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

An exception is the more recently discovered subclass of the photolyase⁄ Keywords cryptochrome; DNA repair; ENDOR; EPR; ESR; flavoprotein; paramagnetic intermediates; photolyase; photore

New roles of flavoproteins in molecular cell biology:

Blue-light active flavoproteins studied by electron

paramagnetic resonance

Erik Schleicher1, Robert Bittl2 and Stefan Weber1

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) photolyasphotolyas-es Both enzymphotolyas-es 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.

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 tradiapplica-tionally 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

giso= 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, ge 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

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 vENDOR¼ vj N A=2j, either: (a) equally spaced

about the magnetic field-dependent nuclear Larmor

frequency, mN, and separated by the

(orientation-dependent) hyperfine coupling constant A (for the case

vN>jA=2j); or (b) centered around A ⁄ 2 and separated

by 2mN(for vN<jA=2j) 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].

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 (Ax6¼ Ay 6¼ Az) 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.

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)9s 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 ‘DMS¼ 2’ 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].

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 pKa 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.

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.

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.

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 (FADox) 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 FADox, 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

FADox 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 FADox 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¢) in X laevis (6-4) photolyase upon pH variations.

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 FADoxphotoreaction 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.