Vibrational Assignment of the Flavin-Cysteinyl Adduct in a Signaling State of the LOV Domain in FKF1 Sadato Kikuchi, † Masashi Unno,* ,†,‡ Kazunori Zikihara, § Satoru Tokutomi, § and Seigo Yamauchi | Department of Chemistry and Applied Chemistry, Faculty of Science and Engineering, Saga UniVersity, Saga 840-8502, Japan; PRESTO, JST, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan; Department of Biological Science, Graduate School of Science, Osaka Prefecture UniVersity, 1-1 Gakuencho, Sakai, Osaka 599-8531, Japan; and Institute of Multidisciplinary Research for AdVanced Materials, Tohoku UniVersity, Sendai 980-8577, Japan ReceiVed: September 22, 2008; ReVised Manuscript ReceiVed: December 8, 2008 LOV domains belong to the PAS domain superfamily, which are found in a variety of sensor proteins in organism ranging from archaea to eukaryotes, and they noncovalently bind a single flavin mononucleotide as a chromophore. We report the Raman spectra of the dark state of LOV domain in FKF1 from Arabidopsis thaliana. Spectra have been also measured for the signaling state, where a cysteinyl-flavin adduct is formed upon light irradiation. Most of the observed Raman bands are assigned on the basis of normal mode calculations using a density functional theory. We also discuss implication for the analysis of the infrared spectra of LOV domains. The comprehensive assignment provides a satisfactory framework for future investigations of the photocycle mechanism in LOV domains by vibrational spectroscopy. Blue light photoreceptors that control various biological processes are under intensive investigations. These include photolyases, cryptochromes, BLUF proteins, and phototorpoins. 1 The former three photoreceptors use flavin adenine dinucleotide (FAD) as the chromophoric molecules, whereas flavin mono- nucleotide (FMN) is involved for the latter. The phototropins mediate diverse physiological responses to blue light in plants, including phototropism, 2 chloroplast movements, 3 stomatal opening, 4 and leaf expansion. 5 Phototropin contains two 12 kDa, FMN binding LOV (light, oxygen, voltage) domains (LOV1 and LOV2) in its N-terminal region and a typical serine-threonine kinase domain in its C-terminal region. 6 LOV domains belong to the PER-ARNT-SIM (PAS) domain superfamily, which are found in a variety of sensor proteins in organisms ranging from archaea to eukaryotes. 7 In addition to the phototropin families, several LOV domain-containing proteins have been identified. For example, FKF1 from Arabidopsis thaliana has a LOV domain and plays important roles in the photoregulation of flowering. 8 Upon light excitation, the LOV domains undergo a cyclic photoreaction. 9 The dark state of the FMN in the LOV domains shows typical absorption spectra of flavoproteins with an absorption maximum around 450 nm and called D450. Blue light irradiation excites the FMN chromophore to a triplet state that absorbs light at maximally around 660 nm. 10 Protonation at N5 and attack of a sulfur atom of a nearby cysteine residue at the C4a atom of the FMN produce a flavin-cysteinyl covalent adduct 11,12 absorbing maximally at around 390 nm (designated S390) (Figure 1). Formation of the covalent adduct ultimately leads to structural changes in the protein moiety. In order to understand the photocycle mechanism of LOV domains in atomic details, structural characterizations of the active site are essential. Such information can be provided by vibrational spectroscopy including Raman scattering and Fourier transform infrared (FTIR) absorption methods, which can probe individual functional groups of proteins through their frequencies and intensities. In fact, the latter technique has played a key role in elucidating protein structural changes during the photocycle. 13-16 On the other hand, an application of Raman spectroscopy to LOV domains is limited, 15 though this technique is a powerful method that provides precise information concern- ing the chromophore structure as well as chromophore-protein interactions. A part of the reasons for the limited success of Raman spectroscopy to LOV domains is strong fluorescence from FMN as well as a relatively short lifetime of S390. To overcome these problems, we have measured the Raman spectra of D450 and S390 in the LOV domain of FKF1 under a nonresonance condition (λ ex ) 647.1 nm) to avoid fluorescence * To whom correspondence should be addressed: Tel +81-952-28-8678; Fax +81-952-28-8548; e-mail [email protected]. † Saga University. ‡ JST. § Osaka Prefecture University. | Tohoku University. Figure 1. Structures of the active site of LOV domains in the dark (D450) and signaling (S390) states. J. Phys. Chem. B 2009, 113, 2913–2921 2913 10.1021/jp808399f CCC: $40.75 2009 American Chemical Society Published on Web 02/06/2009
Transcript
Vibrational Assignment of the Flavin-Cysteinyl Adduct in a Signaling State of the LOVDomain in FKF1
Department of Chemistry and Applied Chemistry, Faculty of Science and Engineering, Saga UniVersity,Saga 840-8502, Japan; PRESTO, JST, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan; Department ofBiological Science, Graduate School of Science, Osaka Prefecture UniVersity, 1-1 Gakuencho, Sakai,Osaka 599-8531, Japan; and Institute of Multidisciplinary Research for AdVanced Materials, TohokuUniVersity, Sendai 980-8577, Japan
ReceiVed: September 22, 2008; ReVised Manuscript ReceiVed: December 8, 2008
LOV domains belong to the PAS domain superfamily, which are found in a variety of sensor proteins inorganism ranging from archaea to eukaryotes, and they noncovalently bind a single flavin mononucleotide asa chromophore. We report the Raman spectra of the dark state of LOV domain in FKF1 from Arabidopsisthaliana. Spectra have been also measured for the signaling state, where a cysteinyl-flavin adduct is formedupon light irradiation. Most of the observed Raman bands are assigned on the basis of normal mode calculationsusing a density functional theory. We also discuss implication for the analysis of the infrared spectra of LOVdomains. The comprehensive assignment provides a satisfactory framework for future investigations of thephotocycle mechanism in LOV domains by vibrational spectroscopy.
Blue light photoreceptors that control various biologicalprocesses are under intensive investigations. These includephotolyases, cryptochromes, BLUF proteins, and phototorpoins.1
The former three photoreceptors use flavin adenine dinucleotide(FAD) as the chromophoric molecules, whereas flavin mono-nucleotide (FMN) is involved for the latter. The phototropinsmediate diverse physiological responses to blue light in plants,including phototropism,2 chloroplast movements,3 stomatalopening,4 and leaf expansion.5 Phototropin contains two 12 kDa,FMN binding LOV (light, oxygen, voltage) domains (LOV1and LOV2) in its N-terminal region and a typical serine-threoninekinase domain in its C-terminal region.6 LOV domains belongto the PER-ARNT-SIM (PAS) domain superfamily, which arefound in a variety of sensor proteins in organisms ranging fromarchaea to eukaryotes.7 In addition to the phototropin families,several LOV domain-containing proteins have been identified.For example, FKF1 from Arabidopsis thaliana has a LOVdomain and plays important roles in the photoregulation offlowering.8
Upon light excitation, the LOV domains undergo a cyclicphotoreaction.9 The dark state of the FMN in the LOV domainsshows typical absorption spectra of flavoproteins with anabsorption maximum around 450 nm and called D450. Bluelight irradiation excites the FMN chromophore to a triplet statethat absorbs light at maximally around 660 nm.10 Protonationat N5 and attack of a sulfur atom of a nearby cysteine residueat the C4a atom of the FMN produce a flavin-cysteinyl covalentadduct11,12 absorbing maximally at around 390 nm (designatedS390) (Figure 1). Formation of the covalent adduct ultimatelyleads to structural changes in the protein moiety.
In order to understand the photocycle mechanism of LOVdomains in atomic details, structural characterizations of theactive site are essential. Such information can be provided byvibrational spectroscopy including Raman scattering and Fouriertransform infrared (FTIR) absorption methods, which can probeindividual functional groups of proteins through their frequenciesand intensities. In fact, the latter technique has played a keyrole in elucidating protein structural changes during thephotocycle.13-16 On the other hand, an application of Ramanspectroscopy to LOV domains is limited,15 though this techniqueis a powerful method that provides precise information concern-ing the chromophore structure as well as chromophore-proteininteractions. A part of the reasons for the limited success ofRaman spectroscopy to LOV domains is strong fluorescencefrom FMN as well as a relatively short lifetime of S390.
To overcome these problems, we have measured the Ramanspectra of D450 and S390 in the LOV domain of FKF1 undera nonresonance condition (λex ) 647.1 nm) to avoid fluorescence
* To whom correspondence should be addressed: Tel +81-952-28-8678;Fax +81-952-28-8548; e-mail [email protected].
Figure 1. Structures of the active site of LOV domains in the dark(D450) and signaling (S390) states.
J. Phys. Chem. B 2009, 113, 2913–2921 2913
10.1021/jp808399f CCC: $40.75 2009 American Chemical SocietyPublished on Web 02/06/2009
from the sample. A very long lifetime of S390 for FKF1 (ahalf-life time of 62.5 h at 298 K)17 allows us to measure a high-quality Raman spectrum of a flavin-cysteinyl covalent adductfor the first time. We also present the results of normal modecalculations based on the density functional theory (DFT). Thesestudies allow us to assign the observed Raman bands of D450and S390. The assignment of the Raman spectra offers usefulinformation concerning the structural marker bands for theflavin-cysteinyl covalent adduct. Furthermore, the present studyhas an important implication for the analysis of the IR spectraof LOV domains. With assignments in hand, vibrationalspectroscopy provides a unique approach for studying the proteindynamic processes in LOV domains.
Materials and Methods
Sample Preparations. Production of a LOV domain of FKF1(FKF1-LOV) from Escherichia coli (E. coli) and the subse-quent protein purification were performed as described previ-ously.17,18 Preparation of recombinant LOV-containing polypep-tide of Arabidopsis thaliana FKF1 protein was prepared by anoverexpression system with E. coli. Using Arabidopsis cDNAas a template, a DNA fragment corresponding to the polypeptidewas amplified by the PCR method with primers to provideappropriate restriction sites. The amplified DNA was isolated,digested, and cloned into a pGEX4T1 expression vector(Amersham Bioscience, Uppsala, Sweden) as a fusion proteinwith glutathione S-transferase (GST). A linker sequence(Gly-Ser) was inserted between GST and the LOV polypeptide.
JM109 strain of E. coli transformed by the vector was grownin the dark after the induction by isopropyl-�-D-thiogalactopy-ranoside. The purification was carried out at 273-277 K underdim red light. The supernatant from the lysate of harvestedbacteria was mixed with glutathione-Sepharose 4B (AmershamBioscience). The FKF1-LOV polypeptides were removeddirectly from the gel-bound fusion proteins by a thrombindigestion at the linker sequence. The LOV polypeptide waspurified further by a gel chromatography with Sephacryl S-100HR (Amersham Bioscience) and the buffer solution containing100 mM NaCl, 25 mM Tris-HCl, and 1 mM Na2EDTA (pH7.8). The purified polypeptide was concentrated to 1.0 mM byultrafiltration, and the purity was examined by the sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Raman Spectroscopy. Raman spectra were obtained asdescribed previously.19-21 A liquid nitrogen cooled CCD detector(Instrument S.A., Inc.) recorded the Raman spectra after aTriax190 spectrometer (Instrument S.A., Inc.) removed theexcitation light, and a Spex 500M spectrometer (1800 grooves/mm grating, 0.5 m focal length) dispersed the scattered light.The 647.1 nm line from a krypton ion laser (BeamLok 2065,Spectra-Physics Lasers, Inc.) excited the samples at a 90° anglerelative to the axis of the collection optics. The laser power atthe sample was 170 mW. A polarization scrambler is placed atthe entrance of the spectrometer. The entrance slit width of 0.3mm corresponds to a spectral resolution of ∼5 cm-1. For themeasurement of S390, the samples were illuminated for a fewminutes with a continuous blue light (350-550 nm), and theformation of S390 was confirmed by measuring UV-visabsorption spectra of the sample. All spectra were taken at roomtemperature (∼23 °C), and a homemade software eliminatedthe noise spikes in the spectra caused by cosmic rays. TheRaman spectra were obtained with samples in a 1.5 × 1.5 ×48 mm quartz cuvette. All Raman spectra were calibrated usingneat fenchone.
DFT Calculation. Among the numerous available DFTmethods, we have selected the B3LYP hybrid functional withthe 6-31G** basis set because of its high accuracy for predictingvibrational frequencies. It has been shown that this level of DFTcalculations yield molecular force fields and vibrational frequen-cies in excellent agreement with experiments in a variety ofsystems.22 The optimized geometry, the harmonic vibrationalfrequencies, and Raman and IR intensities were calculated usingthe DFT method mentioned above via the Gaussian03 program.23
The calculated frequencies were scaled using a factor of0.9627.24
Results and Discussion
1. Raman Spectra of FKF1-LOV. Figure 2 shows theRaman spectra of D450 (trace a) and S390 (trace b) forFKF1-LOV, and the frequencies of the observed Raman bandsare listed in Tables 1 and 2. In Table 1, reported frequencies aswell as isotope shifts for free FAD25 are also listed for acomparison. In addition, Figure 1S in the Supporting Informationcompares the Raman spectra of free FMN, D450, and a BLUFdomain of AppA. As can be seen in Figure 1S and Table 1, themain features of the spectrum for D450 resemble closely thosefor free riboflavin, FMN, or FAD,19,25,26 indicating that most ofthe observed bands in the D450 spectrum can be ascribed tovibrational modes for the isoalloxazine ring. A broad Ramanband around 1670 cm-1 is absent in free FMN (not shown) andis ascribed to the amide I mode.27 Analogously, the D450spectrum involves a broad band near 1460 cm-1, which can becharacterized by a deformation mode of a CH group in theprotein moiety.28
Figure 2. Raman and difference spectra of FKF1-LOV (traces a-c)as well as corresponding simulated spectra (traces d-f). The observedspectra were obtained at 647.1 nm excitation. (a) D450, (b) S390, and(c) S390-D450. Simulated Raman and difference spectra of the activesite models illustrated in Figure 4. Vertical bars represent computedfrequencies and intensities, and Gaussian band shapes with a 10 cm-1
width are used to simulate the spectra. The spectra for (d) model 2, (e)model 4, and (f) model 4-model 2 are shown.
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TABLE 1: Observed and Calculated Vibrational Frequency (cm-1) of D450 of FKF1-LOV and Its Modela
νobs νcal
FKF1-LOVb FADc Neo1-LOV2d model 1e model 2e g h assignment f
a This table summarizes the observed and calculated vibrational frequencies for D450. b Observed values for D450 of FKF1-LOV.c Observed values for free FAD. The numbers in parentheses are the isotope shifts of the [4,10a-13C2]-na and [2-13C]-na, respectively, where nais the natural abundance. These data are taken from ref 25. d Observed values for D450 of Neo1-LOV2. The numbers in parentheses are theisotope shifts of the [4,10a-13C2]-na and [2-13C]-na, respectively. These are the FTIR data reported by ref 13. e Calculated vibrationalfrequencies of models 1 and 2. The numbers in parentheses are the isotope shifts of the [4,10a-13C2]-na and [2-13C]-na, respectively. f Theobserved Raman and IR bands are assigned to the calculated normal modes. Approximate descriptions of the calculated modes are alsodescribed. ν and δ stand for stretching and bending vibrations, respectively. R indicates vibrations of the ribityl moiety. g Mode numbers for ref29. h Mode numbers for ref 32. i Not determined.
TABLE 2: Observed and Calculated Vibrational Frequency (cm-1) of S390 of FKF1-LOV and Its Modela
νobs νcal
FKF1-LOVb Neo1-LOV2c model 3e model 4e assignment e
a This table summarizes the observed and calculated vibrational frequencies for S390. b Observed values for S390 of FKF1-LOV.c Observed values for S390 of Neo1-LOV2. The numbers in parentheses are the isotope shifts of the [4,10a-13C2]-na and [2-13C]-na,respectively. These are the FTIR data reported by ref 13. e Calculated vibrational frequencies of models 3 and 4. The numbers in parenthesesare the isotope shifts of the [4,10a-13C2]-na and [2-13C]-na, respectively. f The observed Raman and IR bands are assigned to the calculatednormal modes. Approximate descriptions of the calculated modes are also described. R indicates vibrations of the ribityl moiety.
Raman Spectrum of S390 in LOV Domain J. Phys. Chem. B, Vol. 113, No. 9, 2009 2915
Figure 2 also illustrates that the Raman spectra of FKF1-LOVexhibit significant changes between D450 and S390. Thesedifferences are clearly seen in the S390 minus D450 differencespectrum (trace c). The fraction of the signaling state estimatedfrom the intensity loss of the Raman band around 1350 cm-1
of D450 is larger than 90%. The observed spectral changesaccompanying the formation of S390 are summarized in thefollowing four points. (i) A small band at 1715 cm-1 upshiftsby ∼10 cm-1. (ii) The bands at 1583, 1548, 1407, and 1353cm-1 reduce its intensity and/or is shifted. (iii) A new bandappears at 1430 cm-1 in S390. (iv) The Raman bands below1300 cm-1 change their intensities and/or frequencies.
In Figure 3, we compare the Raman spectra of FKF1-LOVwith the light-induced difference FTIR spectrum of a LOVdomain. Since the FTIR data for FKF1-LOV are not availableat present, we use the data for Neo1-LOV2 in the figure. Thespectra shown in Figure 3 reveal that many of the negative bandsin the difference FTIR spectrum coincide with the Raman bandsof D450. This implies that the negative IR bands are due to thechromophore vibrations of D450. These observations alsosuggest that the environment of the FMN chromohore in D450is similar between FKF1-LOV and Neo1-LOV2. Analogously,several positive features in the difference FTIR spectrum canbe correlated to the Raman bands for S390. In order to interpretthe Raman and difference IR spectra, we perform normal modecalculations described below.
2. Normal Mode Calculations Based on DFT. Vibrationalspectra of the oxidized form of flavin have been extensivelystudied.19,29-32 However, the assignment of the spectra was notcomplete. A part of the reasons for the incomplete assignmentis due to a relatively large size of flavin molecules. For instance,although lumiflavin (R ) CH3 in Figure 1) has been frequentlyused for a vibrational analysis, the replacement of the ribityl
moiety of FMN with methyl group distinctly affects normalmodes below 1400 cm-1.19 Thus, in this study, we use 7,8-dimethyl-10-glycerylisoalloxazine as a chromophore model forD450 (model 1 in Figure 4). In addition, model 2 containsacetamide to mimic the hydrogen bonds of the isoalloxazinering with the nearby amino acid residues. Methanol is alsoincluded as a model of Ser930 to consider a hydrogen bond atthe carboxyl group of Gln1029. Although the crystal structureof FKF1-LOV is not available, amino acid residues in the activesite are conserved.18 Thus, these components were arranged onthe basis of the crystal structure of D450 in Neo1-LOV233 andsubsequently optimized to yield the structures illustrated inFigure 4. Table 1S in the Supporting Information gives theoptimized geometries along with the experimental parametersof Neo1-LOV2 in crystal.33 A comparison between models 1and 2 shows that the formation of hydrogen bonds around thering III moiety (Figure 1) causes the single-double bondalternation to be less significant. For example, the change ofmodel 1 to 2 shortens the N3-C4 bond length (1.3825f 1.3718Å), whereas the C4dO bond length is lengthened (1.2171 f1.2277 Å).
In the case of S390, we use ethanethiol as a model of theconserved cysteine residue (Cys966 for Neo1-LOV2), whichforms a flavin-cysteinyl adduct (model 3 in Figure 4). We havealso considered an active site model that incorpolates threeacetamide molecules to mimic hydrogen bonds with the sur-rounding amino acid residues (model 4). Similar to the case ofmodel 2, the crystal structure of S390 in Neo1-LOV212 wasused as an initial structure for the subsequent geometryoptimization. Table 1S reports the optimized geometries, andthe main geometrical difference between D450 and S390 modelscan be found in the vicinity of the C4a position. For example,the change from model 2 to 4 significantly increases the bonddistance between the C4a and N5 positions (1.3023 f 1.4178Å).
Next we have calculated vibrational frequencies as well asRaman intensities of the chromophore models described above.Tables 1 and 2 summarize the computed frequencies as well asapproximate descriptions of the vibrational modes for D450 andS390, respectively. The calculated isotope shifts for the 4,10a-13C2 and 2-13C flavin chromophores are also shown in the tables.Figure 2S in the Supporting Information and Figure 5 illustrateatomic displacements for the important normal modes for D450and S390, respectively. Note that we have chosen to retain themode labels for lumiflavin19 in the present study, while Table1 also lists mode numbers used in Abe and Kyogoku29 andEisenberg and Schelvis32 for comparison.
The lower part of Figure 2 shows the simulated Ramanspectra of models 2 (trace d) and model 4 (trace e) as well astheir difference spectra (trace f). Although the agreementsbetween the experiments and calculations are not perfect, thesimulated spectra, especially simulated difference spectrum(trace f), capture main features of the observed spectra. Forexample, negative difference bands around the 1550, 1400, 1350,and 1250 cm-1 as well as a positive band around 1425 cm-1
are reproduced in the simulated difference spectrum. Theseresults allow us to assign most of the Raman bands of D450and S390. In addition to the analysis of the Raman spectra, thepresent DFT calculations are also useful to interpret the IRspectra of LOV domains. The upper parts of Figure 6 are thedifference FTIR spectra of Neo1-LOV2 reconstituted withunlabeled FMN (a, black line; b and c, green lines), [4,10a-13C2]FMN (b, black line), and [2-13C]FMN (c, black line). Thesespectra are taken from ref 13. To analyze these difference FTIR
Figure 3. Comparison of the Raman spectra of FKF1-LOV and thelight-induced difference FTIR spectrum of Neo1-LOV2. Ramanspectrum for S390 of FKF1-LOV (a), S390 - D450 difference Ramanspectrum of FKF1-LOV (b), S390 - D450 difference FTIR spectrumof Neo1-LOV2 at 150 K (c), and vertically inverted Raman spectrumfor D450 of FKF1-LOV (d). Trace c is adapted from ref 13.
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spectra, we have used models 2 and 4 for D450 and S390,respectively. The lower parts of Figure 6 are the simulateddifference IR spectra for the unlabeled (d, black line; e and f,green lines) as well as [4,10a-13C2] (e, black line) or [2-13C](f, black line) labeled species. Comparison of the observed andcalculated spectra demonstrates satisfactory agreements betweenthe experiment and calculation.
3. Assignments for D450. Several earlier studies providedassignment of Raman and infrared spectra for flavin.19,29-32 Herewe improve the confidence level in the assignments by applyingthe DFT method to extended molecular models shown in Figure4. Although most of our results are consistent with thecalculations of previous studies, some of the assignments areupdated as described below.
A. CdO Stretching Mode; ν10- ν11. The weak but distinctRaman band at 1715 cm-1 for D450 in Figure 2 (trace a) isassigned to the carbonyl C4dO stretching vibrations ν10 of theisoalloxazine ring based on the observed and calculated (1722cm-1) frequencies. This assignment is consistent with the FTIRdata for Neo1-LOV2 as well as Raman data for free FAD25
and a BLUF protein.19
The DFT calculation using model 2 predicts the C2dOstretching mode ν11 of the isoalloxazine ring below 1700 cm-1
(Table 1). Unfortunately, a broad amide I band around 1670cm-1 makes the observation of ν11 difficult. However, the S390- D450 difference Raman spectrum (Figure 2, trace c) showsa negative feature at 1678 cm-1, which is assignable to ν11. This
Figure 4. Optimized geometry of four active site models. 7,8-Dimethyl-10-glycerylisoalloxazine is used to mimic FMN, and its adduct withethanethiol is employed as the model adduct compound.
Figure 5. Atomic displacement vectors for some vibrational modes for S390 (model 4 in Figure 4). The Asn998, Asn1008, and Gln1029 moietiesare removed from the figure.
Raman Spectrum of S390 in LOV Domain J. Phys. Chem. B, Vol. 113, No. 9, 2009 2917
assignment agrees with a FTIR study on Neo1-LOV2, whereν11 was detected at 1677 cm-1.13
B. CdC or CdN Stretching Mode; ν12-ν18. The Ramanbands at 1633, 1583, and 1548 cm-1 for FKF1-LOV cor-respond to those observed at 1627, 1582, and 1545 cm-1 forfree FAD,25 respectively. As summarized in Table 1, the FADbands are sensitive to the 13C substitutions and are assigned toCdC and/or CdN stretching vibrations of the isoalloxazine ν13,and ν14, respectively (Figure 2S). The ν13 and ν14 modes involvea larger contribution of a motion of C10a atom compared tothat of ν12, accounting for larger 4,10a-13C2 shifts in ν13 andν14. The ν15 mode involves C4adN5 and C10adN1 stretchingvibrations like ν13 but with different phase (Figure 2S). TheDFT calculations using model 2 predict ν15 at 1512 cm-1, andthis mode is assigned to a Raman band at 1510 cm-1. Thisassignment is supported by the FTIR data for Neo1-LOV2;13
i.e., a negative IR band at 1507 cm-1 exhibits a -13 cm-1 shiftupon 4,10a-13C2 substitution, which is consistent with a -17cm-1 shift for model 2. The ν16 mode is mainly due to ring Ivibrations including the C-H bending and methyl deformation,and this mode has been assigned to the band around 1500 cm-1
for a BLUF protein.19 The present DFT calculations confirmthe assignment because of a -5 cm-1 4,10a-13C2 shift for freeFAD,25 which corresponds to a -3 cm-1 shift at 1477 cm-1 formodel 2. Note that the ring I vibration of ν16 corresponds tomode 11 of Abe and Kyogoku,29 although the mode 11 wasassigned to a band around 1465 cm-1.
C. C-C Stretching/C-H Rocking Modes; ν17-ν31. Asdescribed above, a Raman band around 1465 cm-1 was assignedto the ring I vibration (mode 11) in Abe and Kyogoku.29
However, the present and previous normal mode calculationsbased on DFT assigned this band to methyl deformation modesν17 and ν28.19,31 This assignment is supported by a recent study,32
which reports the Raman spectra of 8-methyl-deuterated FMN.Note that the 1465 cm-1 band for FKF1-LOV and AppA126is very broad compared to that for free FMN (Figure 1S) becauseof an overlap of CH2 scissoring modes of protein moiety.28
The band observed at 1407 cm-1 was ascribed to theisoalloxazine ring I and II vibrations ν21-ν24 in previousstudies,19,31,32 where lumiflavin was used as a model of FMNor FAD. However, replacement of the methyl group with theglyceryl group in models 1 and 2 induces a significant changein their normal mode composition. These modes are calculatedto be mainly methyl deformation vibrations of the isoalloxazinering (Figure 2S), and the 1407 cm-1 band can be ascribed to anoverlap of these modes on the basis of moderate 4,10a-13C2 and2-13C shifts for the corresponding band at 1406 cm-1 for freeFAD.25 The overlapping ν21-ν24 band is also observed in thedifference FTIR spectrum of Neo1-LOV2 at 1407 cm-1 (Figure3). The strong Raman band at 1353 cm-1 has been assignedto an overlap of the chromophore skeleton vibrations ν26 andν27 for a BLUF protein.19 This assignment is confirmed bythe present analysis; i.e., the observed -8 cm-1 shift upon4,10a-13C2 substitution for free FAD25 correlates well withthe calculated shifts for ν26 and ν27 (-10 and -4 cm-1) formodel 2.
Raman bands below 1350 cm-1 contain significant contribu-tions from protein moieties. Thus, the interpretation of thespectra is difficult unless we have data for isotopically labeledsamples. On the other hand, the FTIR data for Neo1-LOV2that contains isotopically labeled FMN are available,13 so thatseveral IR bands can be assigned. As shown in Figure 6, anegative IR band at 1272 cm-1 exhibits -2 and -4 cm-1 shiftsupon 4,10a-13C2 and 2-13C substitutions, respectively. We assignthis band to the overlapping ν30a and ν30b modes, since the lattermode shows -2 cm-1 4,10a-13C2 and -4 cm-1 2-13C shifts formodel 2. Although these modes are mainly allocated to theC6-H/C9-H bending coordinates (Figure 2S), a small contri-bution from the N1-C2 and N3-C4 stretching motions in ν30b
accounts for the 12C/13C isotope shifts. These modes are notclearly seen in the Raman spectra of FKF1-LOV (Figure 3)as well as algal Phot-LOV1.15 As illustrated in Figure 6, anegative IR band at 1250 cm-1 shows large isotope shifts forthe 4,10a-13C2 (-7 cm-1) and 2-13C (-11 cm-1) substitutions.Simulated difference IR spectra (traces e and f) reproduce similarisotope effects around 1210 cm-1, leading to an assignment ofthe band to an overlap of ν31a and ν31b. These modes consistprimarily of N1-C2 and/or N3-C4 stretching vibrations(Figure 2S).
4. Assignments for S390. A. CdO Stretching Mode;ν10-ν11. Figure 2 shows a weak Raman band at 1725 cm-1 forS390 (trace b). This band is assigned to the carbonyl C4dOstretching vibrations ν10 on the basis of the calculated frequency(1729 cm-1) for model 4. This assignment is supported by theFTIR spectra for Neo1-LOV2, where ν10 is observed at 1723cm-1.13 On the other hand, the C2dO stretching mode ν11, whichis predicted at 1703 cm-1 for model 4, is not detected in theRaman spectra.
B. CdC or CdN Stretching Mode; ν12-ν16. For D450, the1500-1650 cm-1 region contains normal modes that involvemotions of C4a atom. Thus, these modes exhibit significantchanges upon formation of the C4a-cysteinyl adduct in S390.The CdC and CdN stretching modes ν12 and ν14 for S390 areexpected to be shifted by about -5 and +45 cm-1, respectively,
Figure 6. Observed and simulated difference IR spectra of LOVdomains. The light-induced difference FTIR spectra of Neo1-LOV2reconstituted with unlabeled FMN (a, black line; b and c, green lines),[4,10a-13C2]FMN (b, black line), and [2-13C]FMN (c, black line) andcorresponding simulated difference IR spectra of model 4 minus model2 (d-f). Vertical blue and red bars represent computed frequenciesand intensities for models 4 and 2, respectively. Gaussian band shapeswith a 10 cm-1 width are used to simulate the spectra. The FTIR spectra(traces a-c) are adapted from ref 13.
2918 J. Phys. Chem. B, Vol. 113, No. 9, 2009 Kikuchi et al.
compared to those for D450 (Tables 1 and 2). A small positivefeature at 1626 cm-1 in the difference Raman spectrum (Figure2, trace c) is assigned to ν12 for S390 based on a 7 cm-1
downshift from 1633 cm-1 for D450. The difference IRspectrum of Neo1-LOV2 exhibits corresponding bands at 1633cm-1 (D450) and 1626 cm-1 (S390) (Figures 3 and 6). Althoughthese bands were mainly ascribed to the protein moiety,13 ν12
may partially contribute to the spectrum. The difference Ramanspectrum shows a clear positive band at 1597 cm-1, and weassign this band to ν14 on the basis of a 49 cm-1 upshift from1548 cm-1 in D450 (Figure 2). Although the ν14 band is clearlyseen in the Raman spectrum, the corresponding IR band hasnot been observed.
Both ν13 and ν15 modes involve C10adN1/C4adN5 stretchingvibrations for D450 (Figure 2S). The ν15 mode for S390 ismainly allocated to stretching of the C10adN1 bond (Figure5). The Raman band observed at 1549 cm-1 for S390 can beassigned to ν15 because of its frequency and ca. +39 cm-1 shiftupon formation of S390 (Tables 1 and 2). In the difference IRspectrum, the ν15 mode was observed as a prominent positiveband at ca. 1540 cm-1 for various LOV domains (Figure3).14-16,33 The assignment of this IR band to ν15 is confirmedby the present DFT calculations; i.e., the IR band at 1540 cm-1
for Neo1-LOV exhibits a -25 cm-1 4,10a-13C2 shift,13 whichis consistent with a -31 cm-1 shift at 1542 cm-1 for model 4(Figure 6). On the other hand, ν13 is not present for S390 becausethe adduct formation at the C4a position of the isoalloxazinering makes the C4adN5 double bond to a single bond.
The DFT calculations using model 4 predict ν16, the combinedCdC stretching and C-H bending vibrations of the ring I, at1495 cm-1. As shown in Figure 2, we tentatively locate ν16
around 1500 cm-1 based on its frequency.C. C-C Stretching/C-H Rocking Modes; ν17-ν31. Figure
2 demonstrates that the most intense positive band at 1430 cm-1
in the difference Raman spectrum (trace c) is well reproducedin the simulated difference spectrum (trace f). We assign this1430 cm-1 band to the ring II vibrations including N5-Hbending ν20, which is also observed at 1432 cm-1 in thedifference IR spectrum for Neo1-LOV213 (Figure 3, trace c).The assignment of ν20 is supported by the IR data; i.e., theobserved -7 cm-1 4,10a-13C2 shift is consistent with a -7 cm-1
shift at 1413 cm-1 for model 4 (Figure 6). Similar to the caseof D450, the Raman spectrum of S390 exhibits a band at 1408cm-1 (Figure 2, trace b). This band can be assigned to methyldeformation vibrations ν21-ν24 of the chromophore based onits frequency.
As described in the previous section, the ν26 mode for D450involves the CC and CN stretching vibrations of rings I, II, andIII of the isoalloxazine (Figure 2S). The adduct formation inS390, however, decouples the ring I and ring III vibrations,providing two modes of ν26a and ν26b (Figure 5, Table 2). Theν26a mode is mainly allocated to the CN stretching coordinatesof ring III and is predicted at 1352 cm-1 for model 4. The DFTcalculations indicate that this mode is expected to have amoderate IR intensity, and a positive IR band at 1374 cm-1 forNeo1-LOV213 is assigned to ν26a based on the -15 cm-1
isotopic shift with 4,10a-13C2 substitution (Figure 6b). The ringI vibration ν26b is calculated at 1315 cm-1 for model 4 and hasa moderate intensity in the simulated Raman spectrum (Figure2, trace e). We assign ν26b to a Raman band at 1314 cm-1
because of its frequency. The corresponding band was notdetected in the difference IR spectra for Neo1-LOV2 (Figure3).13
Figure 6 shows a small positive band at 1258 cm-1 in thedifference IR spectrum. This IR band can be assigned to ν30a
because of its -2 cm-1 4,10a-13C2 and -7 cm-1 2-13C shifts,which correspond to -4 and -8 cm-1 shifts, respectively, at1239 cm-1 for model 4. As illustrated in Figure 5, ν30a is partiallyallocated to CH bending motions of the reactive cysteine residue.Note that the C-S stretching vibrations are expected below 800cm-1 and are not detected in the present study.
5. Implications. The present study reports the Ramanspectrum of FKF1-LOV in the D450 state, and Figure 1Scompares the spectrum with that of a BLUF domain fromAppA.19 A light illumination causes a formation of a covalentadduct in a LOV domain, whereas the photoreaction of theBLUF domain involves a rearrangement of hydrogen bondsaround the C4dO moiety of FAD.36 In spite of a strikingdifference in their primary photochemistry, however, theirRaman spectra under the dark state are quite similar as illustratedin Figure 1S. For example, frequency differences of the Ramanbands due to isoalloxazine ring are within (4 cm-1.37 In somesense, this observation is reasonable because of a notablesimilarity in the flavin binding interactions of these two flavin-containing photoreceptors.33,38-40 In fact, Suzuki et al.41 recentlyshowed that the light-induced formation of a cysteinyl-flavinadduct can be reproduced in the BLUF domain by introducinga cysteine residue near the isoalloxazine ring.
In addition to D450, we report the Raman spectra ofFKF1-LOV in the S390 state. The very long lifetime of S390for FKF1-LOV17 allows us to measure the Raman spectrumof S390 for the first time. The observation of the Raman spectraleads to the assignment of vibrational modes in the 1200-1700cm-1 region, and we characterize useful marker bands for theactive site structure of LOV domains. It also has an importantimplication for the analysis of IR spectra of LOV domains.
A noticeable change in the Raman spectrum upon formationof S390 is a ∼10 cm-1 upshift of the C4dO stretching vibrationν10 (Figure 2). A similar upshift of ν10 was observed in thedifference FTIR spectra of various LOV domains.14-16,34,35
Possible explanations of the observed upshifts in ν10 are aweakening of the hydrogen bonds at the CdO groups13 and anadduct formation of the FMN chromophore.14,15 The presentDFTcalculationsdemonstratethattheformationofacysteinyl-flavinadduct without changing a hydrogen bond (models 1 f 3)accounts for a 6 cm-1 upshift of ν10, which is roughly half ofthe observed shift. Thus, the light-induced upshift of ν10 can beascribed to changes in the hydrogen bonds as well as chro-mophore structural changes associated with adduct formation.
As illustrated in Figure 3 (trace c), the difference IR spectrumof LOV domains shows several bands that arise from S390. Aprominent positive band around 1540 cm-1 is assigned to theC10adN1 stretching vibration ν15. In the adduct, the C4a carbonacquires a nonplanar sp3 tetrahedral configuration, leading todramatic changes in the vibrational modes that involve the C4aatom. In Table 2, a comparison of the ν15 frequencies betweenmodels 3 and 4 demonstrates that the formation of hydrogenbonds at the C2dO, N3-H, and C4dO positions downshiftsν15 by 15 cm-1 (1557 f 1542 cm-1). Thus, the ν15 band in thedifference IR spectra could reflect chromophore-protein inter-actions in S390. For instance, a FTIR study on full-length YtvAand its isolated LOV domain showed that the positive IR bandaround 1540 cm-1 is significantly perturbed by the presence ofthe signaling domain.42 This observation may indicate thestructural changes in the active site of the LOV domain in thepresence of the downstream effector domain.
Raman Spectrum of S390 in LOV Domain J. Phys. Chem. B, Vol. 113, No. 9, 2009 2919
The positive feature at ∼1260 cm-1 in the difference IRspectrum is assigned to ν30a of S390. Because this mode involvesthe C-H bending motions of the reactive cysteine residue, itmay act as a structural probe around the C-S covalent bond.In fact, previous FTIR studies on LOV domains showed thatthis region of the spectra is sensitive to the protein structures;e.g., Iwata et al.34 reported that the difference FTIR spectraaround 1260 cm-1 distinctly differ between LOV1 and LOV2domains from Neo1. Although we cannot rule out a contributionof the vibrational modes from a protein moiety, the observedspectral differences could reflect structural differences aroundthe C-S bond.
Finally, we discuss a functional implication of the measure-ment of the Raman spectra of FKF1-LOV. One of thecharacteristic features of FKF1-LOV is its formation of a stablephotointermediate S390. As mentioned above, Zikihara et al.17
demonstrated that the dark recovery of S390 to D450 with ahalf-life time of 62.5 h, which is much longer than the timeconstants from several seconds to a few minutes for the LOVdomains of phototropin.43 This extremely slow recovery mayderive from a nine amino acid insertion between the R′(A)-helix having the conserved cysteine and R′(C)-helix as comparedto those of phototropin families. In spite of this structuraldifference, however, Figure 3 indicates little spectral differencesbetween FKF1-LOV and Neo1-LOV2. This observationsuggests that the active site structure of FKF1-LOV is notsignificantly different from that of Neo1-LOV2. It is thereforelikely that the slower recovery of S390 in FKF1-LOV thanthat in Neo1-LOV2 originates from differences in proteinstructure and/or protein-chromophore interactions between thetwo LOV domains.
In summary, this study presents the Raman investigation ofFKF1-LOV, and we report the Raman spectrum of S390 forLOV domains for the first time. In addition, most of the observedRaman bands are assigned with the aid of DFT calculations.Furthermore, the present study has an important implication forthe analysis of the IR spectra of LOV domains. With theseassignments in hand, vibrational spectroscopy provides animportant approach for studying the photocycle mechanism inLOV domains.
Acknowledgment. We appreciate Drs. H. Kandori and T.Iwata (Nagoya Institute of Technology) for allowing us to usetheir FTIR data in Figures 3 and 6. A part of the computationswas performed using Research Center for ComputationalScience, Okazaki, Japan.
Supporting Information Available: Optimized geometriesalong with the experimental parameters of Neo1-LOV2 incrystal, Raman spectra of free FMN, FKF1-LOV, and a BLUFdomain of AppA, and atomic displacement vectors for somevibrational modes for D450. This material is available free ofcharge via the Internet at http://pubs.acs.org.
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Raman Spectrum of S390 in LOV Domain J. Phys. Chem. B, Vol. 113, No. 9, 2009 2921
