see the Supporting Information, Figure S1), and thus
converting the protein into the far-red light absorbing Pfr state
(λmax ∼ 702 nm for Cph1). The Pfr state reverts to Pr either in
the dark (on a time scale of weeks in the case of Cph1) at
ambient temperature or instantaneously by a light-induced
sequence of reactions.3 Recently, the three-dimensional structure
of the cyanobacterial phytochrome Cph1 in the Pr state has been
solved.4 In addition, Yang et al. presented the crystal structure
Figure 1. Chromophore binding pocket in Cph1∆2 phytochrome (pdb 2
† Leiden Institute of Chemistry.
VEA).
‡ Justus-Liebig-Universita¨t.
§ Max-Planck-Institut fu¨r Bioanorganische Chemie.
(1) Rockwell, N. C.; Su, Y. S.; Lagarias, J. C. Ann. ReV. Plant Biol. 2006,
57, 837.
of the bacteriophytochrome of Pseudomonas aeruginosa
PaBphP in its unusual Pfr ground state.5 Based on these X-ray
studies, the geometry of the tetrapyrrole methine bridges has
been shown to be ZZZssa and ZZEssa in the Pr and Pfr states,
respectively (Figure S1). Magic-angle spinning (MAS) NMR
(2) Scha¨fer, E.; Nagy, F. Photomorphogenesis in Plants and Bacteria:
Function and Signal Transduction Mechanisms (3rd edition); 3rd ed.;
Springer: Dordrecht, The Netherlands, 2006.
(3) Braslavsky, S. E.; Ga¨rtner, W.; Schaffner, K. Plant Cell EnViron. 1997,
20, 700.
(4) Essen, L. O.; Mailliet, J.; Hughes, J. Proc. Natl. Acad. Sci. U.S.A.
2008, 105, 14709.
(5) Yang, X.; Kuk, J.; Moffat, K. Proc. Natl. Acad. Sci. U.S.A. 2008,
105, 14715.
9
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A R T I C L E S
Rohmer et al.
has revealed that the forward phototransformation (Pr f Pfr)
is linked with a change of the hydrogen-bonding interaction on
the ring D carbonyl and an increase of the length of the
conjugated π-system.6 Very recently, however, it has been
claimed that in the ∼20-kDa GAF-domain fragment of “SyB-
Cph1” phytochrome from the thermotolerant cyanobacterium
Synechococcus OSB’ the photoisomerisation occurs on the C5
methine group.7
In both Pr f Pfr and Pfr f Pr phototransformations,
intermediate states can be distinguished (for review, see ref 3).
Their structural characterization may allow detailed insight into
the mechanism of the phototriggered processes. The photochro-
mic photocycle of various phytochromes has been studied by
absorption and vibrational spectroscopy. For the Pr f Pfr
conversion, absorption spectroscopy has identified at least two
intermediates called Lumi-R and Meta-R.8,9 For the Pfr f Pr
back-reaction, two intermediates (Lumi-F and Meta-F) have
been observed8 (for review see ref 10). These intermediates in
various phytochromes have also been studied by vibrational
spectroscopy.11-14 For the Pr f Pfr transformation it has been
shown that the formation of Lumi-R is linked to the double-
bond isomerization, whereas the single-bond rotation occurs
upon formation of Pfr from Meta-R.11,13,15-17
Because both the parent states absorb significantly in the red
region, the maximal Pfr occupancy attainable at photoequilib-
rium is 70-80%.18 The remaining >20% Pr complicates studies
of Pfr photolysis. Consequently much less is known about the
back-reaction. It has been suggested that the back-reaction
occurs essentially in a single step, directly after the photoex-
citation, followed by minor relaxation steps rearranging the
cofactor-protein interactions.13,19 The occurrence of such con-
certed double-bond and single-bond rotation has been proposed.20
In any case, forward- and back-reactions are different processes
and not simply mirror-symmetric transformations. The intermedi-
ates of the back-reaction, Lumi-F and Meta-F, have been shown
in phytochrome of the cyanobacterium Calothrix to absorb at
635 and 640 nm at low temperatures, respectively,14 although
the specific characteristics of both of these intermediates has
not yet been described.
In order to explore these transformation processes, we
investigated the intermediates of the back-reaction of phyto-
chrome Cph1 by cross-polarization (CP) MAS NMR, exploiting
our ability to prepare Pfr at 100% occupancy via size-exclusion
chromatography. The intermediates were trapped directly in the
magnet under illumination at low temperature. Light-triggered
low-temperature MAS NMR investigations of intermediates
have recently also been presented on rhodopsin,21 bacteriorho-
dopsin,22 and adenylyl transfer reaction of T4 DNA ligase.23
Experimental Section
Sample Preparation for MAS NMR Spectroscopy. Preparation
of u-[13C,15N]-PCB and Cph1∆2 is described in ref 24. Mixtures
at the Pfr/Pr photoequilibrium mixtures (∼70% Pfr) were produced
by saturating irradiation at 660 nm using appropriate LEDs
(Roithner Lasertechnik GmbH, Vienna, Austria). Pfr Cph1∆2
phytochrome at 100% occupancy was obtained by size-exclusion
chromatography of the photoequilibrium mixture using Superdex
200 (Pharmacia/GE).25 The intermediates were produced from
Cph1∆2 in the Pfr state directly in the magnet by irradiating with
light filtered through a far-red cutoff filter (λmax ) 730 nm).
Chemical synthesis of the 15N21-PCB-Cph1∆2 sample is described
in the Supporting Information.
MAS NMR Spectroscopy. About 15 mg of u-[13C,15N]-PCB-
Cph1∆2 protein in the Pfr state were placed in a 4 mm zirconium
rotor. The Lumi-F and Meta-F intermediate states were thermally
trapped in the magnet by irradiation from a xenon lamp via a far-
red interference filter (λmax ) 730 nm) at 173 and 203 K,
respectively. The illumination setup used has been specially
designed for a Bruker MAS probe.26
(6) Rohmer, T.; Lang, C.; Hughes, J.; Essen, L. O.; Ga¨rtner, W.; Matysik,
J. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 15229.
All 2D 13C-13C dipolar-assisted rotational resonance (DARR)
experiments27 were performed in a field of 17.6 T with an Avance-
WB750 spectrometer, equipped with a 4-mm triple resonance CP/
MAS probe (Bruker, Karlsruhe, Germany). The 13C-1H dipolar
interaction has been recovered by continuous wave irradiation on
1H radio frequency field intensity to satisfy the n ) 1 condition.
(7) Ulijasz, A. T.; Cornilescu, G.; Cornilescu, C. C.; Zhang, J. R.; Rivera,
M.; Markley, J. L.; Vierstra, R. D. Nature 2010, 463, 250.
(8) Eilfeld, P.; Ru¨diger, W. Z. Naturforsch. C 1985, 40, 109.
(9) (a) Eilfeld, P.; Vogel, J.; Maurer, R. Photochem. Photobiol. 1987, 45,
825. (b) Scurlock, R. D.; Evans, C. H.; Braslavsky, S. E.; Schaffner,
K. Photochem. Photobiol. 1993, 58, 769.
1
Typical H π/2 and 13C π pulse lengths were set at 3.1 and 5 µs,
(10) Ga¨rtner, W.; Braslavsky, S. E. The Phytochromes: Spectroscopy and
Function; Batschauer, A., Ed.; Royal Society of Chemistry: Cambrige,
U.K., 2003; p 137.
(11) Andel, F.; Lagarias, J. C.; Mathies, R. A. Biochemistry 1996, 35,
15997.
respectively. The 1H power was ramped 80-100% during CP.
Mixing times of 5 and 50 ms were used to maximize homonuclear
1
recoupling between 13C nuclei. H decoupling was about 80 kHz
continuous wave (CW) during the proton mixing and about 43 kHz
TPPM during acquisition. The DARR spectra were recorded with
1536 scans and with 8 µs evolution in the indirect dimension,
leading to experimental times of about 80 h. The data were
(12) (a) Andel, F.; Murphy, J. T.; Haas, J. A.; McDowell, M. T.; van der
Hoef, I.; Lugtenburg, J.; Lagarias, J. C.; Mathies, R. A. Biochemistry
2000, 39, 2667. (b) Foerstendorf, H.; Benda, C.; Ga¨rtner, W.; Storf,
M.; Scheer, H.; Siebert, F. Biochemistry 2001, 40, 14952. (c)
Foerstendorf, H.; Mummert, E.; Scha¨fer, E.; Scheer, H.; Siebert, F.
Biochemistry 1996, 35, 10793. (d) Kneip, C.; Hildebrandt, P.;
Schlamann, W.; Braslavsky, S. E.; Mark, F.; Schaffner, K. Biochem-
istry 1999, 38, 15185. (e) Remberg, A.; Lindner, I.; Lamparter, T.;
Hughes, J.; Kneip, C.; Hildebrandt, P.; Braslavsky, S. E.; Ga¨rtner,
W.; Schaffner, K. Biochemistry 1997, 36, 13389.
(21) Concistre`, M.; Gansmu¨ller, A.; McLean, N.; Johannessen, O. G.;
Montesinos, I. M.; Bovee-Geurts, P. H. M.; Verdegem, P.; Lugtenburg,
J.; Brown, R. C. D.; DeGrip, W. J.; Levitt, M. H. J. Am. Chem. Soc.
2008, 130, 10490.
(13) Matysik, J.; Hildebrandt, P.; Schlamann, W.; Braslavsky, S. E.;
Schaffner, K. Biochemistry 1995, 34, 10497.
(22) Mak-Jurkauskas, M. L.; Bajaj, V. S.; Hornstein, M. K.; Belenky, M.;
Griffin, R. G.; Herzfeld, J. Proc. Natl. Acad. Sci. U.S.A. 2008, 105,
883.
(14) Schwinte´, P.; Ga¨rtner, W.; Sharda, S.; Mroginski, M. A.; Hildebrandt,
P.; Siebert, F. Photochem. Photobiol. 2009, 85, 239.
(23) Cherepanov, A. V.; Doroshenko, E. V.; Matysik, J.; de Vries, S.; de
Groot, H. J. M. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, E85.
(24) (a) Lamparter, T.; Esteban, B.; Hughes, J. Eur. J. Biochem. 2001, 268,
4720. (b) Mozley, D.; Remberg, A.; Ga¨rtner, W. Photochem. Photo-
biol. 1997, 66, 710. (c) Strauss, H. M.; Hughes, J.; Schmieder, P.
Biochemistry 2005, 44, 8244.
(15) Dasgupta, J.; Frontiera, R. R.; Taylor, K. C.; Lagarias, J. C.; Mathies,
R. A. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 1784.
(16) Durbeej, B.; Borg, O. A.; Eriksson, L. A. Phys. Chem. Chem. Phys.
2004, 6, 5066.
(17) Matysik, J., Universita¨t Essen, 1995.
(18) Lamparter, T.; Mittmann, F.; Ga¨rtner, W.; Bo¨rner, T.; Hartmann, E.;
Hughes, J. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 11792.
(19) Foerstendorf, H.; Lamparter, T.; Hughes, J.; Ga¨rtner, W.; Siebert, F.
Photochem. Photobiol. 2000, 71, 655.
(25) Strauss, H. M.; Schmieder, P.; Hughes, J. FEBS Lett. 2005, 579, 3970.
(26) Daviso, E.; Jeschke, G.; Matysik, J. In Biophysical Techniques in
Photosynthesis II; Aartsma, T. J., Matysik, J., Eds.; Springer: Dor-
drecht, The Netherlands, 2008; p 385.
(27) Takegoshi, K.; Nakamura, S.; Terao, T. Chem. Phys. Lett. 2001, 344,
631.
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259.
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Phytochrome as Molecular Machine
A R T I C L E S
Figure 2. 13C chemical shifts for selected carbon atoms during the Pfr f
Pr back-reaction. For sake of clarity, the C13 resonance is noted in blue.
processed with the Topspin software version 2.0 (Bruker, Karlsruhe)
and subsequently analyzed using the program Sparky version 3.100
(T. D. Goddard and D. G. Kneller, University of California).
1D 15N CP/MAS spectra were recorded using an AV-750
1
spectrometer, equipped with 4-mm CP/MAS probe. H-15N het-
eronuclear experiments were performed at the Avance-WB750,
using a frequency switched Lee-Goldburg pulse sequence.28 The
proton π/2 pulse was set to 3.1 µs, and the spinning frequency was
8 kHz.
For the liquid-state NMR, 500 µmol of u-[13C,15N]-PCB was
dissolved in 0.7 mL methanol-d4 and placed in a 5-mm NMR tube.
The 15N-1H HMBC spectrum was obtained with a 600 MHz DMX
spectrometer.
Figure 3. Changes in 13C and 15N chemical shift of the u-[13C,15N]-PCB
chromophore in Cph1∆2 during the Pfr f Lumi-F (A), Lumi-F f Meta-F
(B), and Meta-F f Pr (C) transitions. For each transition, the starting state
is taken as reference and the size of the circles and squares refers to the
difference in 13C and 15N chemical shift in the next state.
Results
the thermally driven single bond transformation following the
light-induced double bond isomerization affects the electronic
structure even stronger. The A/B ring unit shows less extensive
effects, those that are seen affecting the C31 thioether linkage
to Cys-259 (Cph1 numbering of residues is used throughout
the article). In fact, perhaps the biggest surprise is that only
very weak shifts were found to be associated with C15.
Conformational Changes of the Cofactor Carbon Atoms. In
a previous study, we investigated the two parent states of the
1
phytochrome system Pr and Pfr by H, 13C, and 15N CP/MAS
NMR.6 Here we report the complementary information on the
intermediates of the back-reaction from Pfr to Pr, i.e., Lumi-F
and Meta-F. Figures S2 and S3 show the two-dimensional (2D)
13C-13C DARR spectra of the Lumi-F and Meta-F intermedi-
ates. From these data, almost complete sets of 13C assignments
have been obtained for both intermediate states (Table S1).
Calculations of D-ring models confirm the assignments (see the
SI). The carbon atoms C9 and C11 as well as C14 and C16
overlap in both intermediate states (for numbering, see Figure
S1).
Nitrogen Atom Assignments. The one-dimensional (1D) 15
N
spectra of 15N21- and u-[13C,15N]-PCB-Cph1∆2 labeled phy-
tochrome in the Pr, Pfr, and intermediates states are presented
in Figure 4, columns A and B, respectively. 15N21 resonates at
around 158 ppm and appears to be weakly affected during the
Pfr to Pr back-reaction. This evidence, forcing us to correct
previous assignments,6,29 is in line with the weak changes along
ring A during the Pfr f Pr conversion deduced from the 13C
chemical shifts. Further insights into the 15N chemical shift
assignment are obtained by liquid-state NMR, which reveals
that the ring D nitrogen (N24) resonance occurs at 130.9 ppm,
supporting our assignment of the 15N signals at 131.9 and 138.0
ppm to N24 in the Pr and Pfr states, respectively.29 In addition,
we assume that the 15N resonances at 136.5 and 127.1 ppm arise
from N24 in the Lumi-F and Meta-F intermediates states,
respectively. The 15N assignments on ring D have been
confirmed by theoretical calculations (see the SI). The two
remaining 15N signals originate from N22 and N23. Due to the
strong hydrogen-bonding interaction between N22 and the
carbonyl backbone of Asp-207, the resonance of N22 is likely
low-field shifted relative to the one of N23 (see the SI). Hence,
we tentatively assign the peaks at 155.8, 157.9, 161.1, and 160.5
to N22 and the signals at 142.6, 146.0, 144.8, and 147.0 to N23,
in the Pfr, Lumi-F, Meta-F, and Pr states, respectively (Table
S2). This implies that the chemical shifts of the two inner
nitrogens are different in the Pr state despite the fact that the
13C chemical shifts of these rings show minimal differences. It
The changes of chemical shifts during the three transitions
of the back-reaction (Pfr f Lumi-F, Lumi-F f Meta-F, Meta-F
f Pr) for selected carbons are shown in Figure 2. The general
features are as follows: (i) The major changes occur at the
carbons between C13 and C19 and (ii) the transformations
during the two first transitions are significantly larger than during
the third transition. There are unexpectedly large chemical shift
changes during the second transition. Hence, the transition from
Lumi-F to Meta-F appears to be more than small rearrangements
in the chromophore-binding pocket. Spatial details can be better
recognized in Figure 3. At the C19 carbonyl group, the changes
occur during the first two transitions to roughly the same extent.
Since the Pfr state is characterized by stronger hydrogen-bonding
interactions than in Pr,6 it appears that the weakening of these
interactions occurs in two steps. During the first transition, a
significant change occurs selectively at position C18, and also
a change of the chemical shift of the C123 carboxylic group of
ring C occurs already during this transformation. C18 shows
no further changes, whereas during the second transition, in
addition to a large C13 change, both ring carbon neighbors of
the C10 and C15 bridges are strongly affected. It appears that
(28) van Rossum, B. J.; Fo¨rster, H.; de Groot, H. J. M. J. Magn. Reson.
1997, 124, 516.
(29) Rohmer, T.; Strauss, H.; Hughes, J.; de Groot, H.; Ga¨rtner, W.;
Schmieder, P.; Matysik, J. J. Phys. Chem. B 2006, 110, 20580.
9
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A R T I C L E S
Rohmer et al.
Figure 4. 1D 15N spectrum of 15N21-PCB-Cph1∆2 (column A) and u-15N-PCB-Cph1∆2 (column B) in the Pfr, Lumi-F, Meta-F, and Pr recorded at 17.6T
1
with a spinning speed of 8 kHz and at 243, 173, 203, and 243 K, respectively. 2D H-15N FSLG heteronuclear correlation spectra (column C) of Lumi-F
and Meta-F were obtained from a Pfr:Pr (1:1) mixture.
appears that the electronic interaction between the conjugated
π-system and the near-by nitrogens is limited.
tion.14 In addition, the strong effect on C18 might also result
from a mechanical interaction of the C18 ethyl group associated
with the tensed cofactor state. Indeed, the thermal stability of
oat phytochrome A in the Pfr state is improved when the size
of the C18 side chain group is increased.30 In recent X-ray
structure on the Pfr-like state of Pseudomonas aeruginosa
PaBphP,5 however, no residues close to the vinyl group are
observed.
The ring D nitrogen N24 has been shown to be protonated
and hydrogen bound in the Pfr state (Figure 4, column C).
Surprisingly, as demonstrated by the small changes in the 15N
shift at N24 as well as at the 1H resonance of its bound proton,
this hydrogen-bonding interaction remains even after the pho-
toisomerization, implying that the cofactor is not yet fully
relaxed in Lumi-F. The fact that the hydrogen bond of N24
remains almost unchanged in Lumi-F suggests that the hydrogen-
bonding partner is very strong. The crystal structure of Cph1∆2
in the Pr state indicates that the hydrogen-bonding partner is a
water molecule.4 Comparing the 15N chemical shifts of Pfr, Pr,
and the intermediate Meta-F, where this hydrogen bond is
broken, we further conclude that the hydrogen-bonding partner
in Pfr is stronger than water. It has been shown by mutational
studies that Asp-207 is crucial for the formation of the Pfr
state.31 As in the X-ray structure of the PaBphP Pfr state,5 Asp-
207 could indeed be the strong hydrogen-bonding partner of
N24 in the Pfr state of Cph1.
Changes of the Nitrogen Atoms. The Pfr f Lumi-F transition
causes the major effect on N22 and N23 shifting by +2.1 and
-3.4 ppm, respectively (Figures 3 and 4, column B), whereas
changes of the other nitrogen atoms are rather limited. Also,
the proton chemical shifts (Figure 4, column C) do not undergo
any significant change during this step. The Lumi-F f Meta-F
transition shows stronger changes, especially on N24, which
shifts 9.4 ppm downfield. Interestingly, this change is associated
with a significant upfield shift of its pyrrole proton from ∼10
to ∼8 ppm (Figure 4, column C), indicating a modification of
hydrogen-bonding interaction in the Meta-F intermediate state.
In addition, while the signal assigned to N22 shifts from 157.9
to 161.1 ppm, no significant proton shift is observed. The Meta-F
f Pr transition is again associated with strong chemical shift
changes of both N24 and its pyrrole proton, implying that a
hydrogen-bonding interaction at this position is restored. N21
and N23 are also affected during this transition (+2.1 and +2.4
ppm, respectively).
Discussion
Pfr f Lumi-F Transition. It has been proposed that the
chromophore in the Pfr state is conformationally strained,6,13
requiring a strong fixation of the ring D in the pocket. To this
end, a strong hydrogen bond of the ring D carbonyl has been
proposed, leading to enhanced conjugation from ring A to ring
D which increases the stiffness of the cofactor.1,6 Our 13C data
show a very strong chemical shift change at C18 specifically
associated with the Pfr f Lumi-F primary photoreaction. This
can be related to changes in the electronic structure directly
induced by the photoisomarization and the change of hydrogen-
bonding interaction at the C19 carbonyl.6,15 Hence, we expect
that the extended conjugation is already broken in the Lumi-F
state, as also demonstrated by its strongly blue-shifted absorp-
The maintenance of the hydrogen-bond of the ring D nitrogen
is difficult to reconcile with a full 180° rotation. The interruption
of the conjugation in the Pr state and the presence of an extended
conjugation of the π system in the Pfr state also suggest a
rotation less than 180°. Studying a phytochrome model com-
(30) Robben, U.; Lindner, I.; Ga¨rtner, W. G. J. Am. Chem. Soc. 2008, 130,
11303.
(31) Hahn, J.; Strauss, H. M.; Landgraf, F. T.; Gimenez, H. F.; Lochnit,
G.; Schmieder, P.; Hughes, J. FEBS J. 2006, 273, 1415.
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Phytochrome as Molecular Machine
A R T I C L E S
Figure 5. Structures of the tetrapyrrole chromophore and its binding pocket in the Pfr (A), Lumi-F (B), Meta-F (C), and Pr (D) states. The conjugated
system is shown in dark red (Pfr), orange-red (Lumi-F, Meta-F) and red (Pr). Protein residues are indicated in green.
pound in solution, Stanek and Grubmayr32 showed that the full
E/Z isomerization of the C15dC16 double bond dramatically
affects the 13C chemical shift of the C15 carbon (7 ppm upshift
upon E/Z isomerization). For the Pfr f Lumi-F transition,
however, only a very weak effect is observed at C15, although
we cannot rule out that other effects might mask the expected
large change at this position. In any case, the first photochemical
event which has been trapped is definitely not a classical full
180° rotation of ring D associated with a complete E/Z
isomerization as observed in solution.
transition the changes around methine bridges C10 and C15
are stronger than in the Pfr f Lumi-F transition. Since the strong
change in chemical shift observed at C13 specifically at this
step is difficult to rationalize in terms of steric interactions, the
combined upshift of the C13 and C14 resonances, interrupting
the alternating pattern of up- and downshifts, may indicate a
conjugation defect occurring after the initial E-to-Z photo-
isomerization.
Meta-F f Pr Transition. As the chromophore is already
relaxed in the Meta-F state, the changes along the conjugated
system are very minor with respect to the 13C chemical shifts.
Interestingly, the main effects occur at C31, the cofactor-matrix
link, and at N24 which is rebound by a hydrogen-bonding
interaction to a water molecule as seen in the X-ray Pr structure.4
Additional minor changes indicate an overall rearrangement of
the chromophore-protein interactions, potentially involving, not
observable, movements of the protein environment.
Model for the Back-Reaction. We infer from our data that
the tensed chromophore in Pfr is associated with three important
effects (Figure 5A): (i) increased conjugation associated with
C-C single bonds changing to partially higher bond orders and
double bonds to partially lower bond orders, (ii) the strong
hydrogen-bond of the C19 carbonyl to Tyr-263, as shown in
the crystal structure of PaBphP in its Pfr-like ground state, and
(iii) the strong hydrogen-bonding of ring D nitrogen, presumably
to Asp-207, as also implied by the PaBph3 structure,5 although
the latter is not likely to be an entirely reliable model for the
Pfr state in Cph1 and other plant-type phytochromes.
Interestingly, the protein pocket around the C123 carboxylic
group of the ring C propionic side chain appears to be already
rearranged in the Lumi-F intermediate. In particular His-290
may be in its Pr position and already interacts with the ring D
carbonyl group.
Lumi-F f Meta-F Transition. Whereas the Pfr f Lumi-F
transition is characterized by the photochemical E-to-Z C15dC16
double bond isomerization, the Lumi-F f Meta-F transition is
a thermal process. This transition is characterized by the full
release of ring D from its tensed state as indicated by two
significant changes: (i) the loss of hydrogen-bonding interaction
at N24 and (ii) a second stronger change associated with the
methine bridges of C10 and C15. It appears that the chro-
mophore is fully relaxed in Meta-F, whereas in Lumi-F the
hydrogen bridge at N24 still prevents any significant rotation
of ring D. It is surprising that during the Lumi-F to Meta-F
(32) Stanek, M.; Grubmayr, K. Chem.sEur. J. 1998, 4, 1653.
9
J. AM. CHEM. SOC. VOL. 132, NO. 12, 2010 4435

A R T I C L E S
Rohmer et al.
Scheme 1. Changes of the Chromophore during the Photocycle of Phytochrome
The Lumi-F state (Figure 5B) is characterized by (i) a shorter
and weaker conjugation of the π-system, (ii) a partial relaxation
of the cofactor due to incomplete rotation of the ring D caused
by the weakening of the C19 carbonyl hydrogen-bonding
interaction and possibly by the release of the C18 blocking, as
well as (iii) a matrix rearrangement around the carboxylic group
of the ring C propionic side chain.
The Meta-F state (Figure 5C) is described by (i) the full
relaxation of the chromophore after breaking the hydrogen-bond
of the ring D nitrogen to the carboxylic group of the side chain
of Asp-207 allowing rotation around the C15 single bond to be
completed, (ii) the appearance of a conjugation defect localized
around C13 as an effect of shortening of the conjugated system,
and (iii) the formation of a new hydrogen-bond between C19
carbonyl and His-290.
In the Pr state (Figure 5D), in which the cofactor is relaxed
and hydrogen bound, (i) a new hydrogen-bond at N24 is formed
by a water molecule and (ii) protein relaxations have occurred.
Our data show clear evidence that the chromophore isomer-
ization during the back-reaction occurs in two steps and not in
a single concerted step. The first step is linked to the C15dC16
double-bond isomerization, the second occurs at the C14-C15
single bond, and the last step is a pure relaxation process. Hence,
both transformations, Pr f Pfr and Pfr f Pr, are two-step
processes.
neighbor, His-291, is exposed to the solvent in both X-ray
structures, a signaling mechanism is not obvious. Similarly, from
the C123, no molecular signal pathway is apparent although the
interactions implied by the X-ray structures are very different
in the Pr and Pfr states.
On the other hand, our data interpretation suggests a break
of the hydrogen bond between N24 and Asp-207 during the
formation of Meta-F (Figure 5B,C). The release of Asp-207
would allow for the formation of the Asp-207/Arg-472 salt
bridge connecting the PHY domain tongue to the chromophore
region.4 All of these residues are highly conserved, Asp-207
being particularly important for stabilizing the Pfr state.31 The
tongue region comprises a most unusual hairpin structure
inserted into the otherwise GAF/PAS-like PHY domain, making
intimate contact with the extreme N-terminus of the molecule,
itself unusual in forming a knot by passing through a loop in
the GAF domain. Hence, the present data indicate that the Asp-
207/Arg-472 association is likely to couple light-induced
changes in chromophore geometry to the tongue and probably
the N-terminus.
Phytochrome As Molecular Machine. The Pfr f Pr trans-
formation occurs in two separated isomerization steps, and is
linked to a switch of the hydrogen-bonding partner of N24 from
Asp-207 to water (Scheme 1). Although the initial double-bond
isomerization can be interpreted as process to remove the
blockade of the back-reaction, the subsequent single-bond
rotation is related to the release of the tension and the mechanism
of signaling. The formation of the N24-water hydrogen bond
requires both rotations of C14-C15 single and C15dC16
double bond. We have shown that the single-bond rotation is
linked to the break of the hydrogen bond to Asp-207, leading
to an intermediate Meta-F having no hydrogen-bonding interac-
tion at N24.
Model for Photoconversion and Signal Transduction. The
data presented here describe the changes of the Cph1 chro-
mophore from Pfr to the Pr ground state. Thus, the observed
changes of the chromophore and the protein during the transition
from Pfr to Lumi-F, followed by the Meta-F to Pr states, reflect
changes that are connected to both photoconversion and signal
transduction. The Pfr f Pr photoconversion is linked to separate
chromophore-protein interactions at opposite sides of the
chromophore, i.e., at the C123 carboxylic group of ring C and
the entire ring D via its two hydrogen bonds. The highly
As the different absorption properties suggest, the order of
events may be different on the pathway from Pr to Pfr, although
also this well characterized reaction course is initiated by an
isomerization around a double bond and must include a
subsequent single bond rotation as well as the change of the
hydrogen bonding partner of N24 (Scheme 1). It is possible
4
conserved His-290, bound to the C19 carbonyl in Pr and to
the C123 carboxylic group in the Pfr-like ground state of
PaBph35, faces inward from a ꢀ-sheet at the surface of the
molecule. Although the imidazole of its equally well conserved
9
4436 J. AM. CHEM. SOC. VOL. 132, NO. 12, 2010

Phytochrome as Molecular Machine
A R T I C L E S
that the formation of Meta-R is not only linked to a break of a
hydrogen bond to water but to a deprotonation process on N24
leading to a neutral chromophore and therefore a bleached first
electronic transition.33 The single bond rotation occurs during
the final transformation, the formation of Pfr from Meta-R.17
Thus, after double-bond isomerization, the reaction course from
Pr to Pfr is driven protonically, and the final single bond rotation
seems to be a reaction on this charge reorganization. On the
other hand, in the reaction course from Pfr to Pr, it is a
mechanical force of the single-bond rotation which initiates the
change of the hydrogen bonding partner. Hence, phytochrome
is not a simple switch but appears to be a molecular machine
able to channel light excitation to drive nonlinear processes.
This nonlinearity leads to a unidirectionality of the photocycle.
The directional symmetry break of the photocycle may be
required for signaling and therefore be linked to the difference
in matrix interaction in Pr and Pfr. In particular, the chemical
properties of the hydrogen-bonding partners of N24 are distin-
guished and may prearrange the direction of the reaction course.
The geometrically fixed Asp-207 provides sufficient tension on
the ring D to store mechanical tension on the chromophore in
the Pfr state. The rather mobile water molecule may not be able
to fix ring D in the Pr state to a similar extent. On the other
hand, Asp-207, as negatively charged aspartate, would have
sufficient force to deprotonate N24 on the reaction course to
Pfr. A water molecule would not be able to lead to the same
process.
Acknowledgment. The authors thank Prof. HJM de Groot, Dr.
F. Mark, Dr. P. Schmieder, and J. Mailliet for discussions. F.
Lefeber, J. Hollander, and K. Erkelens are gratefully acknowledged
for support during various stages of the experiments. This work
has been financially supported by the Volkswagen-Stiftung (I/
79979) to J.H., W.G., and J.M. and The Netherlands Organization
for Scientific Research (NWO) through a Vidi award (700.53.423),
a Middelgroot subsidy (700.57.107), and ALW open competition
grant (818.02.019) to J.M.
Supporting Information Available: Figures S1-S4 and
Tables S1 and S2. A description of the 15N tentative assignment,
DFT calculation on model compounds, chemical synthesis of
15N21-PCB, and biochemical synthesis of the Cph1 phyto-
chrome. This material is available free of charge via the Internet
at http://pubs.acs.org.
(33) (a) Lagarias, J. C.; Rapoport, H. J. Am. Chem. Soc. 1980, 102, 4821.
(b) Ru¨diger, W.; Thu¨mmler, F.; Cmiel, E.; Schneider, S. Proc. Natl.
Acad. Sci. U.S.A. 1983, 80, 6244. (c) Schaffner, K.; Braslavsky, S. E.;
Holzwarth, A. R. AdV. Photochem. 1990, 15, 229. (d) Schaffner, K.;
Braslavsky, S. E.; Holzwarth, A. R. Frontiers in Supramolecular
Organic Chemistry and Photochemistry; Verlag Chemie: Weinheim,
Germany, 1991.
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