J. Phys. Chem. B 2009, 113, 2913–2921
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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 mononucleotide (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 adduct11,12 absorbing maximally at around 390 nm (designated S390) (Figure 1). Formation of the covalent adduct ultimately leads to structural changes in the protein moiety. * 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.
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 concerning 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 (λex ) 647.1 nm) to avoid fluorescence
10.1021/jp808399f CCC: $40.75 2009 American Chemical Society Published on Web 02/06/2009
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Kikuchi et al.
from the sample. A very long lifetime of S390 for FKF1 (a half-life time of 62.5 h at 298 K)17 allows us to measure a highquality Raman spectrum of a flavin-cysteinyl covalent adduct for the first time. We also present the results of normal mode calculations based on the density functional theory (DFT). These studies allow us to assign the observed Raman bands of D450 and S390. The assignment of the Raman spectra offers useful information concerning the structural marker bands for the flavin-cysteinyl covalent adduct. Furthermore, the present study has an important implication for the analysis of the IR spectra of LOV domains. With assignments in hand, vibrational spectroscopy provides a unique approach for studying the protein dynamic 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 subsequent protein purification were performed as described previously.17,18 Preparation of recombinant LOV-containing polypeptide of Arabidopsis thaliana FKF1 protein was prepared by an overexpression system with E. coli. Using Arabidopsis cDNA as a template, a DNA fragment corresponding to the polypeptide was amplified by the PCR method with primers to provide appropriate restriction sites. The amplified DNA was isolated, digested, and cloned into a pGEX4T1 expression vector (Amersham Bioscience, Uppsala, Sweden) as a fusion protein with 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 grown in the dark after the induction by isopropyl-β-D-thiogalactopyranoside. The purification was carried out at 273-277 K under dim red light. The supernatant from the lysate of harvested bacteria was mixed with glutathione-Sepharose 4B (Amersham Bioscience). The FKF1-LOV polypeptides were removed directly from the gel-bound fusion proteins by a thrombin digestion at the linker sequence. The LOV polypeptide was purified further by a gel chromatography with Sephacryl S-100 HR (Amersham Bioscience) and the buffer solution containing 100 mM NaCl, 25 mM Tris-HCl, and 1 mM Na2EDTA (pH 7.8). The purified polypeptide was concentrated to 1.0 mM by ultrafiltration, and the purity was examined by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). Raman Spectroscopy. Raman spectra were obtained as described previously.19-21 A liquid nitrogen cooled CCD detector (Instrument S.A., Inc.) recorded the Raman spectra after a Triax190 spectrometer (Instrument S.A., Inc.) removed the excitation 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° angle relative to the axis of the collection optics. The laser power at the sample was 170 mW. A polarization scrambler is placed at the entrance of the spectrometer. The entrance slit width of 0.3 mm corresponds to a spectral resolution of ∼5 cm-1. For the measurement of S390, the samples were illuminated for a few minutes with a continuous blue light (350-550 nm), and the formation of S390 was confirmed by measuring UV-vis absorption spectra of the sample. All spectra were taken at room temperature (∼23 °C), and a homemade software eliminated the noise spikes in the spectra caused by cosmic rays. The Raman spectra were obtained with samples in a 1.5 × 1.5 × 48 mm quartz cuvette. All Raman spectra were calibrated using neat fenchone.
Figure 2. Raman and difference spectra of FKF1-LOV (traces a-c) as well as corresponding simulated spectra (traces d-f). The observed spectra were obtained at 647.1 nm excitation. (a) D450, (b) S390, and (c) S390-D450. Simulated Raman and difference spectra of the active site models illustrated in Figure 4. Vertical bars represent computed frequencies 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.
DFT Calculation. Among the numerous available DFT methods, we have selected the B3LYP hybrid functional with the 6-31G** basis set because of its high accuracy for predicting vibrational frequencies. It has been shown that this level of DFT calculations yield molecular force fields and vibrational frequencies in excellent agreement with experiments in a variety of systems.22 The optimized geometry, the harmonic vibrational frequencies, and Raman and IR intensities were calculated using the DFT method mentioned above via the Gaussian03 program.23 The calculated frequencies were scaled using a factor of 0.9627.24 Results and Discussion 1. Raman Spectra of FKF1-LOV. Figure 2 shows the Raman spectra of D450 (trace a) and S390 (trace b) for FKF1-LOV, and the frequencies of the observed Raman bands are listed in Tables 1 and 2. In Table 1, reported frequencies as well as isotope shifts for free FAD25 are also listed for a comparison. In addition, Figure 1S in the Supporting Information compares the Raman spectra of free FMN, D450, and a BLUF domain of AppA. As can be seen in Figure 1S and Table 1, the main features of the spectrum for D450 resemble closely those for free riboflavin, FMN, or FAD,19,25,26 indicating that most of the observed bands in the D450 spectrum can be ascribed to vibrational modes for the isoalloxazine ring. A broad Raman band around 1670 cm-1 is absent in free FMN (not shown) and is ascribed to the amide I mode.27 Analogously, the D450 spectrum involves a broad band near 1460 cm-1, which can be characterized by a deformation mode of a CH group in the protein moiety.28
Raman Spectrum of S390 in LOV Domain
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TABLE 1: Observed and Calculated Vibrational Frequency (cm-1) of D450 of FKF1-LOV and Its Modela νobs b
c
νcal d
e
model 2e
FKF1-LOV
FAD
Neo1-LOV2
model 1
1715 1678 1633 1583 1548 1510 1498 1464
1711 (-51,-3)
1710 (-48, 0) 1677 (0, -36)
1745 (-44,-1) 1734 (0, -43) 1619 (-2, 0) 1573 (-8, 0) 1531 (-8, 0) 1517(-19,-1) 1476 (-3, 0) 1455 (-1, 0) 1450 (0, 0) 1417 (-2, 0) 1384 (0, 0) 1382 (0, 0) 1376 (-2, 0) 1371 (0, 0) 1348 (-1, 0)
1407
1353
1249
1627 (-2, 0) 1582 (-4, 0) 1545 (-10,-3) 1501 (-5, -1) 1463 (-5, -1) 1406 (-2, -1)
1351 (-8, -2)
1254 (-4, -19)
1584 (nd,i 0) 1551 (-14,-3) 1507 (-13, 0)
1407 (-6, -2)
1353 (-5, 0)
1333 1324 1312 1293 1268 1256 1245 1199
1272 (-2, -4) 1250 (-7, -11)
(-10,-2) (-5, -1) (-6, -4) (-7, -2) (0, 0) (-1, 0) (-2, -2) (-3, -1)
1722 1698 1619 1568 1529 1512 1477 1455 1450 1417 1387 1383 1381 1370 1443 1436 1347 1326 1319 1298 1276 1258 1252 1212 1198
(-56, 0) (0, -41) (-2, 0) (-9, -1) (-8, -1) (-17, 0) (-3, 0) (-2, -1) (0, 0) (-3, -1) (-1, 0) (-5, -1) (-2, 0) (0, 0) (-1, -1) (-3, 0) (-10,-3) (-4, -1) (-6, -1) (-6, -1) (0, 0) (-1, 0) (-2, -4) (-7, -6) (0, -5)
ν10 ν11 ν12 ν13 ν14 ν15 ν16 ν17 ν18 ν20 ν21 ν22 ν23 ν24 ν25a ν25b ν26 ν27 ν28a ν28b ν29 ν30a ν30b ν31a ν31b
g
h
assignment f
4 5 6 8 9 10 11
75 74 73 72 71
νC4dO νC2dO νCC(ring I, 8b) νCN, νCC(ring I, 8b) νCC(ring I, 8a) νCN, νCC(ring I, 8a) νCC(ring I), Me-deform. Me-deform. Me-deform. Me-deform., νCC(ring I II III) Me-deform., R Me-deform., R Me-deform., R Me-deform., R δN3-H R,δN3-H νCC,νCN(ring I II III), R νCC,νCN(ring I II III), R νCC,νCN(ring I II III), R νCC,νCN(ring I II III), R νCC, νCN(ring II) δC6-H,δC9-H δC6-H,δC9-H νCC(ring I), νCN(ring III) R
69 67 62
13 14 15 16
56
18
55 54 53
19
51
a This table summarizes the observed and calculated vibrational frequencies for D450. b Observed values for D450 of FKF1-LOV. 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 na is the natural abundance. These data are taken from ref 25. d Observed values for D450 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 1 and 2. The numbers in parentheses are 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 calculated normal modes. Approximate descriptions of the calculated modes are also described. ν and δ stand for stretching and bending vibrations, respectively. R indicates vibrations of the ribityl moiety. g Mode numbers for ref 29. h Mode numbers for ref 32. i Not determined. c
TABLE 2: Observed and Calculated Vibrational Frequency (cm-1) of S390 of FKF1-LOV and Its Modela νobs FKF1-LOVb 1725 1626 1597 1549
νcal Neo1-LOV2c 1723 (-50, 0) 1687 (0, -33) 1541 (-25, -3)
1499 1464 1430 1408
1432 (-7, -1)
1374 (-15, -4) 1314
1258 (-2, -7)
model 3e 1751 1743 1614 1569 1557 1478 1497 1455 1443 1403 1398 1388 1383 1376 1366 1355 1352 1331 1307 1293 1267 1255 1227 1211 1194 1189
(-49, -5) (5, -39) (-1, 0) (-1, 0) (-33, -1) (0, 0) (-1, 0) (-1, 0) (-1, 0) (-4, 0) (-3, -1) (0, 0) (0, 0) (-3, 0) (-3, 0) (-3, -1) (-7, 0) (-3, -1) (-6, -1) (-6, 0) (-3, 0) (-1, 0) (-1, -1) (-1, -3) (-2, -2) (0, -1)
model 4e 1729 1703 1613 1573 1542 1499 1495 1455 1448 1413 1398 1387 1383 1380 1367 1445 1441 1352 1315 1298 1279 1259 1239 1221 1200 1191
(-43, 0) (0, -46) (-1, 0) (-1, -1) (-31, -1) (-1, 0) (-1, 0) (0, 0) (-1, 0) (-7- 1) (0, 0) (-1, 0) (-1, 0) (-3, 0) (-3, -1) (-1, -1) (-1, 0) (-15, -8) (1, 0) (-6, -1) (-2, 0) (-1, -1) (-4, -8) (-1, -2) (-1, -1) (0, -1)
assignment e ν10 ν11 ν12 ν14 ν15 δN5-H ν16 ν17 ν18 ν20 ν21a ν21b ν22 ν23 ν24 ν25a ν25b ν26a ν26b ν27 ν28 ν29 ν30a ν30b ν31a ν31b
νC4dO νC2dO νCC(ring I, 8b) νCC(ring I, 8a) νC10adN1 δN5-H, Me-deform. νCC(ring I), Me-deform. Me-deform. Me-deform. νCN(ring II) R R Me-deform., R Me-deform., R Me-deform., R δN3-H, R δN3-H, R νCC, νCN(ring III), R νCC, νCN(ring I), R νCC, νCN(ring I II III), R νCC(ring I) C-H bending R R R R
a This table summarizes the observed and calculated vibrational frequencies for S390. b Observed values for S390 of FKF1-LOV. 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 parentheses are 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 calculated normal modes. Approximate descriptions of the calculated modes are also described. R indicates vibrations of the ribityl moiety.
c
2916 J. Phys. Chem. B, Vol. 113, No. 9, 2009
Figure 3. Comparison of the Raman spectra of FKF1-LOV and the light-induced difference FTIR spectrum of Neo1-LOV2. Raman spectrum for S390 of FKF1-LOV (a), S390 - D450 difference Raman spectrum of FKF1-LOV (b), S390 - D450 difference FTIR spectrum of Neo1-LOV2 at 150 K (c), and vertically inverted Raman spectrum for D450 of FKF1-LOV (d). Trace c is adapted from ref 13.
Figure 2 also illustrates that the Raman spectra of FKF1-LOV exhibit significant changes between D450 and S390. These differences are clearly seen in the S390 minus D450 difference spectrum (trace c). The fraction of the signaling state estimated from the intensity loss of the Raman band around 1350 cm-1 of D450 is larger than 90%. The observed spectral changes accompanying the formation of S390 are summarized in the following four points. (i) A small band at 1715 cm-1 upshifts by ∼10 cm-1. (ii) The bands at 1583, 1548, 1407, and 1353 cm-1 reduce its intensity and/or is shifted. (iii) A new band appears at 1430 cm-1 in S390. (iv) The Raman bands below 1300 cm-1 change their intensities and/or frequencies. In Figure 3, we compare the Raman spectra of FKF1-LOV with the light-induced difference FTIR spectrum of a LOV domain. Since the FTIR data for FKF1-LOV are not available at present, we use the data for Neo1-LOV2 in the figure. The spectra shown in Figure 3 reveal that many of the negative bands in the difference FTIR spectrum coincide with the Raman bands of D450. This implies that the negative IR bands are due to the chromophore vibrations of D450. These observations also suggest that the environment of the FMN chromohore in D450 is similar between FKF1-LOV and Neo1-LOV2. Analogously, several positive features in the difference FTIR spectrum can be correlated to the Raman bands for S390. In order to interpret the Raman and difference IR spectra, we perform normal mode calculations described below. 2. Normal Mode Calculations Based on DFT. Vibrational spectra of the oxidized form of flavin have been extensively studied.19,29-32 However, the assignment of the spectra was not complete. A part of the reasons for the incomplete assignment is due to a relatively large size of flavin molecules. For instance, although lumiflavin (R ) CH3 in Figure 1) has been frequently used for a vibrational analysis, the replacement of the ribityl
Kikuchi et al. moiety of FMN with methyl group distinctly affects normal modes below 1400 cm-1.19 Thus, in this study, we use 7,8dimethyl-10-glycerylisoalloxazine as a chromophore model for D450 (model 1 in Figure 4). In addition, model 2 contains acetamide to mimic the hydrogen bonds of the isoalloxazine ring with the nearby amino acid residues. Methanol is also included as a model of Ser930 to consider a hydrogen bond at the carboxyl group of Gln1029. Although the crystal structure of FKF1-LOV is not available, amino acid residues in the active site are conserved.18 Thus, these components were arranged on the basis of the crystal structure of D450 in Neo1-LOV233 and subsequently optimized to yield the structures illustrated in Figure 4. Table 1S in the Supporting Information gives the optimized geometries along with the experimental parameters of Neo1-LOV2 in crystal.33 A comparison between models 1 and 2 shows that the formation of hydrogen bonds around the ring III moiety (Figure 1) causes the single-double bond alternation to be less significant. For example, the change of model 1 to 2 shortens the N3-C4 bond length (1.3825 f 1.3718 Å), whereas the C4dO bond length is lengthened (1.2171 f 1.2277 Å). In the case of S390, we use ethanethiol as a model of the conserved cysteine residue (Cys966 for Neo1-LOV2), which forms a flavin-cysteinyl adduct (model 3 in Figure 4). We have also considered an active site model that incorpolates three acetamide molecules to mimic hydrogen bonds with the surrounding amino acid residues (model 4). Similar to the case of model 2, the crystal structure of S390 in Neo1-LOV212 was used as an initial structure for the subsequent geometry optimization. Table 1S reports the optimized geometries, and the main geometrical difference between D450 and S390 models can be found in the vicinity of the C4a position. For example, the change from model 2 to 4 significantly increases the bond distance between the C4a and N5 positions (1.3023 f 1.4178 Å). Next we have calculated vibrational frequencies as well as Raman intensities of the chromophore models described above. Tables 1 and 2 summarize the computed frequencies as well as approximate descriptions of the vibrational modes for D450 and S390, respectively. The calculated isotope shifts for the 4,10a13 C2 and 2-13C flavin chromophores are also shown in the tables. Figure 2S in the Supporting Information and Figure 5 illustrate atomic displacements for the important normal modes for D450 and S390, respectively. Note that we have chosen to retain the mode labels for lumiflavin19 in the present study, while Table 1 also lists mode numbers used in Abe and Kyogoku29 and Eisenberg and Schelvis32 for comparison. The lower part of Figure 2 shows the simulated Raman spectra of models 2 (trace d) and model 4 (trace e) as well as their difference spectra (trace f). Although the agreements between the experiments and calculations are not perfect, the simulated spectra, especially simulated difference spectrum (trace f), capture main features of the observed spectra. For example, 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. These results allow us to assign most of the Raman bands of D450 and S390. In addition to the analysis of the Raman spectra, the present DFT calculations are also useful to interpret the IR spectra of LOV domains. The upper parts of Figure 6 are the difference FTIR spectra of Neo1-LOV2 reconstituted with unlabeled FMN (a, black line; b and c, green lines), [4,10a13 C2]FMN (b, black line), and [2-13C]FMN (c, black line). These spectra are taken from ref 13. To analyze these difference FTIR
Raman Spectrum of S390 in LOV Domain
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Figure 4. Optimized geometry of four active site models. 7,8-Dimethyl-10-glycerylisoalloxazine is used to mimic FMN, and its adduct with ethanethiol 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 moieties are removed from the figure.
spectra, we have used models 2 and 4 for D450 and S390, respectively. The lower parts of Figure 6 are the simulated difference 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 and calculated spectra demonstrates satisfactory agreements between the experiment and calculation. 3. Assignments for D450. Several earlier studies provided assignment of Raman and infrared spectra for flavin.19,29-32 Here we improve the confidence level in the assignments by applying the DFT method to extended molecular models shown in Figure 4. Although most of our results are consistent with the calculations of previous studies, some of the assignments are updated as described below.
A. CdO Stretching Mode; ν10- ν11. The weak but distinct Raman band at 1715 cm-1 for D450 in Figure 2 (trace a) is assigned to the carbonyl C4dO stretching vibrations ν10 of the isoalloxazine ring based on the observed and calculated (1722 cm-1) frequencies. This assignment is consistent with the FTIR data for Neo1-LOV2 as well as Raman data for free FAD25 and a BLUF protein.19 The DFT calculation using model 2 predicts the C2dO stretching mode ν11 of the isoalloxazine ring below 1700 cm-1 (Table 1). Unfortunately, a broad amide I band around 1670 cm-1 makes the observation of ν11 difficult. However, the S390 - D450 difference Raman spectrum (Figure 2, trace c) shows a negative feature at 1678 cm-1, which is assignable to ν11. This
2918 J. Phys. Chem. B, Vol. 113, No. 9, 2009
Figure 6. Observed and simulated difference IR spectra of LOV domains. The light-induced difference FTIR spectra of Neo1-LOV2 reconstituted 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) and corresponding simulated difference IR spectra of model 4 minus model 2 (d-f). Vertical blue and red bars represent computed frequencies and intensities for models 4 and 2, respectively. Gaussian band shapes with a 10 cm-1 width are used to simulate the spectra. The FTIR spectra (traces a-c) are adapted from ref 13.
assignment agrees with a FTIR study on Neo1-LOV2, where ν11 was detected at 1677 cm-1.13 B. CdC or CdN Stretching Mode; ν12-ν18. The Raman bands at 1633, 1583, and 1548 cm-1 for FKF1-LOV correspond to those observed at 1627, 1582, and 1545 cm-1 for free FAD,25 respectively. As summarized in Table 1, the FAD bands are sensitive to the 13C substitutions and are assigned to CdC and/or CdN stretching vibrations of the isoalloxazine ν13, and ν14, respectively (Figure 2S). The ν13 and ν14 modes involve a larger contribution of a motion of C10a atom compared to that of ν12, accounting for larger 4,10a-13C2 shifts in ν13 and ν14. The ν15 mode involves C4adN5 and C10adN1 stretching vibrations like ν13 but with different phase (Figure 2S). The DFT calculations using model 2 predict ν15 at 1512 cm-1, and this mode is assigned to a Raman band at 1510 cm-1. This assignment 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 shift upon 4,10a-13C2 substitution, which is consistent with a -17 cm-1 shift for model 2. The ν16 mode is mainly due to ring I vibrations 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 confirm the assignment because of a -5 cm-1 4,10a-13C2 shift for free FAD,25 which corresponds to a -3 cm-1 shift at 1477 cm-1 for model 2. Note that the ring I vibration of ν16 corresponds to mode 11 of Abe and Kyogoku,29 although the mode 11 was assigned to a band around 1465 cm-1. C. C-C Stretching/C-H Rocking Modes; ν17-ν31. As described above, a Raman band around 1465 cm-1 was assigned to the ring I vibration (mode 11) in Abe and Kyogoku.29
Kikuchi et al. However, the present and previous normal mode calculations based on DFT assigned this band to methyl deformation modes ν17 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 AppA126 is very broad compared to that for free FMN (Figure 1S) because of an overlap of CH2 scissoring modes of protein moiety.28 The band observed at 1407 cm-1 was ascribed to the isoalloxazine ring I and II vibrations ν21-ν24 in previous studies,19,31,32 where lumiflavin was used as a model of FMN or FAD. However, replacement of the methyl group with the glyceryl group in models 1 and 2 induces a significant change in their normal mode composition. These modes are calculated to be mainly methyl deformation vibrations of the isoalloxazine ring (Figure 2S), and the 1407 cm-1 band can be ascribed to an overlap of these modes on the basis of moderate 4,10a-13C2 and 2-13C shifts for the corresponding band at 1406 cm-1 for free FAD.25 The overlapping ν21-ν24 band is also observed in the difference FTIR spectrum of Neo1-LOV2 at 1407 cm-1 (Figure 3). The strong Raman band at 1353 cm-1 has been assigned to an overlap of the chromophore skeleton vibrations ν26 and ν27 for a BLUF protein.19 This assignment is confirmed by the present analysis; i.e., the observed -8 cm-1 shift upon 4,10a-13C2 substitution for free FAD25 correlates well with the calculated shifts for ν26 and ν27 (-10 and -4 cm-1) for model 2. Raman bands below 1350 cm-1 contain significant contributions from protein moieties. Thus, the interpretation of the spectra is difficult unless we have data for isotopically labeled samples. On the other hand, the FTIR data for Neo1-LOV2 that contains isotopically labeled FMN are available,13 so that several IR bands can be assigned. As shown in Figure 6, a negative IR band at 1272 cm-1 exhibits -2 and -4 cm-1 shifts upon 4,10a-13C2 and 2-13C substitutions, respectively. We assign this band to the overlapping ν30a and ν30b modes, since the latter mode shows -2 cm-1 4,10a-13C2 and -4 cm-1 2-13C shifts for model 2. Although these modes are mainly allocated to the C6-H/C9-H bending coordinates (Figure 2S), a small contribution from the N1-C2 and N3-C4 stretching motions in ν30b accounts for the 12C/13C isotope shifts. These modes are not clearly seen in the Raman spectra of FKF1-LOV (Figure 3) as well as algal Phot-LOV1.15 As illustrated in Figure 6, a negative IR band at 1250 cm-1 shows large isotope shifts for the 4,10a-13C2 (-7 cm-1) and 2-13C (-11 cm-1) substitutions. Simulated difference IR spectra (traces e and f) reproduce similar isotope effects around 1210 cm-1, leading to an assignment of the band to an overlap of ν31a and ν31b. These modes consist primarily 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 for S390 (trace b). This band is assigned to the carbonyl C4dO stretching vibrations ν10 on the basis of the calculated frequency (1729 cm-1) for model 4. This assignment is supported by the FTIR spectra for Neo1-LOV2, where ν10 is observed at 1723 cm-1.13 On the other hand, the C2dO stretching mode ν11, which is predicted at 1703 cm-1 for model 4, is not detected in the Raman spectra. B. CdC or CdN Stretching Mode; ν12-ν16. For D450, the 1500-1650 cm-1 region contains normal modes that involve motions of C4a atom. Thus, these modes exhibit significant changes upon formation of the C4a-cysteinyl adduct in S390. The CdC and CdN stretching modes ν12 and ν14 for S390 are expected to be shifted by about -5 and +45 cm-1, respectively,
Raman Spectrum of S390 in LOV Domain compared to those for D450 (Tables 1 and 2). A small positive feature at 1626 cm-1 in the difference Raman spectrum (Figure 2, trace c) is assigned to ν12 for S390 based on a 7 cm-1 downshift from 1633 cm-1 for D450. The difference IR spectrum of Neo1-LOV2 exhibits corresponding bands at 1633 cm-1 (D450) and 1626 cm-1 (S390) (Figures 3 and 6). Although these bands were mainly ascribed to the protein moiety,13 ν12 may partially contribute to the spectrum. The difference Raman spectrum shows a clear positive band at 1597 cm-1, and we assign this band to ν14 on the basis of a 49 cm-1 upshift from 1548 cm-1 in D450 (Figure 2). Although the ν14 band is clearly seen in the Raman spectrum, the corresponding IR band has not been observed. Both ν13 and ν15 modes involve C10adN1/C4adN5 stretching vibrations for D450 (Figure 2S). The ν15 mode for S390 is mainly allocated to stretching of the C10adN1 bond (Figure 5). The Raman band observed at 1549 cm-1 for S390 can be assigned to ν15 because of its frequency and ca. +39 cm-1 shift upon formation of S390 (Tables 1 and 2). In the difference IR spectrum, the ν15 mode was observed as a prominent positive band at ca. 1540 cm-1 for various LOV domains (Figure 3).14-16,33 The assignment of this IR band to ν15 is confirmed by 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 which is 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 because the adduct formation at the C4a position of the isoalloxazine ring makes the C4adN5 double bond to a single bond. The DFT calculations using model 4 predict ν16, the combined CdC stretching and C-H bending vibrations of the ring I, at 1495 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 reproduced in the simulated difference spectrum (trace f). We assign this 1430 cm-1 band to the ring II vibrations including N5-H bending ν20, which is also observed at 1432 cm-1 in the difference IR spectrum for Neo1-LOV213 (Figure 3, trace c). The assignment of ν20 is supported by the IR data; i.e., the observed -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 case of D450, the Raman spectrum of S390 exhibits a band at 1408 cm-1 (Figure 2, trace b). This band can be assigned to methyl deformation vibrations ν21-ν24 of the chromophore based on its frequency. As described in the previous section, the ν26 mode for D450 involves the CC and CN stretching vibrations of rings I, II, and III of the isoalloxazine (Figure 2S). The adduct formation in S390, however, decouples the ring I and ring III vibrations, providing two modes of ν26a and ν26b (Figure 5, Table 2). The ν26a mode is mainly allocated to the CN stretching coordinates of ring III and is predicted at 1352 cm-1 for model 4. The DFT calculations indicate that this mode is expected to have a moderate IR intensity, and a positive IR band at 1374 cm-1 for Neo1-LOV213 is assigned to ν26a based on the -15 cm-1 isotopic shift with 4,10a-13C2 substitution (Figure 6b). The ring I vibration ν26b is calculated at 1315 cm-1 for model 4 and has a moderate intensity in the simulated Raman spectrum (Figure 2, trace e). We assign ν26b to a Raman band at 1314 cm-1 because of its frequency. The corresponding band was not detected in the difference IR spectra for Neo1-LOV2 (Figure 3).13
J. Phys. Chem. B, Vol. 113, No. 9, 2009 2919 Figure 6 shows a small positive band at 1258 cm-1 in the difference 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, at 1239 cm-1 for model 4. As illustrated in Figure 5, ν30a is partially allocated to CH bending motions of the reactive cysteine residue. Note that the C-S stretching vibrations are expected below 800 cm-1 and are not detected in the present study. 5. Implications. The present study reports the Raman spectrum of FKF1-LOV in the D450 state, and Figure 1S compares the spectrum with that of a BLUF domain from AppA.19 A light illumination causes a formation of a covalent adduct in a LOV domain, whereas the photoreaction of the BLUF domain involves a rearrangement of hydrogen bonds around the C4dO moiety of FAD.36 In spite of a striking difference in their primary photochemistry, however, their Raman spectra under the dark state are quite similar as illustrated in Figure 1S. For example, frequency differences of the Raman bands due to isoalloxazine ring are within (4 cm-1.37 In some sense, this observation is reasonable because of a notable similarity in the flavin binding interactions of these two flavincontaining photoreceptors.33,38-40 In fact, Suzuki et al.41 recently showed that the light-induced formation of a cysteinyl-flavin adduct can be reproduced in the BLUF domain by introducing a cysteine residue near the isoalloxazine ring. In addition to D450, we report the Raman spectra of FKF1-LOV in the S390 state. The very long lifetime of S390 for FKF1-LOV17 allows us to measure the Raman spectrum of S390 for the first time. The observation of the Raman spectra leads to the assignment of vibrational modes in the 1200-1700 cm-1 region, and we characterize useful marker bands for the active site structure of LOV domains. It also has an important implication for the analysis of IR spectra of LOV domains. A noticeable change in the Raman spectrum upon formation of S390 is a ∼10 cm-1 upshift of the C4dO stretching vibration ν10 (Figure 2). A similar upshift of ν10 was observed in the difference FTIR spectra of various LOV domains.14-16,34,35 Possible explanations of the observed upshifts in ν10 are a weakening of the hydrogen bonds at the CdO groups13 and an adduct formation of the FMN chromophore.14,15 The present DFTcalculationsdemonstratethattheformationofacysteinyl-flavin adduct without changing a hydrogen bond (models 1 f 3) accounts for a 6 cm-1 upshift of ν10, which is roughly half of the observed shift. Thus, the light-induced upshift of ν10 can be ascribed to changes in the hydrogen bonds as well as chromophore structural changes associated with adduct formation. As illustrated in Figure 3 (trace c), the difference IR spectrum of LOV domains shows several bands that arise from S390. A prominent positive band around 1540 cm-1 is assigned to the C10adN1 stretching vibration ν15. In the adduct, the C4a carbon acquires a nonplanar sp3 tetrahedral configuration, leading to dramatic changes in the vibrational modes that involve the C4a atom. In Table 2, a comparison of the ν15 frequencies between models 3 and 4 demonstrates that the formation of hydrogen bonds at the C2dO, N3-H, and C4dO positions downshifts ν15 by 15 cm-1 (1557 f 1542 cm-1). Thus, the ν15 band in the difference IR spectra could reflect chromophore-protein interactions in S390. For instance, a FTIR study on full-length YtvA and its isolated LOV domain showed that the positive IR band around 1540 cm-1 is significantly perturbed by the presence of the signaling domain.42 This observation may indicate the structural changes in the active site of the LOV domain in the presence of the downstream effector domain.
2920 J. Phys. Chem. B, Vol. 113, No. 9, 2009 The positive feature at ∼1260 cm-1 in the difference IR spectrum is assigned to ν30a of S390. Because this mode involves the C-H bending motions of the reactive cysteine residue, it may act as a structural probe around the C-S covalent bond. In fact, previous FTIR studies on LOV domains showed that this region of the spectra is sensitive to the protein structures; e.g., Iwata et al.34 reported that the difference FTIR spectra around 1260 cm-1 distinctly differ between LOV1 and LOV2 domains from Neo1. Although we cannot rule out a contribution of the vibrational modes from a protein moiety, the observed spectral differences could reflect structural differences around the C-S bond. Finally, we discuss a functional implication of the measurement of the Raman spectra of FKF1-LOV. One of the characteristic features of FKF1-LOV is its formation of a stable photointermediate S390. As mentioned above, Zikihara et al.17 demonstrated that the dark recovery of S390 to D450 with a half-life time of 62.5 h, which is much longer than the time constants from several seconds to a few minutes for the LOV domains of phototropin.43 This extremely slow recovery may derive from a nine amino acid insertion between the R′(A)helix having the conserved cysteine and R′(C)-helix as compared to those of phototropin families. In spite of this structural difference, however, Figure 3 indicates little spectral differences between FKF1-LOV and Neo1-LOV2. This observation suggests that the active site structure of FKF1-LOV is not significantly different from that of Neo1-LOV2. It is therefore likely that the slower recovery of S390 in FKF1-LOV than that in Neo1-LOV2 originates from differences in protein structure and/or protein-chromophore interactions between the two LOV domains. In summary, this study presents the Raman investigation of FKF1-LOV, and we report the Raman spectrum of S390 for LOV domains for the first time. In addition, most of the observed Raman bands are assigned with the aid of DFT calculations. Furthermore, the present study has an important implication for the analysis of the IR spectra of LOV domains. With these assignments in hand, vibrational spectroscopy provides an important approach for studying the photocycle mechanism in LOV domains. Acknowledgment. We appreciate Drs. H. Kandori and T. Iwata (Nagoya Institute of Technology) for allowing us to use their FTIR data in Figures 3 and 6. A part of the computations was performed using Research Center for Computational Science, Okazaki, Japan. Supporting Information Available: Optimized geometries along with the experimental parameters of Neo1-LOV2 in crystal, Raman spectra of free FMN, FKF1-LOV, and a BLUF domain of AppA, and atomic displacement vectors for some vibrational modes for D450. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Van der Horst, M.; Hellingwerf, K. J. Acc. Chem. Res. 2004, 37, 13–20. (2) Briggs, W. R.; Beck, C.; Cashmore, A. R.; Christie, J. M.; Hughes, J.; Jarillo, J. A.; Kagawa, T.; Kanegae, H.; Liscum, E.; Nagatani, A. Plant Cell. 2001, 13, 993–997. (3) Sakai, T.; Kagawa, T.; Kasahara, M.; Swartz, T., E.; Christie, J. M.; Briggs, W. R.; Wada, M.; Okada, K. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 6969–6074. (4) Kinoshita, T.; Doi, M.; Suetugu, N.; Kagawa, T.; Wada, M.; Shimazaki, T. Nature (London) 2001, 414, 656–660. (5) Sakamoto, K.; Briggs, W. R. Plant Cell 2002, 14, 1723–1735.
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