8695
2007, 111, 8695-8697 Published on Web 07/04/2007
Comparison between Ultrafast Fluorescence Dynamics of FMN Binding Protein from DesulfoWibrio Wulgaris, Strain Miyazaki, in Solution vs Crystal Phases Haik Chosrowjan,† Seiji Taniguchi,† Noboru Mataga,† Fumio Tanaka,*,† Daisuke Todoroki,§ and Masaya Kitamura§ Institute for Laser Technology, Utsubo-Honmachi 1-8-4, Nishiku, Osaka 550-0004, Japan, SC1-413 Department of Chemistry, Faculty of Science, Mahasarakham UniVersity, Mahasarakham 44150, Thailand, and Department of Applied and Bioapplied Chemistry, Graduate School of Engineering, Osaka City UniVersity, Sumiyoshiku, Osaka 558-8585, Japan ReceiVed: May 14, 2007; In Final Form: June 18, 2007
Ultrafast fluorescence dynamics of FMN binding protein (FBP) from DesulfobiVrio Vulgaris, strain Miyaxaki F, were compared in solution and crystal phases. Fluorescence lifetimes of FBP were 167 fs (96%) and 1.5 ps (4%) in solution (τav ) 220 fs), and 730 fs (60%) and longer than 10 ps (40%) in crystals (τav ) 4.44 ps). The quenching of the fluorescence of flavin in the protein was considered to be due to photoinduced electron transfer (ET) from Trp or Tyr to the excited isoalloxazine (Iso) nearby. The average lifetime was 20 times longer in crystal vs in solution. Averaged distances between Iso and nearby Trp-32, Tyr-35, and Trp-106 were 8.42, 7.36, and 8.15 Å in solution, respectively (obtained by NMR spectroscopy), and 7.05, 7.72, and 8.49 Å in crystal, respectively (obtained by X-ray crystallography). The prolonged lifetime in crystal cannot be elucidated by the change in the distances between the states. It was suggested that the longer lifetime in crystal was ascribed to the absence of water molecules around FBP with rapid motional freedom, which may be the driving force for the ET in flavoproteins.
Introduction
Materials and Methods
Bright fluorescence of flavins is often almost completely quenched when they bind to proteins. In these flavoproteins, Trp, Tyr, or both always exist near the isoalloxazine ring (Iso) of flavins.1-4 It was demonstrated that the remarkable quenching of flavin fluorescence in flavoproteins was ascribed to photoinduced electron transfer (ET) from Trp or Tyr to the excited Iso.3,5-7 The photoinduced ET process in flavoproteins plays an important role in photoreceptors.8 Although many kinds of ET theories for bulk solution have been reported,9-11 the precise mechanism of ET in proteins is still unclear. It is considered that a driving force for the photoinduced ET is vibrational motions of solvent molecules around the donor and acceptor in the bulk solution.9-12 The driving force for the ET in flavoproteins, however, has not been identified, since the microenvironment around the donor and acceptor in the proteins is always heterogeneous. It is of interest to obtain fluorescence lifetimes of a flavoprotein in the crystal form, because it is expected to reveal the role of water molecules for the photoinduced ET. In the present letter, we report comparison of the fluorescence lifetimes of FMN binding protein (FBP) from DesulfoVibrio Vulgaris, strain Miyazaki, between the solution and crystal phases.
Materials Preparation. FMN binding protein from DesulfoVibrio Vulgaris (Miyazaki F) was prepared basically according to the method described elsewhere.13 Crystallization of FBP was performed according to the method by Suto et al.14 Fluorescence Up-Conversion Techniques. The femtosecond time-resolved fluorescence decays of FMN binding protein in solution were measured using a conventional fluorescence upconversion apparatus, as described elsewhere.1-3 To measure the fluorescence decay of FMN binding protein crystal, we introduced the second harmonics of the Ti:Sapphire laser (∼0.5 mW) into an inverted confocal microscope (IX71, Olympus) from the back port. The fluorescence from the sample, positioned on the stage center plate, was collected in the backscattering geometry and guided to the outside of the microscope from the right side port. Then the fluorescence was focused into a nonlinear BBO crystal (0.4 mm), where it was mixed with the time-delayed fundamental laser pulses and upconverted. The signal registration part was identical to the one used in the conventional up-conversion measurements described above. We used a Schwartzshield refractive objective lens (×40) and a special design of the microscope’s inner light-guiding optics, achieving a time resolution of ∼200 fs. The spatial resolution was estimated to be ∼2 µm. Results and Discussions
†
Institute for Laser Technology. ‡ Mahasarakham University. § Osaka City University.
10.1021/jp073702k CCC: $37.00
Fluorescence decay of the Iso of FBP in aqueous solution is shown in Figure 1. The decay curve was analyzed with a two© 2007 American Chemical Society
8696 J. Phys. Chem. B, Vol. 111, No. 30, 2007
Letters
Figure 1. Fluorescence decay of FMN binding protein in aqueous solution measured at 500 nm (O) and biexponential fitting (s). The upper panel shows the fitting residuals.
Figure 3. The structure of FMN binding protein in the liquid state obtained by NMR spectroscopy. One of 20 structures were drawn by a software of Rasmol with atomic coordinates obtained from Protein Data Bank (code 1AXJ).
Figure 2. Fluorescence decay of FMN binding protein in the crystal phase measured at 500 nm (O) and biexponential fitting (s). The upper panel shows the fitting residuals.
exponential decay function, as described in previous works.1-3 The lifetimes were 167 fs (96%) and 1.5 ps (4%) in solution (τav ) 220 fs). The decay curve of FBP in crystal is shown in Figure 2. The lifetimes were 730 fs (60%) and longer than 10 ps (40%) in crystal (τav ) 4.44 ps). The averaged lifetime in crystal was more than 20 times longer than that in solution. The longer lifetime is not ascribed to free FMN, because it cannot dissociate from the protein moiety in crystal. Twenty different structures of FBP in solution were reported by Liepinsh et al.15 by means of NMR spectroscopy. In solution, FBP is a monomer, whereas in crystal, it forms a dimer. Figure 3 shows one of these structures near an Iso binding site. Averaged distances between the Iso and Trp-32, the Iso and Tyr-35, and the Iso and Trp-106 were 8.42, 7.36, and 8.15 Å, respectively. The averaged distances here were obtained by averaging distances over all pairs of aromatic atoms in Iso and aromatic atoms in Trp or Tyr and then averaging them over 20 structures. Figure 4 shows the structure of FBP near the Iso binding site in crystal obtained by means of the X-ray diffraction method.14 Averaged distances between the Iso and Trp-32, the Iso and Tyr-35, and the Iso and Trp-106 in crystal were 7.05, 7.72, and 8.49 Å, respectively. The average distances here mean distances averaged over all pairs between atoms in the Iso and aromatic atoms in Trp or Tyr and then further averaged over those between both subunits. In the liquid phase, the shortest distance was 7.36 Å between the Iso and Tyr-35. In crystal, the shortest distance was 7.06 Å between the Iso and Trp-32. We have shown that the logarithm of the ET rate decreases rather slowly with an averaged distance shorter than 7 Å and does not correlate with edge-to-edge (shortest) distances in flavoproteins.16 The shortest averaged distance in crystal (Trp32-Iso, 7.06 Å) was shorter than the shortest average distance in solution (Tyr35-Iso, 7.36 Å); nevertheless, the average lifetime in crystal
Figure 4. The structure of FMN binding protein in the crystal phase obtained by X-ray crystallography. The structures were drawn by a software of Rasmol with atomic coordinates obtained from Protein Data Bank (code 1FLM).
is 20 times longer than that in solution. The ET rate in flavoproteins was rather insensitive to the interplanar angle between the donor and acceptor, as well.16 Accordingly, the very long lifetime of FBP in crystal compared to that in solution cannot be elucidated by the difference in the donor-acceptor distance or the change in the interplanar angles in solution vs crystal phases. The most significant difference in the environment of the ET donor and acceptor of FBP in liquid vs solid state is the presence of water molecules with motional freedom around the donor and acceptor in solution. The solvent-accessible surface area of FBP in solution is 34%, whereas it is 13% in crystal as other flavoproteins.14 These facts suggest that the driving force of the photoinduced ET in flavoproteins could be the rapid rotational and vibrational motions of water molecules. Fluorescence lifetimes of FBP in deuterium oxide could be modified from those in water, because vibrational frequencies are smaller in deuterium oxide than in water. Rapid wobbling motions of ionic amino acid residues in FBP may also influence ET processes.17 Glu-13 is located between Trp-32 and Tyr-35 in crystal.14 Motional freedom of these ionic charges may be restricted in crystal, which may also contribute to the longer lifetime in crystal.
Letters References and Notes (1) Mataga, N.; Chosrowjan, H.; Shibata, Y.; Tanaka, F. J. Phys. Chem. B 1998, 102, 7081-7084. (2) Mataga, N.; Chosrowjan, H.; Shibata, Y.; Tanaka, F.; Nishina, Y.; Shiga, K. J. Phys. Chem. B 2000, 104, 10667-10677. (3) Mataga, N.; Chosrowjan, H.; Taniguchi, S.; Tanaka, F.; Kido, N.; Kitamura, M. J. Phys. Chem. B 2002, 106, 8917-8920. (4) Tanaka, F.; Mataga, N. Trends Chem. Phys. 2004, 11, 59-74. (5) Karen, A.; Ikeda, N.; Mataga, N.; Tanaka, F. Photochem. Photobiol. 1983, 45, 495-502. (6) Karen, A.; Sawada, M. T.; Tanaka, F.; Mataga, N. Photochem. Photobiol. 1987, 45, 49-54. (7) Zhong, D.; Zewail, A. H. Proc. Natl. Acad. Sci. 2001, 98, 1186711872. (8) Sancar, A. Annu. ReV., Biochem. 2000, 69, 31. (9) Marcus, R. A. Annu. ReV. Phys. Chem. 1964, 15, 155-196.
J. Phys. Chem. B, Vol. 111, No. 30, 2007 8697 (10) Bixon, M.; Jortner, J.; Cortes, J.; Heitele, H.; Michel-Beyerle, M. E. J. Phys. Chem. 1994, 98, 7289-7299. (11) Kakitani, T.; Matsuda, N.; Yoshimori, A.; Mataga, N. Prog. React. Kinet. 1995, 20, 347-375. (12) Bicout, D. J.; Szabo, A. J. Chem. Phys. 1997, 106, 10292-10298. (13) Kitamura, M.; Kojima, S.; Ogasawara, K.; Nakaya, T.; Sagara, T.; Niki, K.; Miura, K.; Akutsu, H.; Kumagai, I. J. Biol. Chem. 1994, 269, 5566-5573. (14) Suto, K.; Kawagoe, K.; Shibata, N.; Morimoto, K.; Higuchi, Y.; Kitamura, M.; Nakaya; T.; Yasuoka, N. Acta Crystallogr., Sect. D 2000, 56, 368-371. (15) Liepinsh, L.; Kitamura, M.; Murakami, T.; Nakaya, T.; Otting, G. Nat. Struct. Biol. 1997, 4, 975-979. (16) Tanaka, F.; Chosrowjan, H.; Taniguchi, S.; Mataga, N.; Sato, K.; Nishina, Y.; Shiga, K. J. Phys. Chem. B 2007, 111, 5694-5699. (17) Callis, P. R.; Liu, T. Chem. Phys. 2006, 326, 230-239.
