Photochemically Induced Dynamic Nuclear Polarization in a C450A Mutant of the LOV2 Domain of the Avena sativa Blue-Light Receptor Phototropin.

Full text

Page 1

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-selectively13C-labeled FMN and the13C 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-polarized13C-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.1,2The protein specified by the
phot1 gene3comprises a protein kinase domain and two
topologically similar domains carrying noncovalently bound
FMN chromophores.4Blue-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,4accompanied 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
(see Figure 1) on the basis of13C NMR spectroscopy.5A
subsequent X-ray diffraction structural analysis of the respective
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.6The photoadduct undergoes spontaneous
fragmentation in the dark with a rate constant of 2.55 × 10-2
s-1at room temperature.7
The flavin triplet state (3FMN) has been proposed as a reactive
intermediate in the primary blue-light induced reaction of LOV
domains.8More specifically,3FMN has been suggested to be
†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.
(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.
(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.
(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.
Figure 1. Photocycle of wild-type LOV domains. R denotes the ribityl
side chain of FMN.
Published on Web 11/18/2005
10.1021/ja053785n CCC: $30.25 © 2005 American Chemical Society
J. AM. CHEM. SOC. 2005, 127, 17245-17252 9 17245

Page 2

generated via intersystem crossing (ISC) from an excited singlet-
state precursor,9and to decay within a few microseconds by
generating the FMN-cysteinyl photoadduct.7The primary pho-
toreaction has been claimed to proceed (i) via an ionic
mechanism,8or (ii), in close analogy to the photooxidation of
amino acids by3FMN,10via a radical-pair mechanism with a
triplet-configured radical pair converting to a singlet-configured
radical pair as a precursor for covalent-bond formation.11,12A
wealth of information was obtained from LOV mutants in which
the functional cysteine was replaced by alanine, serine or
methionine.11,13,14The 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,7to 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.
Experimental Procedures
Materials. [U-13C17]Riboflavin was prepared by fermentation
of a riboflavin-producing Bacillus subtilis strain using [U-13C6]-
glucose as carbon source.15Other13C-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;5for 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.13C
NMR spectra were recorded at 125.8 MHz using a 5-mm1H/
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.
Composite pulse decoupling was used. All spectra were
recorded using a flip angle of 30°. The repetition rate was 3 s.
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 Gaussian03.19Two
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
Results
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.7After
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
recorded in the dark and under continuous blue-light irradiation
(see Figure 2) as described earlier.5NMR 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
13C-labeled FMN samples were
(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.
(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.
(17) van Schagen, C. G.; Mu ¨ller, F. Eur. J. Biochem. 1981, 120, 33-39.
(18) Dwyer, T. M.; Mortl, S.; Kemter, K.; Bacher, A.; Fauq, A.; Frerman, F. E.
Biochemistry 1999, 38, 9735-9745.
(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.
(20) Schaftenaar, G.; Noordik, J. H. J. Comput.-Aided Mol. Design 2000, 14,
123-134.
A R T I C L E S
Richter et al.
17246 J. AM. CHEM. SOC.9VOL. 127, NO. 49, 2005

Page 3

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.
In the NMR spectra of recombinant LOV2 C450A domains
recorded in the dark, most of the13C-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 two13C 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, all13C 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
neighboring13C atoms. Due to simultaneous coupling to two
adjacent13C 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 adjacent13C atoms. The multiplet signals of C(4a),
C(5a) and C(9a) overlap in the NMR spectra of the LOV2
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
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
Figure 2.
spectrum “light”-minus-“dark”.
13C NMR spectra of [U-13C17]FMN-reconstituted LOV2 C450A domain. (A) “Dark” spectrum. (B) Scaled “light” spectrum. (C) Difference
Nuclear-Spin Polarization in Phototropin
A R T I C L E S
J. AM. CHEM. SOC. 9 VOL. 127, NO. 49, 2005 17247

Page 4

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 a13C atom situated in the C450A apo-protein,
although the amino acid carbon atoms are not13C-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 two13C nuclei. The NMR-data analysis of
these samples is simpler due to the lack of13C-13C nuclear
spin interactions that lead to multiplet signals in LOV2 C450A
containing universally labeled FMN. Furthermore, the polariza-
tions of the13C 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
Figure 3.
[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.
13C NMR spectra of LOV2 C450A domains reconstituted (from top to bottom) with [4,10a-13C2]FMN (first row), [4a-13C1]FMN (second row),
A R T I C L E S
Richter et al.
17248 J. AM. CHEM. SOC.9VOL. 127, NO. 49, 2005

Page 5

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
to13C 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-polarization21-23which transfers magnetization from the
strongly emissive polarized
13C(8R) plays an important role in the uniformly13C-labeled
sample. Such polarization transfer effects are negligible in
protein samples reconstituted with FMN isotopologs carrying
either only one13C or two13C nuclei that are not at close
distance.
According to the radical-pair theory of photochemically
induced nuclear-spin polarization,24,25the 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:26,27negative 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.
From analyses of the NMR spectra of LOV2 C450A samples
containing selectively13C-labeled FMN, two types of amplitude
13C(8) to the directly adjacent
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)26,27that
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.28,29The 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-
(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) Garcı ´a, J. I.; Medina, M.; Sancho, J.; Alonso, P. J.; Go ´mez-Moreno, C.;
Mayoral, J. A.; Martı ´nez, 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.
Figure 4. Density-functional calculations of the unpaired electron-spin
density in a flavin anion radical, Fl•-(A), and a neutral radical, FlH•(B).
Nuclear-Spin Polarization in Phototropin
A R T I C L E S
J. AM. CHEM. SOC. 9 VOL. 127, NO. 49, 2005 17249