Search

Impulsive excitation of molecular vibration is known to induce wave packets in both the ground state and excited state. Here, the ultrafast dynamics of PYP was studied by pump-probe spectroscopy using a sub-8 fs pulse laser at 400 nm. The broadband spectrum of the UV pulse allowed us to detect the pump-probe signal covering 360-440 nm. The dependence of the vibrational phase of the vibrational mode around 1155 cm(-1) on the probe photon energy was observed for the first time to our knowledge. The vibrational mode coupled to the electronic transition observed in the probe spectral ranges of 2.95-3.05 and 3.15-3.35 eV was attributed to the wave packets in the ground state and the excited state, respectively. The frequencies in the ground state and excited state were determined to be 1155 ± 1 and 1149 ± 1 cm(-1), respectively. The frequency difference is due to change after photoexcitation. This means a reduction of the bond strength associated with π-π* excitation, which is related to the molecular structure change associated with the primary isomerization process in the photocycle in PYP. Real-time vibrational modes at low frequency around 138, 179, 203, 260, and 317 cm(-1) were also observed and compared with the Raman spectrum for the assignment of the vibrational wave packet.

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 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 E13T and tau(1) = 134 fs (alpha(1) = 0.85), alpha(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 alpha(2) = 1.4 ps (alpha(2) = 0.05). Average lifetimes (tau(AV) = Sigma(i=1)(2or3)alpha(i)tau(i)) 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 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.

The reductase unit of p-hydroxyphenylacetate hydroxylase contains flavin mononucleotide (FMN) as a cofactor. Fluorescence decay curves measured by fluorescence up-conversion method were remarkably dependent on monitored emission wavelength. The fluorescence lifetime was shorter at the shorter emission wavelengths and longer at the longer wavelengths. Spectral shift correlation function of p-coumaric acid in water and FMN in C1 protein in buffer solution were expressed by two-exponential functions. Correlation times, phi1 and phi2, of p-coumaric acid were 0.053 and 0.650 ps, respectively, which was similar to previous works. phi1 and phi2 of C1 were 0.455 and 250 ps, respectively. The Stokes shift from t=0 to t=infinity was 2200 cm(-1), while it is 500 cm(-1) in the static Stokes shift obtained by the solvent effect of the fluorescence spectrum under static excitation. This suggests that the isoalloxazine ring of FMN in C1 is exposed in hydrophilic environment. Such large Stokes shift was unusual among flavoproteins. The biphasic decay of the spectral correlation function in C1 was discussed and compared to the biphasic decay of tryptophan in proteins.