Photochemically induced dynamic nuclear polarization in a C450A mutant of the LOV2 domain of the Avena sativa blue-light receptor phototropin

Article abstract of DOI:10.1021/ja053785n

Phototropin is a blue-light receptor involved in the phototropic response of higher plants. The photoreceptor comprises a protein kinase domain and two structurally similar flavin-mononucleotide (FMN) binding domains designated LOV1 and LOV2. Blue-light irradiation of recombinant LOV2 domains induces the formation of a covalent adduct of the thiol group of a functional cysteine in the cofactor-binding pocket to C(4a) of the FMN. Cysteine-to-alanine mutants of LOV domains are unable to form that adduct but generate an FMN radical upon illumination. The recombinant C450A mutant of the LOV2 domain of Avena sativa phototropin was reconstituted with universally and site-selectively ¹³C-labeled FMN and the ¹³C NMR signals were unequivocally assigned. ¹³C NMR spectra were acquired in darkness and under blue-light irradiation. The chemical shifts and the coupling patterns of the signals were not affected by irradiation. However, under blue-light exposure, exceptionally strong nuclear-spin polarization was developed in the resonances belonging to certain carbons of the FMN's isoalloxazine moiety. An enhancement of the NMR absorption was observed for the signals of C(5a), C(7), and C(9). NMR lines in emission were detected for the signals belonging to C(2), C(4), C(4a), C(6), C(8), and C(9a). The signal of C(10a) remained in absorption but was slightly attenuated. In contrast, the intensities of the NMR signals belonging to the carbons of the ribityl side chain of FMN were not affected by light. The observation of spin-polarized ¹³C-nuclei in the NMR spectra of the mutant LOV2 domain is clear evidence for radical-pair intermediates in the reaction steps following optical sample excitation.

Full text of DOI:10.1021/ja053785n

Published on Web 11/18/2005
Photochemically Induced Dynamic Nuclear Polarization in a
C450A Mutant of the LOV2 Domain of the Avena sativa
Blue-Light Receptor Phototropin
Gerald Richter,*,†,§ Stefan Weber,*,‡ Werner Ro¨misch,† Adelbert Bacher,†
Markus Fischer,† and Wolfgang Eisenreich*,†
Technische UniVersita¨t Mu¨nchen, Lehrstuhl fu¨r Organische Chemie und Biochemie,
Lichtenbergstrasse 4, 85747 Garching, Germany, and Freie UniVersita¨t Berlin, Fachbereich
Physik, Arnimallee 14, 14195 Berlin, Germany
Received June 9, 2005; E-mail: wolfgang.eisenreich@ch.tum.de; Stefan.Weber@physik.fu-berlin.de; G.Richter@exeter.ac.uk
Abstract: Phototropin is a blue-light receptor involved in the phototropic response of higher plants. The
photoreceptor comprises a protein kinase domain and two structurally similar flavin-mononucleotide (FMN)
binding domains designated LOV1 and LOV2. Blue-light irradiation of recombinant LOV2 domains induces
the formation of a covalent adduct of the thiol group of a functional cysteine in the cofactor-binding pocket
to C(4a) of the FMN. Cysteine-to-alanine mutants of LOV domains are unable to form that adduct but
generate an FMN radical upon illumination. The recombinant C450A mutant of the LOV2 domain of Avena
sativa phototropin was reconstituted with universally and site-selectively 13C-labeled FMN and the 13C NMR
signals were unequivocally assigned. 13C NMR spectra were acquired in darkness and under blue-light
irradiation. The chemical shifts and the coupling patterns of the signals were not affected by irradiation.
However, under blue-light exposure, exceptionally strong nuclear-spin polarization was developed in the
resonances belonging to certain carbons of the FMN’s isoalloxazine moiety. An enhancement of the NMR
absorption was observed for the signals of C(5a), C(7), and C(9). NMR lines in emission were detected for
the signals belonging to C(2), C(4), C(4a), C(6), C(8), and C(9a). The signal of C(10a) remained in absorption
but was slightly attenuated. In contrast, the intensities of the NMR signals belonging to the carbons of the
ribityl side chain of FMN were not affected by light. The observation of spin-polarized 13C-nuclei in the
NMR spectra of the mutant LOV2 domain is clear evidence for radical-pair intermediates in the reaction
steps following optical sample excitation.
Introduction
The phototropic response of higher plants is mediated by the
blue-light receptor phototropin.1,2 The protein specified by the
phot1 gene3 comprises a protein kinase domain and two
topologically similar domains carrying noncovalently bound
FMN chromophores.4 Blue-light irradiation of the recombinant
LOV2 domain (M ) 12.5 kDa) of AVena satiVa phototropin
causes reversible bleaching of the absorption in the range of
400-500 nm,4 accompanied by the appearance of absorption
at 370 nm to 390 nm attributed to the reversible addition of the
thiol group of cysteine-450 to C(4a) of the FMN chromophore
Figure 1. Photocycle of wild-type LOV domains. R denotes the ribityl
side chain of FMN.
LOV2 domain of Adiantum capillus-Veneris has confirmed the
earlier NMR findings and revealed an FMN chromophore with
its isoalloxazine ring strongly deviating from planarity at C(4a)
in the light form.6 The photoadduct undergoes spontaneous
fragmentation in the dark with a rate constant of 2.55 × 10-2
s-1 at room temperature.7
(see Figure 1) on the basis of 13C NMR spectroscopy.5
A
subsequent X-ray diffraction structural analysis of the respective
† Technische Universita¨t Mu¨nchen.
‡ Freie Universita¨t Berlin.
§ Present address: University of Exeter, School of Biological and
Chemical Sciences, Stocker Road, Exeter EX4 4QD, United Kingdom,
E-Mail: G.Richter@exeter.ac.uk.
The flavin triplet state (3FMN) has been proposed as a reactive
intermediate in the primary blue-light induced reaction of LOV
3
domains.8 More specifically, FMN has been suggested to be
(1) Briggs, W. R.; Christie, J. M. Trends Plant Sci. 2002, 7, 204-210.
(2) Kagawa, T. J. Plant Res. 2003, 116, 77-82.
(3) Briggs, W. R. et al. Plant Cell 2001, 13, 993-997.
(4) Christie, J. M.; Salomon, M.; Nozue, K.; Wada, M.; Briggs, W. R. Proc.
Natl. Acad. Sci. U.S.A. 1999, 96, 8779-8783.
(6) Crosson, S.; Moffat, K. Plant Cell 2002, 14, 1067-1075.
(7) Salomon, M.; Christie, J. M.; Knieb, E.; Lempert, U.; Briggs, W. R.
Biochemistry 2000, 39, 9401-9410.
(8) Swartz, T. E.; Corchnoy, S. B.; Christie, J. M.; Lewis, J. W.; Szundi, I.;
Briggs, W. R.; Bogomolni, R. A. J. Biol. Chem. 2001, 276, 36493-36500.
(5) Salomon, M.; Eisenreich, W.; Du¨rr, H.; Schleicher, E.; Knieb, E.; Massey,
V.; Ru¨diger, W.; Mu¨ller, F.; Bacher, A.; Richter, G. Proc. Natl. Acad. Sci.
U.S.A. 2001, 98, 12357-12361.
9
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J. AM. CHEM. SOC. 2005, 127, 17245-17252
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A R T I C L E S
Richter et al.
generated via intersystem crossing (ISC) from an excited singlet-
state precursor,9 and to decay within a few microseconds by
generating the FMN-cysteinyl photoadduct.7 The primary pho-
toreaction has been claimed to proceed (i) via an ionic
mechanism,8 or (ii), in close analogy to the photooxidation of
Free-induction decays were processed using exponential mul-
tiplication. The acquisition of the spectra required 5-20 h.
When required, the samples were irradiated inside the magnet
via an optical fiber whose conical tip was immersed into the
solution of the NMR tube (15 mm above the magnetic center).
The light source was a mercury lamp (Oriel Corporation,
Stamford, CT) operating at a constant power of 100 W. The
emitted light was filtered using a pair of BG7 and GG420 filters
from Schott (Mainz, Germany). Alternatively, a blue-light
emitting photodiode (455 nm, 175 mW, Luxeon Star/O Batwing,
Lumileds Lighting, San Jose, CA) was used as a light source.
DFT Computations. Density-functional computations were
performed using the program package Gaussian03.19 Two
different paramagnetic forms of 7,8,10-trimethyl isoalloxazine
were tested as models for an FMN radical: (i) a neutral radical
form, FlH•, protonated at N(5), and (ii) an anion radical form,
Fl•-, deprotonated at N(5). The geometries of both molecules
were optimized using the B3LYP functional and the 6-31G*
basis set. Subsequently, single-point calculations of the unpaired
electron-spin density were performed on the optimized structures
using the B3LYP functional and the EPR-II basis set. Iso-spin-
density surfaces were obtained using the MOLDEN program
package.20
3
amino acids by FMN,10 via a radical-pair mechanism with a
triplet-configured radical pair converting to a singlet-configured
radical pair as a precursor for covalent-bond formation.11,12
A
wealth of information was obtained from LOV mutants in which
the functional cysteine was replaced by alanine, serine or
methionine.11,13,14 The Cys f Ala and Cys f Ser mutants do
not form covalent adducts but rather undergo spontaneous
photoreduction of the FMN chromophore, which is initially in
the fully oxidized redox state,7 to form a one-electron reduced
FMN radical.
In this study we present NMR experiments on the LOV2
C450A mutant of A. satiVa phototropin in which the FMN
chromophore has been replaced with universally or specifically
13C-labeled FMN isotopologs. We report for the first time the
observation of photochemically induced dynamic nuclear po-
larization (photo-CIDNP) in an integral cofactor-protein system
probed by solution NMR. A semiquantitative analysis of the
polarization phenomenon provides information on the generation
of nuclear-spin polarization in the LOV2 mutant domain.
Results
Experimental Procedures
The recombinant mutant LOV2 C450A domain used in this
study has absorbance maxima at 363 and 447 nm characteristic
of an FMN chromophore in the fully oxidized redox state.7 After
blue-light irradiation, the protein shows absorption maxima at
570 and 605 nm (data not shown) as is typical for FMN-radical
formation. Photoreduction of this LOV2 mutant has been
reported previously to occur in the presence and absence (as is
the case in the present study) of exogenous electron donors.11
In all samples investigated, the flavin radical is completely
reoxidized in the dark on a time scale of minutes.
13C NMR spectra from LOV2 mutant protein that had been
reconstituted with various 13C-labeled FMN samples were
recorded in the dark and under continuous blue-light irradiation
(see Figure 2) as described earlier.5 NMR spectra obtained in
the dark and after preliminary irradiation followed by spontane-
ous reversal to the fully oxidized redox state were essentially
identical with the spectra of unirradiated protein. Notably, the
signals of the FMN chromophore were not affected. However,
some irreversible photodamage was noticed after prolonged
sample irradiation as evidenced by a decrease in the signal-to-
noise ratio and by the appearance of spurious peaks, albeit of
Materials. [U-13C17]Riboflavin was prepared by fermentation
of a riboflavin-producing Bacillus subtilis strain using [U-13C6]-
glucose as carbon source.15 Other 13C-labeled riboflavin isoto-
pologs were prepared by chemical or enzyme-assisted syn-
thesis.15-18
The preparation of the recombinant LOV2 C450A domain
(amino acid residues 405 to 559) of A. satiVa phototropin and
its reconstitution with isotope-labeled FMN follows procedures
that have been reported elsewhere;5 for details, cf. Supporting
Information.
NMR Spectroscopy. NMR spectra were measured at 27 °C
using a four-channel DRX-500 spectrometer (Bruker, Karlsruhe,
Germany) equipped with a pulsed-field gradient accessory. 13
NMR spectra were recorded at 125.8 MHz using a 5-mm H/
13C dual probehead. 13C chemical shifts were referenced to
internal dioxane (67.84 ppm relative to tetramethylsilane, TMS).
The solvent contained 25 mM sodium/potassium phosphate, pH
7, 10% (v/v) 2H2O, and 0.5-1 mM protein; the sample volume
was 0.5 mL.
C
1
Composite pulse decoupling was used. All spectra were
recorded using a flip angle of 30°. The repetition rate was 3 s.
(18) Dwyer, T. M.; Mortl, S.; Kemter, K.; Bacher, A.; Fauq, A.; Frerman, F. E.
Biochemistry 1999, 38, 9735-9745.
(9) Kennis, J. T. M.; Crosson, S.; Gauden, M.; van Stokkum, I. H. M.; Moffat,
K.; van Grondelle, R. Biochemistry 2003, 42, 3385-3392.
(10) Heelis, P. F.; Parsons, B. J.; Phillips, G. O. Biochim. Biophys. Acta 1979,
587, 455-462.
(19) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M.
A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin, K. N.;
Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.;
Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.;
Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa,
J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.;
Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.; Adamo, C.;
Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.;
Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.;
Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich,
S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A.
D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A.
G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.;
Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham,
M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.;
Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian
03, revision B.04; Gaussian, Inc.: Wallingford, CT, 2004.
(11) Kay, C. W. M.; Schleicher, E.; Kuppig, A.; Hofner, H.; Ru¨diger, W.;
Schleicher, M.; Fischer, M.; Bacher, A.; Weber, S.; Richter, G. J. Biol.
Chem. 2003, 278, 10973-10982.
(12) Schleicher, E.; Kowalczyk, R. M.; Kay, C. W. M.; Hegemann, P.; Bacher,
A.; Fischer, M.; Bittl, R.; Richter, G.; Weber, S. J. Am. Chem. Soc. 2004,
126, 11067-11076.
(13) Bittl, R.; Kay, C. W. M.; Weber, S.; Hegemann, P. Biochemistry 2003, 42,
8506-8512.
(14) Kottke, T.; Dick, B.; Fedorov, R.; Schlichting, I.; Deutzmann, R.;
Hegemann, P. Biochemistry 2003, 42, 9854-9862.
(15) Ro¨misch, W.; Eisenreich, W.; Richter, G.; Bacher, A. J. Org. Chem. 2002,
67, 8890-8894.
(16) Sedlmaier, H.; Mu¨ller, F.; Keller, P. J.; Bacher, A. Z. Naturforsch. C 1987,
42, 425-429.
(20) Schaftenaar, G.; Noordik, J. H. J. Comput.-Aided Mol. Design 2000, 14,
123-134.
(17) van Schagen, C. G.; Mu¨ller, F. Eur. J. Biochem. 1981, 120, 33-39.
9
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Nuclear-Spin Polarization in Phototropin
A R T I C L E S
Figure 2. 13C NMR spectra of [U-13C17]FMN-reconstituted LOV2 C450A domain. (A) “Dark” spectrum. (B) Scaled “light” spectrum. (C) Difference
spectrum “light”-minus-“dark”.
relatively low intensity. Hence, to avoid photodamage, spectra
recorded during illumination were collected with fewer acquisi-
tions as compared to the respective “dark” spectra. The signal-
to-noise ratios of the NMR spectra of illuminated samples are
therefore typically slightly lower than those of the previously
recorded spectra of the protein in the dark.
domain reconstituted with [U-13C17]FMN as their chemical shifts
are closely similar. Likewise, the multiplets of C(8) and C(10a)
overlap.
Blue-light illumination of the protein samples, which results
in partial formation of the radical form of FMN, did not affect
the chemical shifts of individual carbon atoms (see Figures 2
and 3). Surprisingly, however, some of the signals now appeared
with negative signal amplitude, whereas other signals remained
in absorption, either with attenuated or enhanced intensity. With
the exception of the signal intensities for carbons C(2′), C(3′),
C(4′), and C(5′) of the ribityl side chain, the intensity ratios
between signals measured in the dark or under blue-light
condition varied over a wide range. Using the integrals of the
ribityl signals as a measure of the amount of diamagnetic (fully
oxidized) FMN, we obtain an approximately 3-fold decreased
concentration of fully oxidized FMN under light conditions as
compared to the sample examined in the dark. Hence, we
conclude that the majority of the initially diamagnetic LOV2
domains rests in paramagnetic states in which the individual
13C NMR signals are broadened beyond detectability due to the
strong hyperfine interaction of the nuclear spins with the
unpaired electron spin which is mainly located on the FMN’s
isoalloxazine ring. This conclusion is consistent with the UV-
vis spectrum of the sample recorded upon photoexcitation and
also with our previous EPR observations of an FMN radical
generated by blue-light irradiation of LOV2 C450A.11
In the NMR spectra of recombinant LOV2 C450A domains
recorded in the dark, most of the 13C-enriched carbon atoms of
the FMN chromophore appear as multiplets in the protein
reconstituted with [U-13C17]FMN (Figure 2), and as singlets in
protein specimens reconstituted with FMN isotopologs carrying
either one or two 13C atoms (Figure 3).
The NMR line widths of signals from protein-bound FMN
were large as compared to those arising from free FMN. A
comparison of the chemical shifts of FMN carbons in wild type
and in mutant LOV domains reveals only minor differences (see
Table in Supporting Information). Apparently, the replacement
of C450 by alanine does not significantly alter the surroundings
of the FMN chromophore.
On the basis of isotopolog editing using single-labeled and
double-labeled FMN in conjunction with the coupling patterns
in samples containing [U-13C17]FMN, all 13C NMR signals of
FMN in the LOV2 domain could be unequivocally assigned.
Specifically, C(2) appears as a singlet in the spectrum of the
protein reconstituted with [U-13C17]FMN. The carbons C(4),
C(7R), C(8R), C(10a), C(1′), and C(5′) appear as doublets with
splittings arising from the spin-spin coupling to the respective
neighboring 13C atoms. Due to simultaneous coupling to two
adjacent 13C atoms, the carbons C(6), C(9), C(2′), C(3′), and
C(4′) appear as pseudo-triplets. Pseudo-quartets are observed
for the carbons C(7) and C(8) due to simultaneous coupling to
three directly adjacent 13C atoms. The multiplet signals of C(4a),
C(5a) and C(9a) overlap in the NMR spectra of the LOV2
A difference NMR spectrum (trace C of Figure 2) is obtained
by subtracting the spectrum measured under dark conditions
(trace A of Figure 2) from the spectrum recorded during
illumination that has been scaled such that the signal intensities
of the carbons C(2′), C(3′), C(4′), and C(5′) match those from
the “dark” state (trace B of Figure 2). In this representation,
strongly enhanced absorptive polarization of the nuclear spins
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A R T I C L E S
Richter et al.
Figure 3. 13C NMR spectra of LOV2 C450A domains reconstituted (from top to bottom) with [4,10a-13C2]FMN (first row), [4a-13C1]FMN (second row),
[5a,8-13C2]FMN (third row), [6,8R-13C2]FMN (fourth row), [7,9a-13C2]FMN (fifth row) and [7R,9-13C2]FMN (sixth row). Left column: “dark” spectra.
Right column: “light” spectra. For details see text.
is observed in the region where the resonances of C(4a), C(5a),
and C(9a) occur. Strong emissive spin polarization is obtained
for C(2) and C(8). The multiplet signal assigned to C(6) is
weakly emissively polarized. Additionally, a strong emissive
NMR line is observed around 110.6 ppm. This signal is not
due to the resonance of any of the carbons within the FMN
chromophore as it is not observed in the NMR spectrum
recorded in the dark. Hence, we conclude that it must be
assigned to a 13C atom situated in the C450A apo-protein,
although the amino acid carbon atoms are not 13C-enriched.
Apparently, the signal of this atom becomes strongly polarized
as a result of the photoreaction of the mutant domain (see
below). There is a notion of other polarized NMR signals in
the range between 120 and 140 ppm that are observed when
the sample is exposed to blue light but are absent in the dark.
These are of weaker intensity than the 110.6-ppm line and some
of them are in emission and others in absorption.
For a semiquantitative analysis of the nuclear-spin polarization
phenomenon observed in the light reaction of the LOV2 C450A
domain, additional NMR experiments were performed with
protein samples, in which the flavin cofactor was selectively
labeled with one or two 13C nuclei. The NMR-data analysis of
these samples is simpler due to the lack of 13C-13C nuclear
spin interactions that lead to multiplet signals in LOV2 C450A
containing universally labeled FMN. Furthermore, the polariza-
tions of the 13C NMR signals are easier to discern due to the
absence of spectral overlap observed for C(4a), C(5a), and C(9a)
as well as for C(8) and C(10a), and C(7R) and C(8R) in spectra
with protein containing [U-13C17]FMN.
Spectra recorded under illumination (“light” spectra) are
compared to the corresponding “dark” spectra in the right-hand
column of Figure 3. All “light” spectra have been scaled using
the same scaling factor that was used to compensate for the
decreasing amount of fully oxidized (diamagnetic) FMN present
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Nuclear-Spin Polarization in Phototropin
A R T I C L E S
under illumination conditions in the protein reconstituted with
[U-13C17]FMN. Whereas the chemical shifts of the individual
carbon atoms are not affected by illumination, the signal
intensities are strongly modulated in the “light” spectra as
compared to those obtained in the dark. From the signals due
to 13C atoms in the pyrimidine ring of the isoalloxazine moiety,
C(4) and C(4a) reverse their sign during sample irradiation, and
the signal of C(10a) becomes slightly attenuated as compared
to that obtained in the dark. Very strong polarization changes
are predominantly observed for carbons situated in the benzene
ring of the isoalloxazine moiety. Specifically, the signals of the
carbons C(6), C(8), and C(9a) change their sign from absorptive
to emissive. The carbons C(5a), C(9), and C(7) remain in
absorption but become slightly more intense. Only small changes
in the NMR signal amplitudes are observed for the absorptively
polarized carbons C(7R) and C(8R). Note, however, that in the
“light” spectrum of LOV2 C450A containing [U-13C17]FMN
the carbon C(8R) is emissively polarized thus suggesting that
cross-polarization21-23 which transfers magnetization from the
strongly emissive polarized 13C(8) to the directly adjacent
13C(8R) plays an important role in the uniformly 13C-labeled
sample. Such polarization transfer effects are negligible in
protein samples reconstituted with FMN isotopologs carrying
either only one 13C or two 13C nuclei that are not at close
distance.
Figure 4. Density-functional calculations of the unpaired electron-spin
density in a flavin anion radical, Fl•- (A), and a neutral radical, FlH• (B).
According to the radical-pair theory of photochemically
induced nuclear-spin polarization,24,25 the net polarizations of
the NMR lines depend on the electron-nuclear hyperfine
couplings which in turn are derived from the unpaired electron-
spin density on the individual halves of an intermediate radical
pair. Therefore, we performed quantum-chemical calculations
on a 7,8,10-trimethyl isoalloxazine radical, which serves as a
model for the FMN radical in LOV2. The truncation of the
ribityl side chain is justified as the spin densities on the carbons
C(2′), C(3′), C(4′), and C(5′) and consequently also their
hyperfine couplings are negligibly small. Two protonation states
have been considered: (i) the anion radical form, Fl•- which is
deprotonated at N(5) (see Figure 4A), and (ii) the neutral radical
form, FlH•, with a protonated N(5) atom (see Figure 4B). The
amplitudes of the unpaired electron-spin densities of Fl•- and
FlH• in the respective highest-occupied molecular orbital are
shown as iso-surfaces, where the blue and red colors represent
positive and negative electron-spin density, respectively. Overall,
both radical species have a closely similar electron-spin density
distribution: positive values are obtained for the carbons C(4),
C(4a), C(6), C(8), and C(9a), and negative values are obtained
for C(5a), C(7), and C(9). The observed differences at position
C(10a) are in agreement with recent quantum-chemical calcula-
tions:26,27 negative spin-density is observed in Fl•-, whereas a
positive value is obtained at the corresponding position in FlH•.
Furthermore, the negative spin densities at C(5a), C(7), and C(9)
are higher in amplitude in the anion radical when compared to
the neutral radical.
changes are observed when comparing “dark” and “light”
states: NMR signals that change the polarization from absorp-
tive to emissive or that are attenuated but still in absorption in
the presence of light have been assigned to atoms C(2), C(4),
C(4a), C(6), C(8), C(9a), and C(10a). Absorptive signals that
are enhanced originate from the atoms C(5a), C(7), and C(9).
According to Kaptein’s rules for the polarization of NMR lines,24
these two groups of nuclei must then have hyperfine couplings
of opposite sign. If we assume (in considering the results from
quantum-chemical calculations of hyperfine couplings)26,27 that
emissive and enhanced absorptive polarized NMR lines arise
from carbons with positive and negative hyperfine couplings,
respectively, we then obtain a hyperfine pattern that correlates
with the FMN anion radical, FMN•-, rather than with the neutral
FMN radical, FMNH•. Hence, we conclude that the nuclear spin-
dependent mixing of singlet and triplet states is more likely to
take place in the anionic rather than in the neutral radical state.
Discussion
Polarized NMR transitions are a signature of CIDNP, a
phenomenon which has been first observed more than 35 years
ago in products of photoinduced radical reactions.28,29 The term
CIDNP refers to non-Boltzmann nuclear spin-state distributions
detected as enhanced absorptive or emissive NMR signals. The
origin of CIDNP lies in the radical-pair mechanism which
postulates that if the outcome of a photochemical reaction
depends on the extent of singlet-triplet mixing in the radical-
pair intermediates and if this mixing is partly driven by the
electron-nuclear hyperfine interaction, then the reaction prod-
From analyses of the NMR spectra of LOV2 C450A samples
containing selectively 13C-labeled FMN, two types of amplitude
(21) Closs, G. L.; Czeropski, M. S. Chem. Phys. Lett. 1977, 45, 115-116.
(22) De Kanter, F. J. J.; Kaptein, R. Chem. Phys. Lett. 1979, 62, 421-426.
(23) Hore, P. J.; Egmond, M. R.; Edzes, H. T.; Kaptein, R. J. Magn. Reson.
1982, 49, 122-150.
(24) Kaptein, R. AdV. Free Rad. Chem. 1975, 5, 319-380.
(25) Closs, G. L. Chemically Induced Dynamic Nuclear Polarization; Academic
Press: New York, 1974; Vol. 7.
(26) Weber, S.; Mo¨bius, K.; Richter, G.; Kay, C. W. M. J. Am. Chem. Soc.
2001, 123, 3790-3798.
(27) Garc´ıa, J. I.; Medina, M.; Sancho, J.; Alonso, P. J.; Go´mez-Moreno, C.;
Mayoral, J. A.; Mart´ınez, J. I. J. Phys. Chem. A 2002, 106, 4729-4735.
(28) Bargon, J.; Fischer, H. Z. Naturforsch. 1967, 22A, 1556-1562.
(29) Ward, H. R.; Lawler, R. G. J. Am. Chem. Soc. 1967, 89, 5518-5519.
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A R T I C L E S
Richter et al.
ucts could have strongly polarized NMR lines.24,30-32 Despite
the fact that CIDNP was extended with great success to
biopolymers33,34 and is nowadays used as a technique to probe
the surface structure and folding of proteins using exogenous
photosensitizers to induce radical-pair reactions,34-36 photo-
CIDNP in integral protein-cofactor systems has so far only
been observed in photosynthetic reaction centers of bacteria37-39
and plants.40,41 These protein complexes, however, are so large
that solid-state NMR techniques such as magic-angle spinning
need to be applied to obtain narrow NMR signals. The details
of the mechanism of producing photo-CIDNP in the solid state
are currently under discussion.42,43 Although a variety of
flavoproteins were examined earlier in the search of nuclear
polarization effects, in no case they have been observed without
adding either “external” flavins, or redox-active amino acids
such as tryptophan or tyrosine.44
Nuclear-spin polarization in solution NMR arises from the
remarkable fact that the spin state of magnetic nuclei may
influence chemical reactivity. The spin-sorting process in a
radical-pair reaction does not necessarily need to be very
efficient to nevertheless lead to strong enhancements of NMR
intensities. A potential reaction mechanism for the origin of
nuclear polarization in the LOV2 C450A protein is required to
be consistent with our present findings as well as with recent
observations of an FMN radical in this protein: (i) Strong triplet
populations in wild-type and mutant LOV2 have been observed
by time-resolved optical spectroscopy8 and EPR.12 This is
indicative of a high quantum yield for triplet formation via ISC.
(ii) Compared to NMR spectra obtained from LOV2 C450A in
the dark, strongly polarized signals in enhanced absorption and
emission are observed when NMR spectra are recorded in the
presence of blue light. (iii) While the intensities of the individual
13C NMR signals of FMN in LOV2 C450A strongly depend
on the irradiation conditions, the chemical shift values of the
NMR resonances are unchanged when comparing “dark” and
“light” spectra. Therefore, we conclude that both the unpolarized
NMR lines recorded in the dark and the polarized NMR lines
obtained in the presence of blue light originate from 13C nuclei
in FMN of the same (diamagnetic) fully oxidized redox state.
(iv) If the integrated signal intensities of the carbons in the ribityl
side chain of FMN are taken as a measure for the amount of
fully oxidized FMN in [U-13C17]FMN-containing samples, then
a considerably decreased amount of FMN in this redox state is
detected under blue-light illumination. (v) In the “light” spectra,
Figure 5. Proposed cyclic reaction scheme for the production of photo-
CIDNP in LOV2 C450A. A is a redox-active amino acid side-chain in the
protein. R and â denote the nuclear-spin state(s) of a 13C nucleus in FMN
and A (for details, see text).
an additional strongly emissive NMR line at 110.6 ppm and
some more weakly polarized lines in the range between 120
and 140 ppm are observed. These signals cannot be assigned
to any of the carbons in the FMN chromophore and hence must
originate from spin-polarized 13C nuclei (occurring at natural
abundance) in the apoprotein. (vi) The observed NMR-intensity
modulations that distinguish the “dark” and “light” spectra
correlate with the electron-spin density distribution of an FMN
anion radical, FMN•-. On the other hand, a neutral radical
(FMNH•) in LOV2 C450A has been detected by optical
spectroscopy and EPR (see below).11 The lifetime of this radical
was determined to be in the range of several minutes to hours
depending on the presence or absence of oxygen and/or
exogenous reductants.
In Figure 5 we present a cyclic reaction scheme that is
consistent with the above-mentioned experimental observations.
This scheme originally proposed by Kaptein has previously been
applied to rationalize the production of nuclear-spin polarization
in the photosensitized reaction of exogenous FMN with surface-
exposed redox-active amino acid residues in proteins.33,35 The
ground-state FMN in the fully oxidized (diamagnetic, and hence
NMR-observable) redox state is promoted into its excited singlet
1
state, FMN, by absorption of blue light. This is followed by
3
ISC to the triplet state, FMN, which is generated with high
quantum yield. 3FMN is a potent oxidant for redox-active amino
acid residues within the LOV2 domain, and may thus abstract
an electron from a nearby redox-active amino acid residue
(species A in Figure 5) to form a geminate radical pair,
3[FMN•-‚‚‚A•+], in a spin-correlated triplet electron-spin con-
figuration. If the distance between the two radical-pair halves
is too close (less than about 1 nm), then the electron-electron
exchange interaction would be too large to permit triplet-to-
singlet interconversion. However, if the radicals are well
separated (and hence the exchange interaction, and in rigid
systems also the dipolar interaction, are comparable in size to
the isotropic hyperfine couplings), then hyperfine interactions
may induce triplet-to-singlet interconversion in the radical-pair
state. The initially generated triplet radical pair has then two
(30) Closs, G. L. J. Am. Chem. Soc. 1969, 91, 4552-4554.
(31) Lawler, R. G. J. Am. Chem. Soc. 1967, 89, 5519-5521.
(32) Lawler, R. G. Acc. Chem. Res. 1972, 5, 25-33.
(33) Hore, P. J.; Broadhurst, R. W. Prog. Nucl. Magn. Reson. Spectrosc. 1993,
25, 345-402.
(34) Hore, P. J.; Winder, S. L.; Roberts, C. H.; Dobson, C. M. J. Am. Chem.
Soc. 1997, 119, 5049-5050.
(35) Mok, K. H.; Hore, P. J. Methods 2004, 34, 75-87.
(36) Maeda, K.; Lyon, C. E.; Lopez, J. J.; Cemazar, M.; Dobson, C. M.; Hore,
P. J. J. Biomol. NMR 2000, 16, 235-244.
(37) Zysmilich, M. G.; McDermott, A. J. Am. Chem. Soc. 1994, 116, 8362-
8363.
(38) Zysmilich, M. G.; McDermott, A. Proc. Natl. Acad. Sci. U.S.A. 1996, 93,
6857-6860.
3
possible fates: (i) [FMN•-‚‚‚A•+] may either evolve into an
(39) Zysmilich, M. G.; McDermott, A. J. Am. Chem. Soc. 1996, 118, 5867-
5873.
1
electronic singlet state, [FMN•-‚‚‚A•+], which subsequently
(40) Alia; Roy, E.; Gast, P.; van Gorkom, H. J.; de Groot, H. J. M.; Jeschke,
G.; Matysik, J. J. Am. Chem. Soc. 2004, 126, 12819-12826.
(41) Matysik, J.; Alia; Gast, P.; van Gorkom, H. J.; Hoff, A. J.; de Groot, H. J.
M. Proc. Natl. Acad. Sci. USA 2000, 97, 9865-9870.
(42) Polenova, T.; McDermott, A. J. Phys. Chem. B 1999, 103, 535-548.
(43) Jeschke, G.; Matysik, J. Chem. Phys. 2003, 294, 239-255.
(44) Mu¨ller, F.; van Schagen, C. G.; Kaptein, R. Methods Enzymol. 1980, 66,
385-416.
may undergo spin-allowed back electron transfer to regenerate
the ground-state reactants (FMN and A), or (ii) so-called escape
products are formed directly from 3[FMN•-‚‚‚A•+] because
radical recombination is spin-forbidden from the triplet spin
configuration. If we assume that the frequency of the coherent
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Nuclear-Spin Polarization in Phototropin
A R T I C L E S
interconversion of singlet and triplet radical-pair states depends
on the spin state of the 13C nuclei in the FMN chromophore
via the electron-nuclear hyperfine interaction, and if we
(arbitrarily) further suppose that this interconversion is faster
when one (or several) 13C atoms are in a â-spin state (mI )
-1/2), then triplet radical pairs containing 13C in the â
configuration are more likely to cross over to the singlet state
and recombine (see Figure 5). On the other hand, triplet radical
pairs containing the same carbons in the opposite spin config-
uration (R, i.e., mI ) +1/2) are then more likely to yield
uncorrelated radicals as escape products. These may ultimately
also recombine to regenerate the same ground-state reactants,
FMN and A, thus leading to a cancellation of the opposite
recombination and escape nuclear polarizations unless the
recombination reaction of the free radicals is considerably slower
carbon that carries the highest (positive) electron-spin density
(0.556) in the neutral radical state.48 Using DFT, a large and
positive isotropic hyperfine-coupling constant of 1.58 mT with
pronounced dipolar anisotropy has been calculated for 13C(3).
More than 2-fold lower spin densities and hence smaller
hyperfine couplings have been calculated for the other carbons
in the tryptophan indole ring. Specifically, electron-spin densities
of -0.105, 0.194, 0.151, and 0.063, and isotropic hyperfine
coupling constants of -0.8 mT, 0.53 mT, 0.36 mT, and 0.02
mT, have been computed for C(3a), C(4), C(6), and C(7a),
respectively.48 In the high-magnetic-field limit (|∆g B0| . |Ai|,
where ∆g is the g-value difference g(FMN•-) - g(A•+), B0 is
the magnetic field, and Ai is the hyperfine coupling constant of
a nucleus i), the nuclear-spin polarization of a nucleus should
be roughly proportional to its isotropic hyperfine coupling
constant in the radical state. Hence, the NMR signals of C(3a),
C(4), C(6), and C(7a) are expected to show considerably weaker
nuclear-spin polarization than C(3) but should nevertheless be
observed in the “light” spectra. That the polarization of C(3) is
overwhelmingly strong could be due to differences in the nuclear
spin-lattice relaxation times of the various nuclei that determine
the degree of cancellation of recombination and escape polariza-
tions. Slow relaxation in the diamagnetic ground state in
combination with fast relaxation in the radical states of FMN
and the amino acid residue A leads to a reduced cancellation
of the opposite escape and recombination polarizations. The
quarternary carbons C(3), C(3a), and C(7a) of the aromatic side
chain of tryptophan exhibit long relaxation times in the
diamagnetic ground state of tryptophan as compared to the other
13C nuclei. In the radical state, however, the relaxation times
of these nuclei might be quite different. If nuclear-spin relaxation
is governed by of hyperfine anisotropies due to molecular
tumbling of A, then C(3) will have fast relaxation due to the
large anisotropy of its hyperfine coupling, whereas C(3a) and
C(7a) will show slower relaxation due to their smaller hyperfine
anisotropy. This will then lead to enhanced cancellation of
recombination and escape polarizations in C(3a) and C(7a) as
compared to C(3). In combination with the continuous sample
illumination, the large net polarization of C(3) in A could be
further enhanced through repetitive sample excitation leading
to a delicate balance of CIDNP pumping and decay.
In the LOV2 domain under examination, there are two
tryptophans, W491 and W557, which are both well separated
(>1.3 nm in a calculated structure of A. satiVa LOV2 C450A
based on Adiantum LOV249 as a model) from the FMN
chromophore thus fulfilling the requirements for the generation
of nuclear-spin polarization governed by nuclear hyperfine
interactions. Future point-mutational studies are aimed at an
unambiguous identification of the amino acid interacting with
FMN in a photoinitiated radical-pair mechanism.
To summarize, we have for the first time observed nuclear
polarization effects in an integral protein using solution 13C
NMR. From a prospective quantitative analysis of the polariza-
tion phenomenon under standardized illumination conditions,
valuable information on the electronic structure of paramagnetic
intermediate states in LOV domains will be obtained through
the nuclear-spin polarization that is detected in the diamagnetic
reaction products. We expect that this method is widely
1
than geminate recombination from [FMN•-‚‚‚A•+] as is typi-
cally the case. In LOV2 C450A, radical recombination takes
place on a time scale of minutes depending on the experimental
conditions.11 Hence, under the influence of the unpaired electron
spins, nuclear spin-lattice relaxation in the escape products is
expected to be faster than radical recombination, thus leading
to an appreciable reduction of the nuclear-spin polarization
generated via the 3[FMN•-‚‚‚A•+] channel. Consequently,
complete cancellation of the recombination and escape nuclear
polarizations is averted as a result of nuclear spin-lattice
relaxation in the one path leading to FMN and A via recom-
bination of the escape products. Note that translational diffusion
(an inherent property of the photo-CIDNP mechanism of
photoreactions of low-molecular-weight substances) of one or
both radicals of the radical-pair state is not required as a
prerequisite for the observation of NMR polarizations in integral-
cofactor proteins as long as the exchange and dipolar electron-
electron interactions in the precursor radical-pair state are not
too large, and two distinct pathways exist where the opposite
nuclear-spin polarizations resulting from singlet-triplet conver-
sion can progress differently. While the nuclear-spin polarization
phenomenon in LOV2 C450A can be described by the radical-
pair mechanism outlined above, we cannot presently exclude
that other mechanisms, such as a triplet mechanism,45 contribute
(at least in part) to the polarization of the NMR lines of FMN.
However, the observation of nuclear-spin polarized NMR
transitions derived from a counter radical (A•+ in Figure 5) leads
us to the conclusion that a triplet-based mechanism can only
play a minor role in the present system.
In the radical-pair reaction scheme described above, the
polarized NMR line at 110.6 ppm and the other more weakly
polarized lines in the 120-140-ppm range stem from a redox-
3
active amino acid, A, that acts as an electron donor to FMN.
In photo-CIDNP studies on the solvent accessibility of proteins
using exogenously added FMN as photosensitizer, typically
tyrosine, tryptophan or histidine radicals are observed.35,44 The
appearance of a signal with a chemical shift of 110.6 ppm in
our experiments is suggestive of a tryptophan residue, where
C(3) (IUPAC numbering scheme for the indole side group of
tryptophan; see Figure in Supporting Information) of the
aromatic side chain typically resonates at 109 to 112 ppm
downfield from TMS as has been shown for tryptophan in a
variety of proteins46,47 and model compounds.47 This is also the
(45) Stehlik, D. Excited States 1977, 3, 203-303.
(46) Eisenreich, W.; Bacher, A. J. Biol. Chem. 1991, 266, 23840-23849.
(47) Sun, H.; Oldfield, E. J. Am. Chem. Soc. 2004, 126, 4726-4734.
(48) Himo, F.; Eriksson, L. A. J. Phys. Chem. B 1997, 101, 9811-9819.
(49) Crosson, S.; Moffat, K. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 2995-
3000.
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A R T I C L E S
Richter et al.
applicable to other medium-sized flavoproteins as well, to
unravel electron-transfer pathways and to identify the redox
partners involved in the protein function.
Hans Fischer Gesellschaft e.V. for generous sponsoring of this
research work.
Supporting Information Available: Experimental details; one
Table with 13C NMR chemical shifts of free FMN, FMN bound
to AVena satiVa LOV2, and FMN bound to AVena satiVa LOV2
C450A; one Figure with IUPAC numbering scheme of tryp-
tophan; complete Ref. (3); complete Ref. (19). This material is
available free of charge via the Internet at http://pubs.acs.org.
Acknowledgment. We thank Professor Peter J. Hore (Uni-
versity of Oxford) for very helpful discussions and comments
on this manuscript. This study was supported by grants from
the Deutsche Forschungsgemeinschaft (SFB 498, project B7,
and SFB 533, project A5) and the Volkswagen Stiftung (grant
I/77100). We also thank Mrs. Ulrike Stier (Bad Soden) and the
JA053785N
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17252 J. AM. CHEM. SOC. VOL. 127, NO. 49, 2005