Light-generated Paramagnetic Intermediates in BLUF Domains

Light-generated Paramagnetic Intermediates in BLUF Domains†

Stefan Weber1, Claudia Schroeder2, Sylwia Kacprzak1, Tilo Mathes3, Radoslaw M. Kowalczyk4§,Lars-Oliver Essen2, Peter Hegemann3, Erik Schleicher*1 and Robert Bittl4

1Albert-Ludwigs-Universitat Freiburg, Institut fur Physikalische Chemie, Freiburg, Germany2Philipps-Universitat Marburg, Fachbereich Chemie, Hans-Meerwein-Straße, Marburg, Germany3Humboldt-Universitat zu Berlin, Fachbereich Biologie, Institut fur Experimentelle Biophysik, Berlin, Germany4Freie Universitat Berlin, Fachbereich Physik, Institut fur Experimentalphysik, Berlin, Germany

Received 24 September 2010, accepted 20 December 2010, DOI: 10.1111/j.1751-1097.2010.00885.x

ABSTRACT

Blue-light sensitive photoreceptory BLUF domains are flavopro-

teins, which regulate various, mostly stress-related processes in

bacteria and eukaryotes. The photoreactivity of the flavin

adenine dinucleotide (FAD) cofactor in three BLUF domains

from Rhodobacter sphaeroides, Synechocystis sp. PCC 6803 and

Escherichia coli have been studied at low temperature using time-

resolved electron paramagnetic resonance. Photoinduced flavin

triplet states and radical-pair species have been detected on a

microsecond time scale. Differences in the electronic structures

of the FAD cofactors as reflected by altered zero-field splitting

parameters of the triplet states could be correlated with changes

in the amino-acid composition of the various BLUF domains’

cofactor binding pockets. For the generation of the light-induced,

spin-correlated radical-pair species in the BLUF domain from

Synechocystis sp. PCC 6803, a tyrosine residue near the flavin’s

isoalloxazine moiety plays a critical role.

INTRODUCTION

The ability to perceive light is pivotal for the survival of most

organisms, enabling them to adapt to changing environmentalconditions. For this purpose, nature has brought up at leasttwo major types of photoreceptors for different wavelength

regions of visible electromagnetic spectrum: red-light and blue-light photoreceptors. With the exception of the photoactiveyellow protein, all to date identified blue-light receptor classesuse either flavin adenine dinucleotide (FAD) or flavin mono-

nucleotide (FMN) as chromophores. Recent studies haveshown that there are three different classes of flavin-basedblue-light receptors (for recent reviews, see Refs. [1–4]):

(i) Phototropin, which comprises two FMN-binding LOVdomains (5,6) and one C-terminal serine ⁄ threonine kinase unit,mediates the blue-light signal for regulation of diverse physi-

ological responses to illumination, including phototropism,chloroplast relocation and stomatal opening. Upon blue-lightirradiation, phototropin undergoes a reversible photocycle

starting with the formation of an FMN–cysteinyl adduct(7–11) presumably via radical-pair (RP) intermediates (12,13).(ii) The FAD-binding domain of plant and mammalian

cryptochromes (14), which are involved in hypocotyl cellelongation, cotyledon ⁄ leaf expansion and flower elongation.Cryptochromes are also known for phasing the circadian clock

in Drosophila melanogaster (15) and mammals (16,17). Recentresults suggest the involvement of redox changes upon blue-light illumination, but their exact role in the proteins’ photo-

cycle remain to be elucidated (18–20). Finally, (iii) theblue-light sensors using FAD, BLUF (‘‘blue-light usingFAD’’), exemplified most recently in the N-terminus of the

AppA protein of the purple bacterium Rhodobacter sphaero-ides. In this organism, AppA acts as a transcriptional antire-pressor and interacts with the photosynthesis repressor proteinPpsR to form a stable AppA–(PpsR)2 complex in the dark and

under low-light conditions (21). The blue-light activated formof AppA can no longer associate with PpsR, and enables PpsRto bind to various promoters of photosynthetic genes to inhibit

their transcription (21,22).Amino acid sequence comparisons showed that AppA was

the first member of a new but widely occurring class of blue-

light photoreceptors, the so-called BLUF domains (23). Thisphotoreceptor class is found in a series of gene productsderived from proteobacteria, cyanobacteria and some eukary-otes (24). It also includes the PAC protein from the flagellate

Euglena gracilis, which utilizes blue light for phototaxis byregulation of its adenylate cyclase activity (25), the cyanobac-terial protein SyPixD (or also named Slr1694), which is a blue-

light receptor for the phototactic regulation of pili-dependentcell motility in Synechocystis sp. PCC 6803 (26), as well asYcgF acting as an antirepressor regulating biofilm formation

by Escherichia coli (27).Three-dimensional single-crystal and NMR structures are

now available for a number of BLUF proteins (28–35). Their

arrangement is unique among flavin-binding proteins, andbears a global similarity to the ferredoxin fold rather than toother photoreceptor modules. A photocycle in which veryshort-lived radical intermediates play a role in controlling the

antirepressor activity of AppA has been suggested; however,no defined structural changes upon light irradiation wereobserved so far (31,33,35).

Light irradiation of BLUF domains induces a small butcharacteristic redshift of the FAD absorption of about

*Corresponding author email: [email protected](Erik Schleicher)

†This paper is part of the Symposium-in-Print on ‘‘Blue Light Effects.’’§Current address: Radoslaw M. Kowalczyk, Department of Physics, Universityof Surrey, Guildford GU2 7XH, UK.

� 2011 The AuthorsPhotochemistry and Photobiology � 2011 The American Society of Photobiology 0031-8655/11

Photochemistry and Photobiology, 2011, 87: 574–583

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10–15 nm in the UV ⁄ visible region. This shift is spontaneouslyreversed in the dark. Lifetimes between a few seconds and upto several minutes have been determined for dark-staterecovery (36–38). The photocycle is unique as compared with

LOV and cryptochrome blue-light photoreceptors, in whichlight absorption induces pronounced changes in the spectro-scopic properties of the chromophores. Although the molec-

ular mechanism of BLUF photochemistry is still under debate(39–41), two conserved amino acids are believed to be essentialfor its light-driven reaction: a tyrosine and a glutamine; both

are located in direct proximity to the N(5) position of theFAD’s 7,8-dimethylisoalloxazine moiety. A reaction mecha-nism, in which a very short-lived biradical species comprising

an FAD radical and a tyrosine radical is generated uponoptical excitation of the flavin, was first proposed by Gaudenand coworkers (42). Radical formation is believed to drive arearrangement of the hydrogen-bonding network around N(5)

of FAD. This proposal is in line with previous FT-IR studies(38,43,44) that, upon illumination, revealed a strengthening ofthe hydrogen bond to O(4) of FAD. In contrast to LOV

domains, in which the triplet state of the FAD chromophore ispopulated prior to adduct formation, the BLUF reaction startsout predominantly from an excited singlet state of the FAD

(45). The light-induced reaction is completed within 1 ns,whereas the reversal of the dark state takes place on atimescale of several tens of seconds.

Despite the reported prominence of the singlet pathway,

flavin triplet initiated reaction channels have also beenobserved in a recent study, which has revealed that triplet-state precursors are similarly capable of inducing hydrogen-

bond network rearrangements in BLUF domains (46). To dateit is still unclear why flavin-triplet initiated photochemistry isprevalent in LOV domains but is only a minority in BLUF

domains, and why their relative contributions vary amongBLUF proteins from different organisms. It is the goal of thisstudy to shed some light on photogenerated triplet states in

BLUF domains, and to identify specific protein–cofactorinteractions that modulate their electronic wavefunctions. Tothis end, we utilized electron paramagnetic resonance (EPR), apowerful tool for identification and characterization of radi-

cals, RPs and other paramagnetic species including moleculartriplet states (47–49), to specifically identify paramagneticstates in BLUF domains. Such paramagnetic centers can be

used as probes for changes in their local surroundings.Time-resolved EPR spectroscopy (tr-EPR) with its temporal

resolution reaching up to 10 ns is the method of choice fordetecting very short-lived reaction intermediates (48,50). Inthis contribution we identify and characterize transient para-magnetic species generated by blue-light irradiation of various

BLUF domains. Our examination extends a recently publishedEPR study on BLUF domains by Nagai and coworkers, whohave characterized reaction intermediates under low-tempera-

ture steady-state conditions (51). By mutational analyses weidentify amino acid residues that are involved in fine-tuningthe electronic structure of the flavin chromophore. With this

contribution we hope to trigger additional experimentsexploiting other spectroscopic methods to corroborate ourobservations. Strong emphasis is given to the assignment of

protein–cofactor interactions capable of modulating BLUFphotochemistry.

MATERIALS AND METHODS

Construction of an AppA-overexpressing plasmid. Cloning, expressionand purification of AppA-BLUF samples were performed by proce-dures published previously (52,53).

Preparation of Slr1694. Wild-type (WT) Slr1694 and a Y8Wmutantwere cloned, expressed and purified as described previously (54,55).

Expression, reconstitution and purification of YcgF(1–137)-BLUFdomain. Cloning of the gene coding for YcgF (1–137) into pET36b(Novagen, Darmstadt, Germany) expression vector was performed asrecently described (56). Site-directed mutagenesis of E. coli YcgF(1–137) was performed using the Phusion Polymerase (Finnzymes, Espoo,Finland) and the primers listed in Table 1. The correctness of thesequences was verified by sequence analyses.

The YcgF-BLUF domain variants were transformed into theE. coli BL21(DE3)Gold strain and recombinantly produced in 2 L LBmedium containing 35 lg mL)1 kanamycin at 37�C. After inductionwith 1 mMM IPTG at OD595 0.5–0.6, cells were harvested after 2 hgrowing and resuspended in buffer I (20 mMM Tris ⁄HCl pH 8.0,200 mMM NaCl). The inclusion bodies were purified, refolded andreconstituted with FAD and FMN using previously described proto-cols (56). The solution comprising the respective YcgF-BLUF domainwas concentrated with centrifuge filter devices (Millipore, Billerica,MA). The addition of glycerol reduces protein aggregation of theYcgF-BLUF domain mutants. Subsequently, the YcgF-BLUFdomain variants were purified by gel filtration using a Superdex 200XK 16 ⁄ 70 column (GE Healthcare, Amersham, UK) equilibrated withbuffer I, concentrated again using centrifuge filter devices (Millipore,Billerica, MA), and then stored at )20�C in the presence of 10–15%glycerol.

EPR sample preparation. All recombinant BLUF domains wereinitially isolated in buffer containing 10 mMM sodium phosphate and10 mMM NaCl. Prior to the EPR measurements, glycerol was added at afinal concentration of 60% (v ⁄ v) to yield a transparent glass in thefrozen state. The concentrations of the protein samples were adjusted

Table 1. A, Oligonucleotides used for site-directed mutagenesis of Escherichia coli YcgF BLUF domain (mismatched bases are printed in boldfaceand introduced restrictions sites are underlined). B, Oligonucleotides used for site-directed mutagenesis of Slr-BLUF (mismatched bases are printedin boldface).

A YcgF-BLUFOligonucleotide Sequence (5¢ fi 3¢)M23I_fw CCTGTCAAAAAAATCGAAGAAATCGTTTCGATCGCAAATCGCAGGM23I_rv CCTGCGATTTGCGATCGAAACGATTTCTTCGATTTTTTTGACAGGM23L_fw GTCAAAAAAATCGAAGAACTGGTTTCGATCGCAAATCGCAGGM23L_rv CCTGCGATTTGCGATCGAAACCAGTTCTTCGATTTTTTTGAC

B Slr-BLUFOligonucleotide Sequence (5¢ fi 3¢)Y8W_fw GTTTGTACCGTTTGATTTGGAGCAGTCAGGGCATTCCCY8W_rv GGGAATGCCCTGACTGCTCCAAATCAAACGGTACAAAC

Photochemistry and Photobiology, 2011, 87 575

to �0.7 mMM using 10 kDa centrifugal concentrators (Millipore).Protein concentrations were controlled by optical absorption spec-troscopy using an optical spectrophotometer (Shimadzu, Kyoto,Japan). All samples were then deoxygenated, transferred into EPRsynthetic-quartz tubes (3 mm inner diameter) under an argon atmo-sphere and rapidly frozen in liquid nitrogen in the dark.

EPR instrumentation. Tr-EPR experiments were performed using alaboratory-built spectrometer described elsewhere (12). Pulsed opticalsample excitation was performed using a Spectra Physics GCR-11Nd:YAG laser (Spectra Physics, Irvine, CA) pumping an opticalparametric oscillator Opta BBO-355-vis ⁄ IR (Opta GmbH, Bensheim,Germany) tuned to a wavelength of 460 nm (pulse width, 6 ns; pulseenergy, 4 mJ).

Basics of transient EPR spectroscopy. In contrast to conventionalcontinuous-wave EPR spectroscopy, tr-EPR is recorded in a ‘‘direct-detection mode’’ (without modulation of the external magnetic field) soas not to constrain the time resolution of the experiment. Conse-quently, positive and negative tr-EPR signals indicate enhancedabsorptive (A) and emissive (E) electron-spin polarization of theEPR transitions, respectively. tr-EPR spectra can be obtained at anyfixed time after pulsed laser excitation. Following published proce-dures, the tr-EPR spectra of all examined BLUF domains have beencorrected to compensate for slow sample degradation or alteration as aresult of prolonged sample irradiation (57).

In general, each spectrum consists of a narrow emissive featurecentered at around 168.5 mT (corresponding to g � 4), and a broadsignal centered at about 348 mT (g � 2), spanning roughly 130 mTwith emissive electron-spin polarization below and enhanced absorp-tive electron-spin polarization above g � 2 respectively. The overallwidths of the EPR spectra, the spectral positions of extreme andinflection points, and the electron-spin polarization patterns arecharacteristic for photoexcited flavin triplet states generated byintersystem crossing (ISC) from excited singlet-state precursors (57).The narrow emissive signals at 168.5 mT are the so-called half-fieldresonances arising from formal ‘‘DMS = ±2’’ transitions (58). Inprinciple, an EPR experiment with sufficiently high time resolutionreveals the full electron-spin polarization of all transitions between thenon-Boltzmann populated (hence, electron-spin polarized) energylevels. Thus, a characteristic spectral powder pattern is observed, inwhich the DMS = ±1 transitions at magnetic-field positions, corre-sponding to canonical orientations of the dipolar coupling tensor(principal axes X, Y and Z) with respect to the direction of the externalmagnetic field, are separated by |D¢| + 3|E¢|, |D¢| ) 3|E¢| and 2|D¢|(with |D¢| = |D| hc ⁄ (gbe) and |E¢| = |E| hc ⁄ (gbe), where D and E arethe zero-field splitting (ZFS) parameters in units of cm)1, be is the Bohrmagneton and h and c are Planck’s constant and the vacuum speed oflight, respectively), and centered about the g-value of the flavin tripletstate.

Simulation of EPR spectra. Electron paramagnetic resonancepowder spectra have been analyzed using a home-written programfor simulation and fitting of spectra with isotropic g-factor anddipolar-coupling tensor D as described previously (57).

RESULTS AND DISCUSSION

Detection of photoexcited triplet states in BLUF domains

UV ⁄ vis spectral features of flavin triplet states in the 600–850 nm range are typically broad and unresolved, thus makingit difficult to distinguish them from flavin radicals. By tr-EPR

spectroscopy, on the other hand, flavin triplets can be easilyidentified by means of their characteristic spectral pattern,which is dominated by the strong mutual dipolar interaction

between the formally two unpaired electron spins that are bothsituated on the 7,8-dimethylisoalloxazine moiety of the flavin(47). By spectral simulation, the ZFS parameters that param-

eterize the dipole–dipole interaction tensor can be extracted(see below). With these values at hand, the electronic structureof the excited flavin chromophore and its modulation by thedifferent surrounding amino acids in the various blue-light

active flavoproteins can be characterized.

Representative proteins from rhodobacteraceae, cyanobac-teria and enterobacteriaceae were chosen to study the extent ofdiversity of photochemical behavior in BLUF domains and toassign structure–function relationships as protein-structure

information is available for all investigated samples. Specifi-cally, BLUF domains from (i) the AppA protein of Rb.sphaeroides (AppA-BLUF), (ii) the Slr1694 protein of Syn-

echocystis sp. (Slr-BLUF) and (iii) the YcgF BLUF protein ofE. coli (reconstituted with either FAD or FMN) (YcgF-BLUF)were studied using tr-EPR in frozen aqueous solution

(T = 80 K).Because in BLUF domains the back reactions from the

signaling states to the ground states are typically too slow for

repetitive photocycling, which is required to achieve a suffi-ciently high signal-to-noise ratio, we decided to block light-induced reactions by lowering the temperature well below thefreezing point of the solvent. Under such conditions, major

structural alterations of the flavin chromophore and neigh-boring amino acids are arrested, allowing us to focus ontriplet-state generation and its deactivation mediated by the

specific amino acid surroundings.Samples were either frozen after dark adaption to investi-

gate the dark state of the photoreceptor, or after 5 min of blue-

light illumination under continuous optical irradiation toinvestigate the signaling state. As a representative example, thetr-EPR spectrum of YcgF-BLUF is shown in Fig. 1 in a three-dimensional representation of the EPR signal amplitude as a

function of the external magnetic field B0, and the time t afterpulsed laser excitation (460 nm).

Triplet tr-EPR spectra reported on various light-excited

phototropin LOV domains showed similar overall electron-spin polarization and ZFS parameters. Significant differenceswere only observed in the intensity ratios at the canonical

orientations (12). In contrast, BLUF domains exhibit muchhigher spectral diversity. While the tr-EPR spectrum of theAppA-BLUF protein (see Fig. 2, upper panel) is very similar

to that of the LOV2 domain from Avena sativa (12), tripletspectra obtained from the YgcF-BLUF protein show acompletely different electron-spin polarization pattern (seeFig. 2, middle panel). To complicate things further, spectra

recorded from the Slr-BLUF protein exhibit an even more

Figure 1. Complete tr-EPR data set of the BLUF domain from YcgF-BLUF measured at 80 K. Each time profile is the average of 25acquisitions recorded with a laser-pulse repetition rate of 1.25 Hz, at amicrowave frequency of 9.68 GHz, a microwave power of 2 mW and adetection bandwidth of 100 MHz. Tr-EPR spectra were generated byintegration of EPR time profiles over a time window of 500 ns centeredat the peak amplitude at around 1 ls after pulsed laser excitation.

576 Stefan Weber et al.

complex spectral shape at g � 2 (see Fig. 2, lower panel). To

rationalize these differences, spectral simulations of flavin-triplet-state tr-EPR spectra have been performed. From least-squares fittings of calculated to experimental data, the ZFS

parameters, |D| and |E|, and the zero-field populations, AX, AY

and AZ, of the three zero-field triplet sublevels were obtained(57). For the three different BLUF domains, these arecompiled in Table 2 and compared with the corresponding

values obtained under similar experimental conditions fromFAD in aqueous solution (57) and from FMN bound to LOVdomains (12).

In general, the ZFS parameters are related to the overalldistribution of the two-center spin contributions from a tripletstate. TheD value allows an estimate of the amount aswell as the

geometrical shape of the electron-spin distribution. Therefore,largerD values correspond to a less delocalized triplet state (forplanar aromatic molecules, D is assumed positive according tothe convention defined in Ref. [59]). The size of the ZFS

parameter E permits an evaluation of the in-plane spin anisot-ropy; the sign of E, however, is not readily understood.

The triplet parameters obtained for AppA-BLUF resemblethose from A. sativa LOV2 obtained in a previous study. The

differences for |D| are close to the experimental error;differences for |E| are insignificant. Only the values for thezero-field populations vary to some extent: A slightly stronger

population of AY is obtained for AppA-BLUF, although thegeneral trend of population probabilities is quite similar: BothLOV domains and AppA-BLUF show relative zero-field

populations AX and AY which are reversed as compared withthose of FAD in frozen aqueous solution (57). This indicatesthat the symmetry of the triplet state is altered such that thesign of E is reversed as compared with that of ‘‘free’’ FAD.

Completely different triplet parameters are obtained fromsimulations of tr-EPR spectra of YcgF-BLUF samples (seeTable 2). As a first result, the ZFS parameters |D| and |E|, as

well as the zero-field populations are identical (within theexperimental error) for YcgF-BLUF domains binding eitherFMN or FAD. This is consistent with the proposal that the

adenine ring of FAD is electronically decoupled from thephotoactive isoalloxazine ring (33). Compared with valuesfrom AppA-BLUF, the |D| and |E| parameters obtained for

YcgF-BLUF are significantly smaller (550 · 10)4 cm)1 and157 · 10)4 cm)1 in YcgF-BLUF as compared to 584 ·10)4 cm)1 and 187 · 10)4 cm)1 in AppA-BLUF, for |D| and|E|, respectively). This reflects a more delocalized flavin triplet

state in YcgF-BLUF domains. Moreover, the populations ofthe three zero-field triplet sublevels are completely different: Incontrast to all flavin triplet states reported so far, the

population of AX is zero, whereas AZ carries nearly 50% ofthe population. The significantly modified triplet populationsare a consequence of changes in the specific ISC rates from the

photoexcited singlet state to the three triplet sublevels. A likelysource for this observation is a weak noncovalent protein–cofactor interaction in YcgF-BLUF domains. Specifically,

interactions of a sulfur atom such as from a cysteine or amethionine residue with an aromatic chromophore are wellknown to influence spin-orbit coupling of the latter, and

Figure 2. Tr-EPR spectrum of wild-type BLUF domains recorded1 ls after pulsed laser excitation. Other experimental parameters are asspecified in Fig. 1. Upper panel: dark-adapted and blue–light-adaptedAppA-BLUF protein. The dashed curve represents a spectral simula-tion of the dark-state sample. Middle panel: dark and blue-lightilluminated YcgF-BLUF samples. The dashed curve represents aspectral simulation of the dark-state sample; the dotted line representsan FMN-exchanged dark-state sample. Lower panel: dark and blue-light illuminated Slr-BLUF protein. The dashed curve represents aspectral simulation of the dark-state sample. For details, see text.

Table 2. Triplet parameters of flavin cofactors bound to variousBLUF domains obtained by simulations of tr-EPR spectra depicted inFigs. 2 and 3, in comparison with published data for FAD in frozenaqueous solution and for FMN in LOV domains of phototropin. Thevalues of |D| and |E| are accurate to within ±6 · 10)4 cm)1; the zero-field populations Ai within ±0.03. Please note that the Slr-BLUFspectra could not be simulated with an automated fitting routine; thevalues for |D| and |E| were obtained by manual simulation of thespectra (error margin of |D| and |E|: ±1 · 10)3 cm)1), but the zero-field populations could not be determined.

|D|(10)4 cm)1)

|E|(10)4 cm)1) AX AY AZ

FAD 559 158 0.39 0.61 0Avena sativa LOV2 570 181 0.62 0.38 0Chlamydomonasreinhardtii LOV1

581 167 0.68 0.38 0

AppA-BLUF 584 187 0.57 0.43 0YcgF-BLUF (FAD) 550 157 0 0.54 0.46YcgF-BLUF (FMN) 552 159 0 0.55 0.45YcgF-BLUF M23L 573 193 0.51 0.27 0.22YcgF-BLUF M23I 575 194 0.44 0.28 0.27Slr-BLUF* 586 167 n.d. n.d. n.d.

*Data were obtained by manual fitting.


consequently, modulate ISC rates. This is called the ‘‘heavy

atom effect.’’

Methionine mutants in YcgF-BLUF domains

We expect that the immediate surroundings of the isoalloxa-zine moiety are responsible for the significant differences

between the YgcF-BLUF and the other BLUF domains.Therefore, we analyzed a multiple amino acid sequencealignment of all investigated BLUF proteins, see Fig. 3.

Within a 0.5 nm distance to the isoalloxazine ring, theBLUF domains only differ in four amino acid positions (theamino acid numbering scheme in Fig. 3 is for AppA-BLUF).

Isoleucine-37 is mostly conserved, but in YcgF-BLUF and afew other BLUF domains it is exchanged to a methionine (24).The highly polarizable sulfur atom of methionine is located ina 0.4 nm distance to the xylene ring of FAD. This amino acid

could in principle alter the symmetry of the triplet state due tosulfur–aromatic interactions. If so, one would expect astronger delocalization of the triplet state resulting in a smaller

D value and modified sublevel population rates. This is indeedobserved, see Table 2. The second replacement, a serine-to-alanine variation at position 41, is not expected to cause any

significant alterations of the wave function of the flavin tripletstate. The residue at position 44, in hydrogen-bonding distance(0.33 nm) to the O(2) atom of FAD, is rather poorly

conserved. This position should be capable of altering theelectron-spin distribution of the pyrimidine ring of the flavin;however, the overall spin density at position O(2) waspredicted to be rather low (60). The same holds true for

position 75, which is located above the pyrimidine ring, andwhich is also not conserved in BLUF domains. Anotheraromatic amino acid (near N(5) of the isoalloxazine moiety),

W104 in AppA-BLUF, has been a matter of discussion in therecent literature (31,32). This residue is conserved in mostBLUF domains. A mechanism for signal propagation was

proposed that involves ‘‘flipping’’ of W104 with the conservedH105. The proposal was based on the observation of differentconformations of this amino acid in crystal structures (28,31).

In YcgF-BLUF, instead of an aromatic amino acid an alanineresidue is observed at the corresponding position. Independentof the putative ‘‘flipping’’ motion, the more proximal aminoacid is either an alanine or a conserved glycine in YcgF-BLUF

(position 91). Therefore, we do not expect major interactionsof this residue with the flavin’s triplet state. In summary, theseamino acid variations are rather conservative, and thus, are

not expected to account for the spectral differences observedby tr-EPR. Only the methionine residue in YcgF-BLUF seems

to be a plausible candidate for the delocalization of the triplet-state wave function and, due to the sulfur heavy-atom effect,for altered ISC rates.

To corroborate the influence of the methionine residue, amutational analysis has been performed: Two mutant proteins,YcgF-BLUF M23I and M23L, in which the methionine is

replaced by either isoleucine or leucine, have been constructed,overexpressed and purified. In Fig. 4, the tr-EPR spectra of thetwo mutant proteins are compared with that of the WT. It

should be noted, however, that both mutants exhibit only

○ ○ ○ ○○ ○ ○ ○ ○AppA BLUF 16 LVSCCYRSLAAPDLTLRDLLDIVETSQAHNARAQLTGALFYSQGVFFQWLEGRPAAVAEV 75Slr BLUF 2 LYRLIYSSQGIPNLQPQDLKDILESSQRNNPANGITGLLCYSKPAFLQVLEGECEQVNET 61YcgF BLUF 2 LTTLIYRSHIRDDEPVKKIEEMVSIANRRNMQSDVTGILLFNGSHFFQLLEGPEEQVKMI 61

○ ○ ○○ ○AppA BLUF 76 MTHIQRDRRHSNVEILAEEPIAKRRFAGWHMQ 107 Slr BLUF 62 YHRIVQDERHHSPQIIECMPIRRRNFEVWSMQ 93 YcgF BLUF 62 YRAICQDPRHYNIVELLCDYAPARRFGKAGME 93

Figure 3. Multiple amino acid sequence alignment of BLUF domains from various organisms. Residues within a distance of 0.5 nm from theisoalloxazine moiety of the respective FAD cofactor are marked with open circles. Conserved amino acid residues are shaded in black; similarresidues (in electronic properties) are shaded in gray. Dissimilarities between residues in BLUF domains that are found within 0.5 nm are markedwith black squares.

Figure 4. Tr-EPR spectrum of wild-type (WT) and mutant YcgF-BLUF domains recorded 2 ls after pulsed laser excitation. Upperpanel: dark-adapted WT AppA-BLUF protein. The dashed curverepresents a spectral simulation of the dark-state sample. Middle andlower panel: dark-adapted mutant YcgF-BLUF samples. The dashedcurve represents spectral simulations. Other experimental parametersare as specified in Fig. 2.


rather limited signal-to-noise ratios even at very low temper-atures approaching 20 K. This could be due to altered ISCrates. However, the exact reasons for this behavior remain tobe clarified. The triplet decay rates of all three investigated

YcgF-BLUF domains are very similar (data not shown)suggesting only small differences in the excited triplet-statestabilities.

Both YcgF-BLUF mutants show a clearly different spectralpattern as compared with the WT: While the outer wings in thetriplet spectra of the mutant proteins show distinct similarities

to those of AppA-BLUF samples, the complex A ⁄E-polarizedpattern near g � 2 has changed dramatically. Both methioninemutants show an A ⁄E inflection at this spectral position;

however, its intensity is decreased. Simulations of the twomutant spectra reveal an increase of the ZFS parameters(|D| = 573 · 10)4 cm)1 and |E| = 193 · 10)4 cm)1 for M23L,and |D| = 575 · 10)4 cm)1 and |E| = 194 · 10)4 cm)1 for

M23I) as compared with the WT (see Table 2). It should benoted that the |D| and |E| values for the two mutant YgcF-BLUF domains are now within the range found for LOV

domains and the other BLUF proteins (see Table 2). More-over, the zero-field populations have changed and are now asfollows: The population AX is roughly 50%, whereas AY and

AZ are almost equal, the difference in populations between thetwo mutants being within the error margin. Hence, the ISCrates are significantly altered by replacing a single amino acidin the immediate surroundings of the flavin chromophore.

Observation and assignment of short-lived paramagnetic states

in Slr-BLUF domains

The triplet state generated in the third type of BLUF samples,

the Slr-BLUF domain from Synechocystis sp. PCC 6803, wasalso investigated using tr-EPR. Its spectrum is very similar tothe one from AppA-BLUF in its outer wings; however, an

intense additional feature (with a spectral width of about20 mT) is observed near g � 2. This signal is absent in theother two BLUF samples. In contrast to the point-symmetric

shape of the triplet spectra from the other BLUF proteins, inSlr-BLUF the central spectral part contains only absorptivesignals. The decays of the signal amplitudes at the outer wingsof the spectrum can be described by monoexponential decay

functions (s1 = 7 ls). A biexponential decay is required tomodel the time trace recorded near g � 2. The observation oftwo clearly different time constants, s1 = 5.5 ls and

s2 = 27 ls, is indicative for two independent species contrib-uting to the tr-EPR signal at this spectral position.

Tr-EPR allows direct observation of laser-flash generated

paramagnetic intermediate states ranging from weakly coupledspin-correlated RPs with relatively large distances between thetwo radicals to triplet states, in which the two unpairedelectron spins are strongly coupled (50). In general, spectra

arising from weakly coupled spin-correlated RPs consist offour resonance lines arranged in two antiphase line pairs, eachcentered at the resonance position of the individual radical

respectively (61–63). The spacing between the lines within eachantiphase doublet is determined by the weak exchange (J) anddipolar (D, E) interactions in the RP respectively. The line pair

of each single RP half may be further split by hyperfineinteractions of magnetic nuclei coupling to the electron spins.In nonoriented rigid samples, interaction anisotropies contrib-

ute to an inhomogeneous spectral broadening of some or alltransitions. Published tr-EPR spectra, e.g. for the coupled RPsin reaction centers of plant and bacterial photosynthesis(64,65), in photolyases (50,66) or in cryptochromes (67) show

rather complicated electron-spin polarization patterns. Intriplet states, on the other hand, the formally two unpairedelectrons are localized on the same molecule and couple to an

overall S = 1 spin state. Here, the exchange interactiondetermines the energy gap between the singlet (S = 0) andthe triplet-state sublevels (S = 1) and does not influence the

tr-EPR spectral pattern, which is dominated by the ZFSparameters (D and E) of the dipolar interaction. Exchange anddipolar interactions between unpaired electron spins have in

general different distance dependences. While the exchangeinteraction decays exponentially with increasing distance rbetween the spin centers, the dipolar interaction falls off withthe inverse cubed distance of r. Hence, in weakly coupled spin-

correlated RPs (separated by more than about 2 nm), thedipolar interaction dominates. Assuming magnetic pointdipoles, the strength of the dipolar coupling between radicals

is given by D(r) (mT) = )2.78 (r (nm))3. The exchange inter-action parameter has a complex dependence on the electronicproperties of the radicals, their spatial separation and the

nature of the intervening medium. It can be approximated byJ(r) = J0 exp()br), where b assumes a positive value and J0may be positive or negative. Given that the distance depen-dence of J is similar to that of the matrix element that couples

the electronic wavefunctions of the reactants and products ofthe electron-transfer reaction, one may use an average value ofb = (14 ± 2) nm)1 for proteins, and a |J0| value in the range

of 1011 mT, the exact values of b and J0 depending strongly onthe specific electronic properties of the protein under exami-nation (68).

We tentatively assign the RP feature observed near g � 2 toa strongly coupled radical pair (stcRP) comprising the flavinchromophore and an electron-donating amino acid both in the

radical form. The different tr-EPR decay rates (see above) andthe lifetime of the excited singlet state of the flavin (42) asdetermined by time-resolved optical spectroscopy suggest thatthe stcRP is generated from an excited triplet state precursor.

Two aromatic amino acid residues are reasonably close to theisoalloxazine ring of FAD to efficiently transfer an electron toFAD upon photoexcitation: Y8 and W91 in Slr-BLUF are

located within 0.5 and 0.6 nm to C(4a) of FAD respectively(measured from the points of highest unpaired electron-spindensity of the respective residue: O(4) and C(3) in tyrosine and

tryptophan, respectively). W91 is assumed in the postulated‘‘in-conformation’’ (28,29,31). At such short distances, theexchange interaction between the flavin radical and the aminoacid radical is expected to be much larger than the dipolar

interaction, and as a consequence, we assume that the twounpaired electron spins (S = ½), one on either Y8 or W91,and the other on FAD, are strongly exchange-coupled such

that an effective S = 1 (triplet) spin state results. For such asystem, the tr-EPR spectrum is dominated by the dipolarinteraction within the triplet RP state (69). From the magnetic-

field position of extreme and inflection points, the ZFSparameters of the dipole–dipole coupling tensor can then beextracted. If perfect axial symmetry (i.e. E = 0) of the stcRP is

assumed, the inflection points of the outer wings are separatedby 2|D|, and the intense inner features by |D|, see Fig. 5. From


the tr-EPR data of Slr-BLUF, a 2|D| spacing between the

maxima of the central signal of (20.7 ± 0.6) mT is extracted.This corresponds to a |D| value of (10.3 ± 0.3) mT (see Fig. 5,upper panel). Using the point-dipole approximation intro-duced above, a distance of about 0.7 nm is calculated as

average distance between the two electron spins in the stcRPstate. This value is close to the distances found between Y8 andFAD (specifically the bond between N(5) and C(4a) of the

flavin’s isoalloxazine ring), or W91 (in the ‘‘in-conformation’’)and FAD. We rule out stcRP delocalization extending fromthe FAD to W91 in the ‘‘out-conformation’’ (28,31): With a

flavin-to-tryptophan distance of around 1.4 nm, a muchsmaller D value (ca )1 mT) than experimentally observed ispredicted.

Origin of the radical pair in Slr-BLUF domains

In contrast to the flavin triplet state, only absorptive featuresare detected by tr-EPR in the central signal arising from thestrongly coupled FAD–amino acid RP species at g � 2 values

(Fig. 2 [lower panel]). However, at present it is impossible todeconvolute the flavin triplet signal from that of the stcRP forthe following reasons: First, assuming a flavin triplet spectrum

with similar shape as that of AppA-BLUF depicted in Fig. 2(upper panel), the ‘‘pure’’ RP spectrum could have someemissive features. Second, some examples of RPs and triplet

states have been reported where spectral shapes deviating fromthe classical ‘‘E ⁄E ⁄A ⁄A’’ polarization pattern were observed(70). This was explained by singlet–triplet mixing in combina-tion with different mixing rates of the singlet state with the

three triplet sublevels, |T+æ, |T0æ and |T)æ. Efficient singlet–triplet mixing can be ruled out in our case, because it isnegligible at very large energy separations of singlet and triplet

states. On the other hand, different spin-lattice relaxationtimes (T1) of the three triplet sublevels, |T+æ, |T0æ and |T)æ,could lead to variable contributions of the individual sublevels

to the overall spectrum and could therefore be responsiblefor the unusually strong absorptive polarization in the

flavin–amino acid RP. A third explanation is a nonequilibratedspin-correlated RP originating from a triplet-state precursor: Ifthe radical-pair precursor is a spin-polarized triplet rather thana singlet state, the initial tr-EPR spectrum may exhibit either

net absorptive or net emissive electron spin polarization (71).This is understood in terms of the three triplet wavefunctions,|T+æ, |T0æ and |T)æ: all three states can be populated. In

contrast to the |T0æ state, which has an equal number of ‘‘spinup’’ and ‘‘spin down’’ electrons, the |T)æ and |T+æ states cancontain different populations because of second-order effects

created by the size of the ZFS parameters D and E relative tothe strength of the external magnetic field B0. If any of the twosublevels are in excess, net polarization occurs.

To assign the amino acid involved in electron transfer to theflavin, we compared the RP signals of the wild-type Slr-BLUFdomain (SlrWT) with that of a mutant where the tyrosineresidue Y8 near the FAD’s isoalloxazine moiety is replaced by

a tryptophan residue (SlrY8W), see Fig. 5. Removal of Y8completely changes the RP feature in the center of thespectrum: In contrast to SlrWT, a RP feature with emissive

and absorptive electron-spin polarization (with extrema at 342and 350 mT) is observed. On the other hand, the lifetime of theRP feature in SlrY8W remains largely unaltered: As depicted

in the right panel of Fig. 5, the decay of the tr-EPR signal isonly slightly faster in the mutant protein as compared with theWT (6.6 ls for SlrWT versus 4.9 ls for SlrY8W, respectively).Surprisingly, the spectral positions of the two prominent

extreme points are unaffected when replacing tyrosine with atryptophan residue. This observation is unexpected as thetheory of spin-correlated RPs predicts that the positions of

extreme and inflection points depend on the g-tensors of thetwo coupled radicals (72). Tryptophan and tyrosine differslightly in their g-values and thus, a noticable change of the RP

signals should have been expected (73).Our results show that Y8 is directly involved in light-

induced electron transfer to the flavin. However, the precise

origin of the RP signal from the SlrY8W sample remainsunclear: The electron-donating residue could be W8, W91 oranother, yet unidentified, aromatic amino acid. From ultrafastoptical spectroscopy studies on the same Slr-BLUF samples a

RP species FADHÆ–W8Æ in SlrY8W has been proposed (55).Clearly, tr-EPR can contribute to solving this puzzle. How-ever, for a quantitative understanding of the RP signature

reported here, further independent information is required. Amore comprehensive inspection of aromatic amino acids andtheir involvement in the photocycle is essential in combination

with application of high-field EPR spectroscopy to betterresolve RP signals by means of the different g-values of thecoupled radicals. As such experiments clearly exceed the goalof this article, we will report on results of studies along these

lines in a subsequent contribution.In summary, examination of light-excited BLUF domains

by tr-EPR reveals a completely different behavior as compared

with previously investigated LOV domains. Tr-EPR spectrafrom WT and mutant YcgF-BLUF domains led us to theassumption that a nearby methionine can alter the wave

function of the flavin’s triplet state. It is quite probable thatthis residue also affects the excited singlet-state wave function.Moreover, Slr-BLUF exhibits a competing electron-transfer

reaction, which clearly deserves further investigation. At thisstage, the RP precursor state, either singlet or triplet, cannot be

Figure 5. Tr-EPR spectrum of wild-type (WT) and Y8W mutation ofSlr-BLUF domains recorded 2 ls after pulsed laser excitation. Upperpanel: dark-adapted WT Slr-BLUF protein. Lower panel: dark-adapted Y8W Slr-BLUF sample. On the right panel, two representa-tive decay curves, recorded at 350.8 mT, are depicted. Dashed linesrepresent fittings of the monoexponential decays.


assigned conclusively. Moreover, it remains unclear why onlySlr-BLUF samples show light-induced electron transfer underthe chosen experimental conditions. The answer to thisquestion may be the key to correlating the electronic structure

of the light-induced excited states with biological signalingactivity, which most likely depends on the stability of the lightstate. On the other hand, based on the rather broad tr-EPR RP

signature, long-range electron transfer (over distances largerthan 1 nm) can be excluded. Here, tr-EPR beautifully shows itspotential for assigning electron-transfer partners even in

molecules with several potential electron donors.

Acknowledgements—This work has been supported in part by Tina

Schiereis for technical assistance and Petra Gnau for cloning and

preparation of YcgF-BLUF. This work was supported by the

Deutsche Forschungsgemeinschaft (project 6 of FOR 526 ‘‘Blue-light

photoreceptors’’ to R.B. and S.W., and ES152 ⁄ 4).

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