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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 C1protein in buffer solution were expressed by two-exponential functions. Correlation times, ϕ1and ϕ2, of p-coumaric acid were 0.053 and 0.650 ps, respectively, which was similar to previous works. ϕ1and ϕ2of C1were 0.455 and 250 ps, respectively. The Stokes shift from t= 0 to t= ∞ was 2200 cm−1, while it is 500 cm−1in 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 C1is exposed in hydrophilic environment. Such large Stokes shift was unusual among flavoproteins. The biphasic decay of the spectral correlation function in C1was discussed and compared to the biphasic decay of tryptophan in proteins. [ABSTRACT FROM AUTHOR]

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 (av220 fs), and 730 fs (60%) and longer than 10 ps (40%) in crystals (av4.44 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. [ABSTRACT FROM AUTHOR]