Báo cáo khoa học: New roles of flavoproteins in molecular cell biology: Blue-light active flavoproteins studied by electron paramagnetic resonance pptx

MINIREVIEW
New roles of flavoproteins in molecular cell biology:
Blue-light active flavoproteins studied by electron
paramagnetic resonance
Erik Schleicher
1
, Robert Bittl
2
and Stefan Weber
1
1 Institut fu
¨
r Physikalische Chemie, Albert-Ludwigs-Universita
¨
t Freiburg, Germany
2 Fachbereich Physik, Freie Universita
¨
t Berlin, Germany
Introduction
Ultraviolet light (k £ 300 nm) is known to induce the
formation of covalent linkages between pairs of thy-
mine and cytosine bases in cellular DNA. The most
common UV damages are cyclobutane pyrimidine
dimers (CPDs) and (6-4) photoproducts (64PPs)
generated from adjacent pyrimidine bases in single- or
double-stranded DNA [1]. These premutagenic lesions
change the conformation of DNA and consequently
can interfere with cellular transcription such that
transcription is arrested or coding mutations are
generated because of misreading of the DNA sequence.
Damage-specific DNA repair enzymes are able to

repair pyrimidine dimers; the most direct ones, the
DNA photolyase enzymes, operate by exploiting
longer wavelength light in the blue spectral region
[2–4]. Photolyases are subdivided into CPD photolyas-
es and (6-4) photolyases. Both enzymes are found in
various organisms, exhibit a 20–30% amino acid
sequence identity [5–7] and share a common chromo-
phore, FAD [8–11], although the two photolyases
differ in DNA substrate specificity and, in parts, in
their repair mechanism.
Cryptochromes are very similar in structure and
cofactor composition to photolyases but lack DNA
repair activity [12–14]. An exception is the more
recently discovered subclass of the photolyase⁄
Keywords
cryptochrome; DNA repair; ENDOR;
EPR; ESR; flavoprotein; paramagnetic
intermediates; photolyase; photoreceptor;
radical pair
Correspondence
S. Weber, Institut fu
¨
r Physikalische Chemie,
Albert-Ludwigs-Universita
¨
t Freiburg,
Albertstr. 21, 79104 Freiburg, Germany
Fax: +49 761 203 6222
Tel: +49 761 203 6213
E-mail: Stefan.Weber@physchem.

uni-freiburg.de
(Received 17 March 2009,
accepted 9 June 2009)
doi:10.1111/j.1742-4658.2009.07141.x
Exploring enzymatic mechanisms at a molecular level is one of the major
challenges in modern biophysics. Based on enzyme structure data, as
obtained by X-ray crystallography or NMR spectroscopy, one can suggest
how substrates and products bind for catalysis. However, from the 3D
structure alone it is very rarely possible to identify how intermediates are
formed and how they are interconverted. Molecular spectroscopy can pro-
vide such information and thus supplement our knowledge on the specific
enzymatic reaction under consideration. In the case of enzymatic processes
in which paramagnetic molecules play a role, EPR and related methods
such as electron-nuclear double resonance (ENDOR) are powerful tech-
niques to unravel important details, e.g. the electronic structure or the pro-
tonation state of the intermediate(s) carrying (the) unpaired electron
spin(s). Here, we review recent EPR ⁄ ENDOR studies of blue-light active
flavoproteins with emphasis on photolyases that catalyze the enzymatic
repair of UV damaged DNA, and on cryptochrome blue-light photorecep-
tors that act in several species as central components of the circadian
clock.
Abbreviations
64PP, (6-4) photoproducts; CPD, cyclobutane pyrimidine dimer; ENDOR, electron-nuclear double resonance; TREPR, transient EPR.
4290 FEBS Journal 276 (2009) 4290–4303 ª 2009 The Authors Journal compilation ª 2009 FEBS
cryptochrome protein family named cryptochrome-
DASH [15] which, to some extent, has repair activity
towards CPD lesions in single-stranded DNA [16,17].
Cryptochromes are blue-light receptors that regulate
the entrainment of circadian rhythms in animals
[18,19], and the regulation of growth and development

in plants [20]. They are also under consideration as
magnetoreceptor molecules in light-dependent mag-
netic sensing in a wide variety of living organisms [21].
The most well-studied example is the case of migratory
birds that use the earth’s magnetic field, as well as a
variety of other environmental cues, to find their way
during migration [22,23]. A feature that distinguishes
cryptochromes from photolyases is an extension of
variable length and sequence that interacts with other
proteins involved in intracellular signaling or localiza-
tion [24,25].
In photolyases and cryptochromes, radicals and
radical pairs play a prominent role in their function.
In nearly all cases, these are generated by illumina-
tion with light in the blue to green ranges of the
electromagnetic spectrum, the excitation wavelength
matching the optical absorption properties of the fla-
vin cofactor in its physiologically relevant redox state
or of the second so-called light-harvesting chromo-
phore which is used to enhance the quantum yield
of the photoreaction by increasing the extinction
coefficient in a certain spectral range [3]. Long-lived
paramagnetic states are favorably probed with EPR
techniques but also with electron-nuclear double res-
onance (ENDOR), by which NMR transitions are
detected via EPR. The results of such studies yield
information on magnetic interactions within the radi-
cal, or between radicals if these are not too far
apart. In some cases, the radical state of the flavin
cofactor can also be used as a probe to study its

immediate surroundings, which may be modulated in
terms of its micropolarity or hydrogen-bonding situa-
tion by the presence or absence of a substrate, such
as the pyrimidine dimer lesion in photolyases. When
very short-lived paramagnetic intermediates are to be
detected, stationary methods quickly reach their lim-
its. In such cases, transient EPR (TREPR), by which
the formation and decay of paramagnetic species can
be directly probed on a nanosecond time scale fol-
lowing pulsed light excitation, is the method of
choice.
EPR and ENDOR investigations of
flavoproteins
In studies of paramagnetic flavin species, the applica-
tion of EPR has traditionally been valuable to distin-
guish the protonation state of flavin semiquinones by
means of the signal width of its typical inhomoge-
neously broadened EPR resonance centered at
g
iso
= 2.0034 [26,27]. Anion flavin radicals (Fl
• –
) show
peak-to-peak linewidths (of the EPR signals in the first
derivative) of $ 1.2–1.5 mT, whereas neutral flavin
radicals (FlH
•
) exhibit significantly larger spectral
widths ($ 1.8–2.0 mT) because of the presence of addi-
tional large hyperfine coupling from the H(5) proton

of the 7,8-dimethyl isoalloxazine moiety [28,29]. How-
ever, because the variable hydrogen bonding strength
of surrounding amino acids to N(5) in Fl
• –
or from
NH(5) in FlH
•
contributes to the EPR signal width,
clear-cut assignment of a flavin semiquinone signal to
either a neutral or anion flavin radical is often not pos-
sible based on the peak-to-peak EPR linewidth alone.
Therefore, recent studies have been targeted on pre-
cisely measuring the g-tensor of protein-bound flavin
radicals in order to correlate this quantity to the chem-
ical structure of flavin semiquinones [30–36]. However,
because the principal values of g deviate only margin-
ally from the free-electron value, g
e
$ 2.00232, rather
large magnetic fields and correspondingly high micro-
wave frequencies are required to resolve the very small
g anisotropies of flavin radicals. With the recent avail-
ability of powerful EPR instrumentation operating at
high magnetic fields and high microwave frequencies,
it is now possible to perform precision measurements
that are not feasible at standard X-band frequencies
(9–10 GHz) where large hyperfine inhomogeneities typ-
ically obscure the g anisotropy. In Fig. 1, characteristic
high magnetic field ⁄ high microwave frequency EPR
spectra of various flavin radical species are depicted;

the ranges of typical g principal values are shown in
Fig. 2. For protein-bound flavin radicals, the g-tensor
reflects the overall electronic structure on the redox-
active isoalloxazine ring, and is thus potentially an
applicable property by which chemically different
flavin radicals, e.g. noncovalently versus covalently
bound at specific isoalloxazine ring positions, and neu-
tral radical versus anion radical, may be distinguished
[34].
ENDOR spectroscopy is derived from EPR spec-
troscopy and is used routinely to determine the geo-
metric and electronic structure of paramagnetic entities
by hyperfine interactions between nuclear magnetic
moments and the magnetic moment of the unpaired
electron spin. In most cases, these are too small to be
resolved in the EPR spectrum. The electron-spin den-
sity at the positions of magnetic nuclei can be evalu-
ated via the hyperfine coupling constant. Several
excellent review articles are available which provide
detailed descriptions of the basics and the application
E. Schleicher et al. Blue-light active flavoproteins
FEBS Journal 276 (2009) 4290–4303 ª 2009 The Authors Journal compilation ª 2009 FEBS 4291
of this technique for structure determination in para-
magnetic proteins and biomolecules [37,38]. In brief,
using ENDOR spectroscopy, hyperfine couplings of a
particular nucleus can be determined directly from
pairs of resonance lines that are, according to the con-
dition v
ENDOR
¼ v

N
Æ A=2
jj
, either: (a) equally spaced
about the magnetic field-dependent nuclear Larmor
frequency, m
N
, and separated by the (orientation-
dependent) hyperfine coupling constant A (for the case
v
N
> A=2
jj
); or (b) centered around A ⁄ 2 and separated
by 2m
N
(for v
N
< A=2
jj
). Traditionally, ENDOR studies
on flavoproteins have been performed using continu-
ous-wave methodology [26,39,40]. In recent years,
however, pulsed ENDOR techniques (primarily based
on the Davies pulse sequence) have become increas-
ingly popular [28,35,36,41–45]. In pulsed ENDOR
experiments, the ENDOR signal is obtained by record-
ing the echo intensity as a function of the frequency of
a radiofrequency pulse. Changes in the echo intensity
occur when the radiofrequency is on resonance with

an NMR transition, thus generating the ENDOR
response [38,46,47]. The pulsed methodology offers
many advantages over continuous-wave ENDOR, in
particular, when protein samples in frozen solution
with a dilute distribution of paramagnetic centers are
examined. Pulsed ENDOR generates practically distor-
tion-less line shapes and is particularly useful when
strongly anisotropic hyperfine interactions are to be
Fig. 1. High magnetic field ⁄ high microwave frequency continuous-wave EPR spectra (first derivatives) of various flavin radicals. Left,
360.04 GHz EPR spectrum of the stable anionic FAD radical of Aspergillus niger glucose oxidase (pH 10) recorded at 140 K [35]. Middle,
360.03 GHz EPR spectrum of the neutral FAD radical of E. coli CPD photolyase [30]. Right, 360.03 GHz EPR spectrum of the flavin radical of
a protein-bound FMN radical of the LOV1 domain (C57M mutant) of Chlamydomonas reinhardtii phototropin [34]. Experimental and calcu-
lated EPR spectra are shown as solid and dashed lines, respectively.
Fig. 2. Principal values of the g-tensor of flavin anion and neutral rad-
icals. The values listed here were compiled from recent EPR experi-
ments on flavoproteins using microwave frequencies of at least
90 GHz [30,31,33–36]. For the neutral FAD radical, the principal axes
X, Y and Z of the g-tensor were determined with respect to the
molecular axes of the 7,8-dimethyl isoalloxazine moiety [30,32].
Blue-light active flavoproteins E. Schleicher et al.
4292 FEBS Journal 276 (2009) 4290–4303 ª 2009 The Authors Journal compilation ª 2009 FEBS
measured [28]. Furthermore, in the pulsed mode, the
ENDOR intensity does not depend on a delicate
balance between electron and nuclear spin relaxation
rates and the applied microwave and radiofrequency
powers, unlike the continuous-wave technique. Its
implementation is therefore much simpler providing
the relaxation times are long enough.
Characteristic proton ENDOR spectra of a flavo-
protein with the flavin cofactor in its neutral radical

form are shown in Fig. 3. In general, the detected reso-
nances can be grouped into five spectral regions
between 1 and $ 37 MHz. (a) The central so-called
matrix-ENDOR signal extends from $ 13 to 16 MHz
and comprises hyperfine couplings from protons whose
nuclear spins are interacting only very weakly with the
unpaired electron spin, e.g. protons from the protein
backbone within the cofactor binding pocket, protons
of water molecules surrounding the flavin, and also
weakly coupled protons attached directly to the
7,8-dimethyl isoalloxazine ring, namely H(3), H(7a)
and H(9). (b) Prominent features of axial shape are
observed in the flanking 10–12 and 17–19 MHz radio-
frequency ranges and arise from the hyperfine
couplings of protons of the methyl group attached to
C(8). In general, signals of this tensor are easily
detected in proton-ENDOR spectroscopy on flavins
[31,48–51] and are considered to be sensitive probes of
the electron-spin density on the outer xylene ring of
the flavin isoalloxazine moiety. Furthermore, theory
shows that the size of this coupling responds sensi-
tively to polarity changes in the protein surroundings
[52]. (c) Flanking the H(8a) signals at $ 9–10 and
19–20 MHz are transitions belonging to one of the
two b-protons, H(1 ¢), attached to C(1¢) in the ribityl
side chain of the isoalloxazine ring [39]. (d) Signals
arising from hyperfine coupling of the H(6) proton
occur at $ 12 and 17 MHz. (e) The broad, rhombic
(A
x

6¼ A
y
6¼ A
z
) feature extending from 21 to 34 MHz
in the pulsed ENDOR spectrum is assigned to the pro-
ton bound to N(5) [28,53]. Its contribution to the over-
all spectrum is easily discriminated from that of other
protons in the isoalloxazine moiety because of the
exchangeability of H(5) with a deuteron upon buffer
deuteration. Observation of this very anisotropic
hyperfine coupling beautifully demonstrates the advan-
tages of pulsed ENDOR over the conventional con-
tinuous-wave methodology. In the latter, the first
derivative of the signal intensity (with respect to the
radiofrequency) is recorded, which becomes very small
when broad spectral features are to be measured, and
such couplings often escape direct detection in continu-
ous-wave ENDOR [28].
A flavin anion radical shows a markedly different
proton ENDOR spectrum compared with that of a
neutral radical (Fig. 4). The most pronounced differ-
ences are the absence of the signal from the H(5) pro-
ton and the larger splittings of the signal pairs arising
from H(8a) and H(6) in the anion radical case. Hence,
in addition to the g-tensor, the hyperfine pattern of a
flavin radical allows for unambiguous discrimination
of the radical’s protonation state [35,36].
With the commercial availability of pulsed EPR
instrumentation, other pulsed methods such as elec-

tron-spin echo envelope modulation or hyperfine sub-
level correlation spectroscopy, which are quite useful
for studying specific hyperfine and quadrupolar cou-
plings, have been applied to flavoproteins [51,54,55].
These studies have been reviewed recently [40].
Short-lived paramagnetic intermediates such as triplet
states or radical pairs generated during (photo-)chemical
reactions can be favorably studied by measuring the
EPR signal intensity as a function of time at a fixed
value of the external magnetic field [56,57]. Typically,
the best-possible time response of a commercial EPR
spectrometer that uses continuous-wave fixed-frequency
lock-in detection is in the order of $ 20 ls. By using
magnetic-field modulation frequencies higher than the
Fig. 3. A comparison of continuous-wave ENDOR with pulsed
ENDOR. E. coli CPD photolyase was investigated with continuous-
wave ENDOR (upper spectrum) [39] and pulsed ENDOR spectro-
scopy [28] (Please note that the upper spectrum is shown as the
first derivative of the signal intensity with respect to the radio
frequency.) Detectable protons are marked accordingly.
E. Schleicher et al. Blue-light active flavoproteins
FEBS Journal 276 (2009) 4290–4303 ª 2009 The Authors Journal compilation ª 2009 FEBS 4293
100 kHz usually employed in commercial instruments,
the time resolution can be increased by one order of
magnitude, which makes the method well suited to the
study of transient free radicals on a microsecond time
scale. By removing the magnetic-field modulation alto-
gether, the time resolution can be pushed into the 10
)8
–

10
)9
s range. A suitably fast data acquisition system
comprising a high-bandwidth microwave frequency
mixer read out by a fast transient recorder or a digital
oscilloscope is used to directly detect the transient EPR
signal as a function of time at a fixed magnetic field. In
TREPR, paramagnetic species are generated on a nano-
second time scale by a short laser flash or radiolysis
pulse, which also serves as a trigger to start signal acqui-
sition. Spectral information can be obtained from a
series of TREPR signals taken at magnetic-field points
covering the total spectral width. This yields a 2D varia-
tion of the signal intensity with respect to both the mag-
netic field and the time axis. Transient spectra can be
extracted from such a plot at any fixed time after the
laser pulse as slices parallel to the magnetic field axis.
In Fig. 5A, the 2D representation of the TREPR
signal from the photo-generated triplet state of FMN
is shown as a function of the magnetic field and the
time after pulsed laser excitation [58,59]. Because of
signal detection in the absence of any effect modula-
tion, the sign of the resonances directly reflects the
emissive or enhanced absorptive polarization of the
EPR transitions, which arises due to the generation
of the electron-spin state with an initial nonequilibrium
energy-level population [60–62]. The width of the
signal reflects the mutual interaction of the unpaired
electron spins in the triplet configuration. Because they
are both localized on the same isoalloxazine moiety,

the spin–spin interactions are quite strong and TREPR
spectra of flavin triplet states are therefore rather
broad [58,59]. The weak transition at low magnetic
fields represents the ‘DM
S
¼Æ2’ transition. In radical
pairs, the average distance between the two unpaired
electron spins is typically much larger. Hence, TREPR
spectra of photo-generated (and electron-spin polar-
ized) radical pair states are narrower because of
reduced mutual dipolar and exchange interactions
A
B
C
Fig. 5. Triplet and radical pair TREPR spectra of flavoproteins.
(A) Complete TREPR data set S(B
0
, t) of the photoexcited triplet
state of FMN in frozen aqueous solution measured at 80 K [58].
(B) TREPR spectrum of the photoexcited triplet state of FMN
extracted from the dataset in (A) at 500 ns after pulsed laser excita-
tion, for details see Kowalczyk et al. [58]. (C) TREPR spectrum of a
photogenerated radical pair comprising a flavin and a tryptophan
radical in E. coli CPD photolyase, measured at 274 K [2].
Fig. 4. ENDOR spectroscopy on neutral versus anionic flavin radi-
cals. Pulsed ENDOR spectra of the flavin radical in Aspergillus niger
glucose oxidase obtained at pH 10 (upper, anionic radical) and pH 5
(lower, neutral radical), recorded at 80 K [35].
Blue-light active flavoproteins E. Schleicher et al.
4294 FEBS Journal 276 (2009) 4290–4303 ª 2009 The Authors Journal compilation ª 2009 FEBS

compared with flavin triplets. This is shown in Fig. 5B,
where the TREPR signal of the FMN triplet state is
compared with that of a flavin-based radical pair in
photolyase (Fig. 5C) [2]. Analysis of the spectral
shapes of TREPR signals yields information on the
chemical nature of the individual radicals of the radi-
cal pair state, and their interaction with each other
and with their immediate surroundings.
EPR ⁄ ENDOR investigations of (6-4)
photolyase
Pulsed ENDOR has been favorably applied to charac-
terize the electronic structure of the FADH
•
cofactor
and its surroundings in (6-4) photolyase. For CPD
photolyase, the proposed repair mechanism includes a
photo-induced single electron-transfer step from the
fully reduced FAD cofactor (FADH
–
) to the CPD,
resulting in the formation of a CPD anion radical and
a neutral FADH
•
radical [63]. The cyclobutane ring of
the putative CPD radical then splits, and subsequently
the electron is believed to be transferred back to the
FADH
•
radical, thus restoring the initial redox states.
Hence, the entire process represents a true catalytic

cycle with net-zero exchanged electrons. By contrast,
(6-4) photolyases are not able to restore the original
bases from 64PP-damaged DNA in one reaction step;
rather, following binding of the DNA lesion, the over-
all repair reaction consists of at least two different
steps, one of which could be light independent,
whereas the other must be light dependent (Fig. 6)
[64–66]. Hitomi and coworkers [67,68] first proposed a
detailed reaction mechanism based on a mutational
study, model geometries calculated on the basis of pre-
viously published CPD photolyase coordinates, and
the important finding that the repair rate of (6-4)
photolyases strongly depends on the pH. In the initial
light-independent step, a 6¢-iminium ion intermediate is
generated from the 64PP aided by two highly con-
served histidines [His354 and His358 in Xenopus laevis
(6-4) photolyase]. The 6¢-iminium ion then spontane-
ously rearranges to an oxetane intermediate by intra-
molecular nucleophilic attack [66]. The oxetane species
was proposed earlier in analogy to the repair mecha-
nism of CPD photolyases, and because it was identi-
fied as an intermediate in the formation of 64PPs
[64,69]. This putative repair mechanism of (6-4) pho-
tolyases requires one histidine acting as a proton
acceptor and the other as a proton donor, which
implies that the two histidines should have markedly
different pK
a
values. However, until recently, it has
not been established which histidine can act as an acid

and which as a base. The subsequent blue-light-driven
(350 < k < 500 nm) reaction splits the oxetane inter-
mediate, presumably via an electron-transfer mecha-
nism similar to the one of CPD photolyases (Fig. 6).
Detailed information on the protonation states of the
two essential amino acids for 64PP repair is crucial for
a thorough understanding of the light-independent
catalytic steps preceding blue-light initiated enzymatic
DNA repair, and the specific structural traits that
distinguish (6-4) photolyase from the related CPD
photolyase.
Support for the oxetane-intermediate mechanism
came from a study with model compounds (a 64PP
containing a 3¢-thymine-4-methylthymine molecule),
Fig. 6. Putative reaction mechanism of (6-4) photolyase.
E. Schleicher et al. Blue-light active flavoproteins
FEBS Journal 276 (2009) 4290–4303 ª 2009 The Authors Journal compilation ª 2009 FEBS 4295
which was irradiated and repaired without the partici-
pation of an enzyme [70]. By careful assignment of the
intermediate structures, the authors concluded that an
oxetane intermediate in the 64PP repair reaction seems
most likely.
The ongoing discussion regarding the detailed repair
mechanism of (6-4) photolyases, the intermediates and
the involvement of functional relevant amino acids led
to the design of an ENDOR study [48] which is
reviewed briefly here. In general, because the function
of a histidine is markedly influenced by its protonation
state, it seems likely that the histidines at the solvent-
exposed active site cause the unusual pH dependence

of the (6-4) photolyase repair activity in vitro [67]. The
principal idea was that the protonation of a histidine
alters its polarity, which may be probed indirectly by
proton-ENDOR spectroscopy using the radical state
of the FAD cofactor as an observer. No 3D structure
of a (6-4) photolyase enzyme was available when these
experiments were performed.
Pulsed proton ENDOR spectra of wild-type
X. laevis (6-4) photolyase have been recorded over a
range of pH values [48]. The spectrum recorded at
pH 8 exhibits the characteristic hyperfine pattern of a
neutral flavin radical, FADH
•
(Fig. 7A). Sections of
the complete ENDOR spectrum (the radiofrequency
region in the 17.8–21.2 MHz range), where the H(8a)
and the H(1¢) protons resonate, are shown in
Fig. 7B,C. It is clearly shown that the intensity of the
H(8a) ENDOR signal changes significantly as a func-
tion of pH (Fig. 7B), whereas the resonances of the
other protons do not depend on pH (data not shown)
[48]. For a detailed data analysis it was taken into
account that the signal of H(8a) overlaps with the one
arising from H(1¢). Using spectral simulation, the indi-
vidual signal contributions of these protons could be
deconvoluted (Fig. 7C). Both the principal values of
the H(8a) hyperfine coupling tensor and the overall
signal intensity (data not shown) are affected by
changes in pH. This is not unexpected because it is
well documented that changes in the micropolarity or

pH of the surroundings of a paramagnetic molecule
may alter both the hyperfine couplings and the relaxa-
tion behavior of magnetic nuclei [71–73]. Furthermore,
changes in pH often cause small but distinct geometri-
cal reorientations of protein side chains. These struc-
tural changes may influence the free rotation of methyl
groups.
In further experiments, two mutant proteins in
which the two important histidines are individually
replaced by alanine (His354Ala and His358Ala) were
also examined at pH 6 and 9.5 to identify the origin of
the pH dependence of the principal values of the
H(8a) hyperfine tensor. Both mutant enzymes are inac-
tive in photorepair [67] but the flavin photoreduction
reaction and binding of the substrate are still possible.
Comparison of the ENDOR spectra for wild-type and
mutant enzymes at different pH revealed characteristic
differences in both the hyperfine principal values and
the signal intensities.
For the wild-type enzyme, the ENDOR signal aris-
ing from the H(8a) protons has axial symmetry, as
expected for a methyl group undergoing rapid (on the
timescale of the ENDOR experiment) rotation about
the C(8)–C(8a) bond in FADH
•
. The H(1¢) hyperfine
coupling tensor, however, is slightly rhombic, as pre-
dicted from quantum chemical calculations [74].
Within the bounds of experimental error, the principal
values of the hyperfine tensors of H(8a) and H(1 ¢)

remain constant from pH 9.5 to 6. By contrast, the
signal intensity of H(8a) is pH dependent with an
observed maximum at pH 7 [48]. The overall shapes
of the ENDOR spectra of the His358Ala mutant
protein largely resemble those of the wild-type at the
respective pH values. In contrast to the wild-type or
A
C B
Fig. 7. X-band frozen-solution pulsed ENDOR spectra of FADH
•
bound to wild-type X. laevis (6-4) photolyase. (A) Complete proton
ENDOR spectrum [48]. (B) Pulsed ENDOR spectra recorded at dif-
ferent pH values: 6 (blue curve), 7 (green curve), 8 (red curve),
9 (turquoise curve) and 9.5 (magenta curve). (C) Experimental (dots)
and simulated (dashed line) pulsed ENDOR spectra of wild-type
X. laevis (6-4) photolyase (pH 8.0). The red and blue curves show
the contributions of the H(8a) and H(1¢) hyperfine couplings to the
overall ENDOR spectrum.
Blue-light active flavoproteins E. Schleicher et al.
4296 FEBS Journal 276 (2009) 4290–4303 ª 2009 The Authors Journal compilation ª 2009 FEBS
the His358Ala mutant, the His354Ala protein exhibits
significant pH-dependent changes in its ENDOR spec-
tra. At pH 9.5, a substantial reduction in H(1¢) hyper-
fine coupling is observed accompanied by a clear
change in the symmetry (from axial to rhombic) of
the H(8a) signal. Furthermore, the principal hyperfine
values of both protons change significantly upon alter-
ing the pH. Thus, replacement of His354 by alanine
leads to significant modification of the cofactor-
binding site at the 8a-methyl group and at the linkage

of the ribityl side chain. Hence, as a first result, struc-
tural information regarding the distance and location
of the two histidines with respect to the flavin obser-
ver was obtained: the strong shift in the isotropic
hyperfine coupling of H(1¢) in His354Ala, compared
with the wild-type or His358Ala protein observed at
all measured pH values, suggested that His354 is close
to H(1¢). Slight geometrical reorientation because of
the histidine-to-alanine replacement results in an
altered direction of the C(1¢)–H bond with respect to
the p-plane of the isoalloxazine ring, thus changing
the dihedral angle, and hence the value of the H(1¢)
hyperfine coupling. However, the shift in the isotropic
hyperfine coupling of H(8a) with respect to the wild-
type was greater in the His358Ala mutant than in
His354Ala. From this finding, it could be concluded
that His358 is closer to the H(8a) protons than is
His354.
Very recently, the long-awaited crystal structures of
Drosophila melanogaster (6-4) photolyase in complex
with DNA containing a 64PP lesion, and in complex
with DNA after in situ repair have been presented [75].
The overall structure of the (6-4) photolyase looks sur-
prisingly similar to the previously published structures
from class I CPD photolyases [76]: the protein consists
of an a ⁄ b-domain and a FAD-binding domain. The
binding pocket, which is smaller but deeper than those
of CPD photolyases, is strictly hydrophobic and con-
tains conserved tryptophans, tyrosines and prolines.
This change in amino acid composition reflects the

altered geometry of the enzyme-bound 64PP and may
be an argument for an alternative repair mechanism.
The previously discussed two conserved histidines were
indeed located in the binding pocket of the substrate,
although only His354 was found to be in direct contact
with the 64PP lesion via hydrogen bonds (Fig. 8). The
lesion was flipped out of the double strand of the
DNA into the substrate-binding pocket by almost
180°. Based on their structural data, the authors pro-
posed a new mechanism for repair of the 64PP lesion
[75]. However, in contrast to previously suggested reac-
tion schemes, this mechanism does not involve an
oxetane intermediate but electron transfer from the
flavin directly to the 64PP. Protonation of the one-
electron reduced 64PP’s 5-OH group by the close-by
histidine then facilitates the elimination of water,
which subsequently attacks the acylimine molecule.
This intermediate is proposed to split into the two
thymines, and after back-electron transfer to the flavin
the intact bases are restored.
Comparing the results from ENDOR spectroscopy
with high resolution X-ray crystallographic data
(Fig. 8), the positions of the two histidines were
assigned surprisingly accurately by ENDOR. His365
(the analog amino acid of His354 in X. laevis)is
indeed located in the binding pocket nearby the H(1¢)
proton. Moreover, His369 (the analog amino acid of
His358 in X. laevis) is found to be closer to H(8a) than
to H(1¢ ). It should be mentioned, however, that all
ENDOR data have been collected from samples with-

out bound substrate. Therefore, changes in the binding
pocket (and in the structural alignment of the two
histidines) upon substrate binding cannot be ruled out.
As a second major result of the ENDOR study, the
H(8a) ENDOR signal in the His354Ala mutant was
also shown to be strongly pH dependent. This is likely
to originate from a change in protonation of His358
when going from pH 9.5 to 6. For steric reasons, it
was concluded that at pH 9.5 the (deprotonated)
His358 residue should move towards the smaller
Ala354 in the His354Ala mutant, which then affects
the axial symmetry of the hyperfine tensor. This
implies that His354 does not change its protona-
tion state when going from pH 9.5 to 6. Hence, the
Fig. 8. Binding pocket of D. melanogaster (6-4) photolyase. The 3D
structure of the active site of (6-4) photolyase including the 64PP
substrate and selected amino acids [75]. Please note that the num-
bering scheme for X. laevis is included in parenthesis.
E. Schleicher et al. Blue-light active flavoproteins
FEBS Journal 276 (2009) 4290–4303 ª 2009 The Authors Journal compilation ª 2009 FEBS 4297
protonated histidine that is proposed to catalyze inter-
mediate formation must be His354 because His358 is
deprotonated at pH 9.5 (Fig. 9) [48].
TREPR studies of reactive
paramagnetic intermediates in
cryptochrome
As in photolyases, redox reactions have been proposed
to play a key role in the light-responsive activities of
cryptochromes [77,78]. Both in vitro and in vivo experi-
ments suggest that the FAD redox state is changed

from fully oxidized (FAD
ox
) to the radical form when
it adopts the signaling state [44,45,77]. The results agree
with the redox activity of photolyases. In the latter,
when starting from FAD
ox
, photoinduced intraprotein
electron transfer produces a radical pair, comprising a
FAD radical and either a tyrosine or tryptophan radi-
cal, which is directly observable by time-resolved EPR
[2,79]. The specific amino acid involved in the photore-
duction of FAD in Escherichia coli CPD photolyase
was first identified by a comprehensive point-muta-
tional study in which each individual tryptophan of the
enzyme was replaced by phenylalanine [80]. Only the
Trp306Phe mutation abolished the photoreduction of
FAD
ox
or FADH
•
. Trp306 is situated at the enzyme
surface at a distance of $ 20 A
˚
from FAD [76].
However, this distance is too great for a rapid direct
electron transfer which is completed within 30 ps, as
determined recently using time-resolved optical
spectroscopy [81]. Hence, a chain of tryptophans
comprising Trp359 and Trp382 was postulated early-on

upon elucidation of the 3D structure [76] to provide an
efficient multistep electron-transfer pathway through
well-defined intermediates between Trp306 and the
FAD [82]. This chain of tryptophans is conserved
throughout all photolyases structurally characterized to
date and is also found in cryptochromes. Although the
relevance of this intraprotein electron transfer for
photolyase function is still under debate [83], the
cascade is believed to be critical for cryptochrome
signaling. For example, it has been shown that substitu-
tions of the surface-exposed tryptophan or the trypto-
phan proximal to FAD reduce in vivo photoreceptor
function of Arabidopsis cryptochrome-1 [84].
Radical pairs generated along the tryptophan chain
by light-induced electron transfer to FAD
ox
in crypto-
chromes have been proposed to function as compasses
for geomagnetic orientation in a large and taxonomi-
cally diverse group of organisms [85]. In principle,
a compass based on radical pair photochemistry
requires: (a) the generation of a spin-correlated radical
pair with coherent interconversion of its singlet and
triplet states in combination with a spin-selective reac-
tion, such as further ‘forward’ reactions that compete
with charge recombination, which regenerates the
ground-state reactants (the latter is only allowed for
the singlet radical pair but not the triplet radical pair
configuration); (b) modulation of the singlet-to-triplet
interconversion by Zeeman magnetic interactions of

the unpaired electron spins with the magnetic field; and
(c) sufficiently small inter-radical exchange and dipolar
interactions such that they do not block the radical
pair’s singlet-to-triplet interconversion [23]. Hence,
understanding the suitability and potential of crypto-
chromes for magnetoreception requires identification of
radical pair states and their origin, and the detailed
characterization of magnetic interaction parameters
and kinetics. TREPR with a time resolution of up to
10 ns allows real-time observation of such spin states
generated by pulsed laser excitation. Cryptochromes of
the DASH type are ideal paradigm systems for such
studies, because these proteins can be expressed from
diverse species, and are stable and available in the
amounts required for spectroscopic studies.
Recently, we presented a TREPR study of light-
induced paramagnetic intermediates from wild-type
cryptochrome-DASH of X. laevis and compared the
results with those from a mutant protein (Trp324Phe)
lacking the terminal tryptophan residue of the con-
served putative electron-transfer chain [86].
In Fig. 10, the TREPR signal of wild-type crypto-
chrome-DASH recorded at a physiologically relevant
temperature is depicted in three dimensions as a func-
tion of the magnetic field and the time after pulsed
laser excitation. Positive and negative signals indicate
enhanced absorptive and emissive electron-spin polari-
zation of the EPR transitions, respectively. The signal
is assigned to a radical pair based on its spectral shape
and narrow width (A spin polarized flavin triplet state

detected under comparable experimental conditions
Fig. 9. Proposed changes in the microenvironment of H(8a) and
H(1¢)inX. laevis (6-4) photolyase upon pH variations.
Blue-light active flavoproteins E. Schleicher et al.
4298 FEBS Journal 276 (2009) 4290–4303 ª 2009 The Authors Journal compilation ª 2009 FEBS
would span > 150 mT because of the large spin–spin
interactions between the two unpaired electrons, see
Fig. 5). Time evolution reveals that the radical pair
state exists for at least 6 ls; a more precise determina-
tion is not possible because the exponential signal
decay is influenced by spin relaxation of the electron-
spin polarization to the Boltzmann equilibrium popu-
lation. The spectrum of X. laevis cryptochrome-DASH
resembles those obtained recently from TREPR on
light-induced short-lived radical pair species in FAD
photoreduction of photolyases [2,79]. The origin of the
radical pair signal in cryptochrome-DASH could be
unraveled by examination of a single-point mutant,
Trp324Phe, which lacks the enzyme surface-exposed
tryptophan (equivalent to Trp306 in E. coli CPD
photolyase) of the conserved electron-transfer cascade.
Under identical experimental conditions, the mutant
protein did not exhibit any TREPR signal. The conclu-
sion, that Trp324 is the terminal electron donor in the
light-induced electron-transfer reaction to the flavin
chromophore in X. laevis cryptochrome-DASH is sup-
ported by spectral simulations, which were performed
on the basis of the correlated coupled radical pair
model and assuming the fixed orientations of the
Trp324

•
radical and the flavin radical given by the 3D
structure of the protein [86]. The strength of the dipolar
interaction between the two radicals was estimated
based on the point-dipole approximation, which yielded
D = )0.36 mT assuming an inter-radical distance of
$ 2.0 nm between Trp324
•
and FADH
•
. Principal val-
ues for the g-tensors of both radicals were taken from
high magnetic field ⁄ high microwave frequency examina-
tions (see above). However, a satisfactory simulation of
the TREPR signal of the Trp324
•
FAD
•
radical pair was
only obtained if a nonzero and positive exchange inter-
action parameter J was taken into account. Together
with recent findings from optical spectroscopy on the
FAD
ox
photoreaction of the related E. coli CPD photol-
yase, from which an electronic singlet precursor state of
Trp306
•
FAD
•

radical pair formation was confirmed
[87], a positive J value indicates that the triplet radical
pair configuration is favored by 2J over its singlet con-
figuration. Both, J and D are rather large compared with
the strength of the geomagnetic field, which is on the
order of $ 50 lT in Europe. Hence, the rather strong
radical–radical interactions may inhibit the magnetic
field dependence of singlet-to-triplet interconversion of
radical pair states, and hence, make radical pairs of the
type of Trp324
•
FAD
•
in cryptochromes unsuitable
as sensors for the earth’s magnetic field unless exchange
and dipolar interactions are of appropriate size and sign
for their effects to be approximately equal and opposite,
as recently suggested by Efimova & Hore [88].
The TREPR results clearly demonstrate that crypto-
chromes (at least of the DASH type) readily form radi-
cal pair species upon photoexcitation. Spin correlation
of such radical pair states (singlet versus triplet), which
is a necessary condition for the magnetoselectivity of
radical pair reactions, manifests itself as electron-spin
polarization of EPR transitions, which can be directly
detected by TREPR in real time. Such observations
support the conservation of photo-induced radical pair
reactions and their relevance among proteins of the
photolyase ⁄ cryptochrome family. The results are of
high relevance for studies of magnetosensors based on

radical pair (photo-)chemistry in general, and for the
assessment of the suitability of cryptochrome radical
pairs in animal magnetoreception in particular.
A
B
C
Fig. 10. TREPR spectrum of a photo-generated radical pair in
X. laevis cryptochrome-DASH. (A) Complete TREPR data set of
X. laevis cryptochrome-DASH measured at 274 K [86]. (B) TREPR
spectrum of wild-type (solid blue curve) and Trp324Phe (solid green
curve) X. laevis cryptochrome-DASH recorded 500 ns after pulsed
laser excitation. The dashed curve shows a spectral simulation of
the EPR data of the wild-type protein using parameters described
in the text and in Biskup et al. [86]. (C) The conserved tryptophan
triad of X. laevis cryptochrome-DASH.
E. Schleicher et al. Blue-light active flavoproteins
FEBS Journal 276 (2009) 4290–4303 ª 2009 The Authors Journal compilation ª 2009 FEBS 4299
Concluding remarks
In recent years, a wealth of information on photoly-
ase-mediated DNA repair and cryptochrome-mediated
blue-light responses has been obtained through the
combined efforts of biologists, chemists and physicists,
both from experimental and theoretical studies. Here,
we have chosen two recent examples of experimental
work from our group to demonstrate the potential of
modern EPR methods to answer mechanism-related
questions and to study reactive intermediates in photo-
induced electron transfer. Nevertheless, some key
aspects of these reactions remain to be solved. Impor-
tant questions are: What are the precise differences

between CPD photolyase and (6-4) photolyase regard-
ing substrate binding and DNA repair? Why are the
cryptochromes, despite their high protein-sequence
homology to (6-4) photolyases, incapable of repairing
UV-induced DNA lesions? Are cryptochromes capable
of sensing and transducing magnetic field information,
and if so, how is this task achieved in detail? Solving
these questions will be a challenge for the next dec-
ade(s). We are confident that application of modern
EPR methods will make an important contribution to
this.
Acknowledgements
We thank our colleagues, collaborators, and cowork-
ers, who contributed substantially to the work that is
reviewed here: Adelbert Bacher (Technical University
Munich), Till Biskup (Free University Berlin), Markus
Fischer (University of Hamburg), Martin Fuchs (SLS,
Paul-Scherrer-Institute Villigen), Elizabeth D. Getzoff
(Scripps Research Institute, La Jolla), Peter Hegemann
(Humboldt-University of Berlin), Kenichi Hitomi
(Scripps Research Institute, La Jolla), Monika Joshi
(University of Iowa), Chris Kay (University College
London), Radoslaw Kowalczyk (University of War-
wick), Gerhard Link (Albert-Ludwigs-University of
Freiburg), Klaus Mo
¨
bius (Free University Berlin),
Asako Okafuji (Albert-Ludwigs-University of Frei-
burg), Gerald Richter (Cardiff University), Alexander
Schnegg (Free University Berlin), and Takeshi Todo

(Osaka University). This work was supported by the
Deutsche Forschungsgemeinschaft (Sfb-498, projects
A2 and B7, and FOR-526).
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