Effects of the Disappearance of One Charge on Ultrafast Fluorescence Dynamics of the FMN Binding Protein

Crystal structures of E13T (G1u13 was replaced by Thr13) and E13Q (G1u13 was replaced by G1n13) 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 lso and Trp32 did not change appreciably (0.705-0.712 nm) among wr, Elyr, and E13Q FiV1Nbps, 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 Is and surrounding amino acids were not modified when Glu 13 was replaced by Thr13 or Gin 13. Effects of elimination of ionic charge at Glu 13 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 tau(1) = 107 fs (alpha(1) = 0.86), tau(2) = 475 fs (alpha(2) = 0.12), and tau(3) = 30 Ps (alpha(3) = 0.02) in El3T and tau(1) = 134 fs (alpha(1) = 0.85), tau(2)= 746 fs (alpha(2) = 0.12), and tau(3) = 30 ps (alpha(3) = 0.03) in E13Q, which are compared to the reported lifetimes in WT, tau(1) = 168 fs (alpha(1) = 0.95) and tau(2) = 1.4 Ps (alpha(2) = 0.05). Average lifetimes (tau(Av) = Sigma(2or3)(i=1)alpha(i)iota(o)) were 0.75 ps in E13T, 1.10 ps in E13Q, and 0.23 ps in WT, which implies that tau(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 H2O to D2O. Static Er 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.