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 Schleicher1, Robert Bittl2 and Stefan Weber1 Institut fur Physikalische Chemie, Albert-Ludwigs-Universitat Freiburg, Germany ă ă Fachbereich Physik, Freie Universitat Berlin, Germany ă Keywords cryptochrome; DNA repair; ENDOR; EPR; ESR; avoprotein; paramagnetic intermediates; photolyase; photoreceptor; radical pair Correspondence S Weber, Institut fur Physikalische Chemie, ă Albert-Ludwigs-Universitat Freiburg, ă 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 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 provide 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 techniques to unravel important details, e.g the electronic structure or the protonation 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 photoreceptors that act in several species as central components of the circadian clock Introduction Ultraviolet light (k £ 300 nm) is known to induce the formation of covalent linkages between pairs of thymine 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 photolyases 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 chromophore, 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 ⁄ 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 E Schleicher et al cryptochrome protein family named cryptochromeDASH [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 magnetic 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 localization [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 illumination with light in the blue to green ranges of the electromagnetic spectrum, the excitation wavelength matching the optical absorption properties of the flavin cofactor in its physiologically relevant redox state or of the second so-called light-harvesting chromophore 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 resonance (ENDOR), by which NMR transitions are detected via EPR The results of such studies yield information on magnetic interactions within the radical, 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 situation 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 limits In such cases, transient EPR (TREPR), by which the formation and decay of paramagnetic species can be directly probed on a nanosecond time scale following pulsed light excitation, is the method of choice EPR and ENDOR investigations of flavoproteins In studies of paramagnetic flavin species, the application of EPR has traditionally been valuable to distin- Blue-light active flavoproteins guish the protonation state of flavin semiquinones by means of the signal width of its typical inhomogeneously 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 additional large hyperfine coupling from the H(5) proton of the 7,8-dimethyl isoalloxazine moiety [28,29] However, 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 possible based on the peak-to-peak EPR linewidth alone Therefore, recent studies have been targeted on precisely measuring the g-tensor of protein-bound flavin radicals in order to correlate this quantity to the chemical structure of flavin semiquinones [30–36] However, because the principal values of g deviate only marginally from the free-electron value, ge $ 2.00232, rather large magnetic fields and correspondingly high microwave frequencies are required to resolve the very small g anisotropies of flavin radicals With the recent availability 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 typically 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 For protein-bound flavin radicals, the g-tensor reflects the overall electronic structure on the redoxactive 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 neutral radical versus anion radical, may be distinguished [34] ENDOR spectroscopy is derived from EPR spectroscopy and is used routinely to determine the geometric 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 density at the positions of magnetic nuclei can be evaluated via the hyperfine coupling constant Several excellent review articles are available which provide detailed descriptions of the basics and the application FEBS Journal 276 (2009) 4290–4303 ª 2009 The Authors Journal compilation ª 2009 FEBS 4291 Blue-light active flavoproteins E Schleicher et al Fig 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 calculated EPR spectra are shown as solid and dashed lines, respectively of this technique for structure determination in paramagnetic 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 condition vENDOR ẳ jvN ặ A=2j, either: (a) equally spaced about the magnetic field-dependent nuclear Larmor frequency, mN, and separated by the (orientationdependent) hyperfine coupling constant A (for the case vN > jA=2j); or (b) centered around A ⁄ and separated by 2mN (for vN < jA=2j) Traditionally, ENDOR studies on flavoproteins have been performed using continuous-wave methodology [26,39,40] In recent years, however, pulsed ENDOR techniques (primarily based on the Davies pulse sequence) have become increasingly popular [28,35,36,41–45] In pulsed ENDOR experiments, the ENDOR signal is obtained by recording 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 distortion-less line shapes and is particularly useful when strongly anisotropic hyperfine interactions are to be 4292 Fig Principal values of the g-tensor of flavin anion and neutral radicals The values listed here were compiled from recent EPR experiments 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] FEBS Journal 276 (2009) 4290–4303 ª 2009 The Authors Journal compilation ª 2009 FEBS E Schleicher et al Fig A comparison of continuous-wave ENDOR with pulsed ENDOR E coli CPD photolyase was investigated with continuouswave ENDOR (upper spectrum) [39] and pulsed ENDOR spectroscopy [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 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 flavoprotein with the flavin cofactor in its neutral radical form are shown in Fig In general, the detected resonances can be grouped into five spectral regions between 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 radiofrequency ranges and arise from the hyperfine couplings of protons of the methyl group attached to Blue-light active flavoproteins 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 sensitively 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 (Ax 6¼ Ay 6¼ Az ) feature extending from 21 to 34 MHz in the pulsed ENDOR spectrum is assigned to the proton bound to N(5) [28,53] Its contribution to the overall 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 advantages of pulsed ENDOR over the conventional continuous-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 continuous-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 differences are the absence of the signal from the H(5) proton 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 electron-spin echo envelope modulation or hyperfine sublevel correlation spectroscopy, which are quite useful for studying specific hyperfine and quadrupolar couplings, 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 FEBS Journal 276 (2009) 4290–4303 ª 2009 The Authors Journal compilation ª 2009 FEBS 4293 Blue-light active flavoproteins E Schleicher et al A B C Fig ENDOR spectroscopy on neutral versus anionic flavin radicals Pulsed ENDOR spectra of the flavin radical in Aspergillus niger glucose oxidase obtained at pH 10 (upper, anionic radical) and pH (lower, neutral radical), recorded at 80 K [35] 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 altogether, 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 nanosecond time scale by a short laser flash or radiolysis pulse, which also serves as a trigger to start signal acquisition 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 variation of the signal intensity with respect to both the magnetic 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 modula4294 Fig Triplet and radical pair TREPR spectra of flavoproteins (A) Complete TREPR data set S(B0, 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 excitation, 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] 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 DMS ẳ ặ2 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 polarized) radical pair states are narrower because of reduced mutual dipolar and exchange interactions FEBS Journal 276 (2009) 4290–4303 ª 2009 The Authors Journal compilation ª 2009 FEBS E Schleicher et al 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 radical 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 characterize 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 overall 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 Blue-light active flavoproteins study, model geometries calculated on the basis of previously 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 conserved histidines [His354 and His358 in Xenopus laevis (6-4) photolyase] The 6¢-iminium ion then spontaneously rearranges to an oxetane intermediate by intramolecular nucleophilic attack [66] The oxetane species was proposed earlier in analogy to the repair mechanism of CPD photolyases, and because it was identified as an intermediate in the formation of 64PPs [64,69] This putative repair mechanism of (6-4) photolyases 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 intermediate, presumably via an electron-transfer mechanism 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 Putative reaction mechanism of (6-4) photolyase FEBS Journal 276 (2009) 4290–4303 ª 2009 The Authors Journal compilation ª 2009 FEBS 4295 Blue-light active flavoproteins E Schleicher et al which was irradiated and repaired without the participation 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 solventexposed 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 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 function of pH (Fig 7B), whereas the resonances of the other protons 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 individual 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 relaxation behavior of magnetic nuclei [71–73] Furthermore, changes in pH often cause small but distinct geometrical reorientations of protein side chains These structural 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 and 9.5 to identify the origin of the pH dependence of the principal values of the 4296 A B C Fig 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 different pH values: (blue curve), (green curve), (red curve), (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 H(8a) hyperfine tensor Both mutant enzymes are inactive 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 arising 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 FADHã The H(1Â) hyperne coupling tensor, however, is slightly rhombic, as predicted 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 By contrast, the signal intensity of H(8a) is pH dependent with an observed maximum at pH [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 FEBS Journal 276 (2009) 4290–4303 ª 2009 The Authors Journal compilation ª 2009 FEBS E Schleicher et al the His358Ala mutant, the His354Ala protein exhibits significant pH-dependent changes in its ENDOR spectra At pH 9.5, a substantial reduction in H(1¢) hyperfine 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 altering the pH Thus, replacement of His354 by alanine leads to significant modification of the cofactorbinding site at the 8a-methyl group and at the linkage of the ribityl side chain Hence, as a first result, structural information regarding the distance and location of the two histidines with respect to the flavin observer 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 wildtype 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 surprisingly 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 contains 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 proposed a new mechanism for repair of the 64PP lesion [75] However, in contrast to previously suggested reaction schemes, this mechanism does not involve an oxetane intermediate but electron transfer from the Blue-light active flavoproteins Fig 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 numbering scheme for X laevis is included in parenthesis flavin directly to the 64PP Protonation of the oneelectron 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 without 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 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 protonation state when going from pH 9.5 to Hence, the FEBS Journal 276 (2009) 4290–4303 ª 2009 The Authors Journal compilation ª 2009 FEBS 4297 Blue-light active flavoproteins E Schleicher et al Fig Proposed changes in the microenvironment of H(8a) and H(1¢) in X laevis (6-4) photolyase upon pH variations protonated histidine that is proposed to catalyze intermediate 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 experiments 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 radical, which is directly observable by time-resolved EPR [2,79] The specific amino acid involved in the photoreduction of FAD in Escherichia coli CPD photolyase was first identified by a comprehensive point-mutational 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 4298 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 substitutions of the surface-exposed tryptophan or the tryptophan 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 cryptochromes have been proposed to function as compasses for geomagnetic orientation in a large and taxonomically 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 reaction, 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 not block the radical pair’s singlet-to-triplet interconversion [23] Hence, understanding the suitability and potential of cryptochromes 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 lightinduced 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 conserved putative electron-transfer chain [86] In Fig 10, the TREPR signal of wild-type cryptochrome-DASH recorded at a physiologically relevant temperature is depicted in three dimensions as a function of the magnetic field and the time after pulsed laser excitation Positive and negative signals indicate enhanced absorptive and emissive electron-spin polarization 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 FEBS Journal 276 (2009) 4290–4303 ª 2009 The Authors Journal compilation ª 2009 FEBS E Schleicher et al 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 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 ls; a more precise determination is not possible because the exponential signal decay is influenced by spin relaxation of the electronspin polarization to the Boltzmann equilibrium population 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 Blue-light active flavoproteins photolyase) of the conserved electron-transfer cascade Under identical experimental conditions, the mutant protein did not exhibit any TREPR signal The conclusion, that Trp324 is the terminal electron donor in the light-induced electron-transfer reaction to the flavin chromophore in X laevis cryptochrome-DASH is supported 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 values for the g-tensors of both radicals were taken from high magnetic field ⁄ high microwave frequency examinations (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 interaction parameter J was taken into account Together with recent findings from optical spectroscopy on the FADox photoreaction of the related E coli CPD photolyase, 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 configuration 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 cryptochromes (at least of the DASH type) readily form radical 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 FEBS Journal 276 (2009) 4290–4303 ª 2009 The Authors Journal compilation ª 2009 FEBS 4299 Blue-light active flavoproteins E Schleicher et al Concluding remarks In recent years, a wealth of information on photolyase-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 photoinduced electron transfer Nevertheless, some key aspects of these reactions remain to be solved Important questions are: What are the precise differences between CPD photolyase and (6-4) photolyase regarding 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 decade(s) We are confident that application of modern EPR methods will make an important contribution to this Acknowledgements We thank our colleagues, collaborators, and coworkers, 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 Warwick), Gerhard Link (Albert-Ludwigs-University of Freiburg), Klaus Mobius (Free University Berlin), ă Asako Okafuji (Albert-Ludwigs-University of Freiburg), 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) References Friedberg EC, Walker GC, Siede W, Wood RD, Schultz RA & Ellenberger T (2006) DNA Repair and Mutagenesis, 2nd edn ASM Press, Washington, DC 4300 Weber S (2005) Light-driven enzymatic catalysis of DNA repair: a review of recent biophysical studies on photolyase Biochim Biophys Acta 1707, 1–23 Essen L-O (2006) Photolyases and cryptochromes: common mechanisms of DNA repair and light-driven signaling? 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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¢)