Effects of the Disappearance of One Charge on Ultrafast Fluorescence Dynamics of the FMN Binding Protein Haik Chosrowjan,* ,† Seiji Taniguchi, † Noboru Mataga, † Takeshi Nakanishi, ‡ Yoshihiro Haruyama, ‡ Shuta Sato, ‡ Masaya Kitamura,* ,‡ and Fumio Tanaka* ,§ Institute for Laser Technology, Utsubo-Hommachi 1-8-4, Nishiku, Osaka 550-0004, Japan, Department of Applied Chemistry and Bioengineering, Graduate School of Engineering, Osaka City UniVersity, 3-3-138 Sugimoto, Sumiyoshiku, Osaka 558-8585, Japan, and Department of Biochemistry, Center for Excellence in Protein Structure and Function, Faculty of Science, Mahidol UniVersity, 272 Rama VI Road, Bangkok, 10400, Thailand ReceiVed: December 23, 2009; ReVised Manuscript ReceiVed: March 2, 2010 Crystal structures of E13T (Glu13 was replaced by Thr13) and E13Q (Glu13 was replaced by Gln13) FMN binding proteins (FMN-bp) from DesulfoVibrio Vulgaris, strain Miyazaki F, were determined by the X-ray diffraction method. Geometrical factors related to photoinduced electron transfer from Trp32, Tyr35, and Trp106 to the excited isoalloxazine (Iso*) were compared among the three forms of FMN-bp. The rate of ET is considered to be fastest from Trp32 to Iso* in FMN-bp and then from Tyr35 and Trp106. The distances between Iso and Trp32 did not change appreciably (0.705-0.712 nm) among WT, E13T, and E13Q FMN- bps, though the distances between Iso and Tyr35 or Trp106 became a little shorter by ca. 0.01 nm in both mutated FMN-bps. The distances between the residue at 13 and the ET donors or acceptor in the mutated proteins, however, changed markedly, compared to WT. Hydrogen bonding pairs and distances between Iso and surrounding amino acids were not modified when Glu13 was replaced by Thr13 or Gln13. Effects of elimination of ionic charge at Glu13 on the ultrafast fluorescence dynamics in E13T and E13Q were investigated comparing to WT, by means of a fluorescence up-conversion method. Fluorescence lifetimes were τ 1 ) 107 fs (R 1 ) 0.86), τ 2 ) 475 fs (R 2 ) 0.12), and τ 3 ) 30 ps (R 3 ) 0.02) in E13T and τ 1 ) 134 fs (R 1 ) 0.85), τ 2 ) 746 fs (R 2 ) 0.12), and τ 3 ) 30 ps (R 3 ) 0.03) in E13Q, which are compared to the reported lifetimes in WT, τ 1 ) 168 fs (R 1 ) 0.95) and τ 2 ) 1.4 ps (R 2 ) 0.05). Average lifetimes (τ AV ) ∑ i)1 2or3 R i τ i ) were 0.75 ps in E13T, 1.10 ps in E13Q, and 0.23 ps in WT, which implies that τ AV was 3.3 times longer in E13T and 4.8 times longer in E13Q, compared to WT. The ultrafast fluorescence dynamics of WT did not change when solvent changed from H 2 O to D 2 O. Static ET rates (inverse of average lifetimes) were analyzed with static structures of the three systems of FMN-bp. Net electrostatic (ES) energies of Iso and Trp32, on which ET rates depend, were 0.0263 eV in WT, 0.322 eV in E13T, and 0.412 eV in E13Q. The calculated ET rates were in excellent agreement with the observed ones in all systems. Introduction Photoinduced electron transfer (ET) in proteins is very important for photosynthetic processes and also for photore- ceptors in plants and bacteria. 1 Recently, a number of new flavin photoreceptors have been found. 2-4 In these flavin photorecep- tors, cryptochrome and BLUF contain aromatic amino acids as Trp and Tyr near isoalloxazine (Iso). ET from Tyr21 to Iso* in BLUF (blue-light sensing using flavins) of AppA is considered to be a first step of its function. 5-7 The ultrafast fluorescence dynamics of various “nonfluores- cent” flavoproteins were investigated by means of an up- conversion method. 8-10 In these nonfluorescent flavoproteins, Trp and/or Tyr always exist near Iso. Femtosecond transient absorption spectra of these flavoprotein systems revealed that the back ET reaction from the excited to the ground state takes place within 8 ps in riboflavin binding protein and 30 ps in glucose oxidase, after ET from Trp and/or Tyr to the excited flavin. 11 Three-dimensional structures of WT FMN-bp were deter- mined by X-ray crystallography 12 and NMR spectroscopy. 13 According to these structures, Trp32 was closest to Iso, followed by Tyr-35 and Trp106. Various ET theories have been modeled for bulk solution. 14-19 It is not yet clear which factor is the most important for ET in proteins. There should be something different between ET processes in protein and ones in bulk solution. The ET rate of FMN-bp was much slower in crystal than in solution, 20 which could not be explained solely by donor-acceptor distance. The results suggest an influential factor for ET in FMN-bp other than donor-acceptor distance, but the reason for it is not yet known. The fluorescence lifetimes were compared among WT, W32Y (Trp-32 was replaced by Tyr-32), and W32A (Trp-32 was replaced by Ala-32) FMN-bps. 21 Fluorescence dynamics of WT, W32Y, and W32A were simultaneously analyzed with KM theory and atomic coordinates obtained by MD. 22,23 In these works it was found that electro- static interaction between the Iso anion and other ionic amino acids and between ET donor cations and other ionic amino acids are important. In the present work, we have determined crystal structures of E13T and E13Q where Glu13 with a negative * Corresponding authors. † Institute for Laser Technology. ‡ Osaka City University. § Mahidol University. J. Phys. Chem. B 2010, 114, 6175–6182 6175 10.1021/jp912137s  2010 American Chemical Society Published on Web 04/16/2010