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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 quenching of flavin in flavodoxin from Megasphaera elsdeniiwas investigated by means of a fluorescence up-conversion method. Fluorescence lifetimes of flavodoxin from M. elsdeniiwere estimated to be 1∼ 165 fs (0.97%) and 2∼ 10 ps (0.03%). Correlation of photoinduced electron-transfer rates (kET) with averaged distances (Dav) between isoalloxazine and nearby tryptophan or tyrosine was examined and obtained an empirical equation of ln kETvs Davby means of a nonlinear least-squares method using reported data together with flavodoxin from M. elsdenii. The values of Davwere calculated from X-ray structures of the flavoproteins. The ln kETwas approximately linear at Davshorter than 7 Å. The model free empirical equation was expressed as ln kET29.7 (−0.327Dav2.84 × 10-5)/(0.698 − Dav2). We also analyzed the observed values of ln kETwith Marcus theory, but could not obtain reasonable results. Our analysis suggests that the average distance, rather than the shortest (edge to edge) distance or interplanar angles between the aromatics rings, is the key factor in the process of the photoinduced electron transfer in these flavoproteins. [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]

Ultrafast fluorescence decays of FMN binding proteins (FBP) from Desulfovibrio vulgaris(Miyazaki F) were analyzed with an electron transfer (ET) theory by Kakitani and Mataga (KM theory). Time-dependent distances among isoalloxazine (Iso) and Trp-32, Tyr-35, and Trp-106 in wild-type FBP (WT), among Iso and Tyr-32, Tyr-35, and Trp-106 in W32Y (Trp-32 was replaced by Tyr-32), and among Iso and Tyr-35 and Trp-106 in W32A (Trp-32 was replaced by Ala-32) were determined by molecular dynamic simulation (MD). Electrostatic energies between Iso anion and all other ionic groups, between Trp-32 cation and all other ionic groups, and between Tyr-32 cation and all other ionic groups were calculated in WT, W32Y, and W32A, from the MD coordinates. ET parameters contained in KM theory, such as frequency (ν 0), a coefficient of the ET process (β), a critical distance of the ET process ( R0), standard free energy related to the electron affinity of the excited Iso ( GIso0), and the static dielectric constant in FBP species (ε 0), were determined with and without inclusion of the electrostatic energy, so as to fit the calculated fluorescence decays with the observed decays of all FBP species, by a nonlinear least-squares method according to the Marquardt algorithm. In the analyses the parameters, ν 0, β, and R0were determined separately between Trp residues and Tyr residues among all FBP species. Calculated fluorescence intensities with the inclusion of the electrostatic energy fit quite well with the observed ones of all WT, W32Y, and W32A. [ABSTRACT FROM AUTHOR]

Abstract: A photosynthetic reaction center (RC) complex was isolated from a purple bacterium, Acidiphilium rubrum. The RC contains bacteriochlorophyll a containing Zn as a central metal (Zn-BChl a) and bacteriopheophytin a (BPhe a) but no Mg-BChl a. The absorption peaks of the Zn-BChl a dimer (PZn), the accessory Zn-BChl a (BZn), and BPhe a (H) at 4 K in the RC showed peaks at 875, 792, and 753 nm, respectively. These peaks were shorter than the corresponding peaks in Rhodobacter sphaeroides RC that has Mg-BChl a. The kinetics of fluorescence from PZn *, measured by fluorescence up-conversion, showed the rise and the major decay with time constants of 0.16 and 3.3 ps, respectively. The former represents the energy transfer from BZn * to PZn, and the latter, the electron transfer from PZn to H. The angle between the transition dipoles of BZn and PZn was estimated to be 36° based on the fluorescence anisotropy. The time constants and the angle are almost equal to those in the Rb. sphaeroides RC. The high efficiency of A. rubrum RC seems to be enabled by the chemical property of Zn-BChl a and by the L168HE modification of the RC protein that modifies PZn. [Copyright &y& Elsevier]