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The rates of photoinduced electron transfer (ET) from Trp168 to the excited isoalloxazine (Iso*) in the pyranose 2-oxidase (P2O) with T169S mutation were studied using ET theory by fitting structural properties calculated from molecular dynamics simulation to the reported fluorescence lifetimes. The obtained ET rates and related physical quantities were compared to those reported for the wild-type (WT) P2O. Experimental fluorescence lifetimes of Iso in T169S have two components of 92–240 fs (depending on the emission wavelength) and 15 ps (independent of the emission wavelength). One of the four T169S subunits (Sub C) displayed a low rate, and the other three (Sub A, B, and D) had high rates. Mean ET rates of the fast components were ca. 10 ps–1 at 480 nm, 7 ps–1 at 500 nm, and 4.2 ps–1 at 530, 555, and 580 nm, which were lower than the reported rates in the WT P2O of ca. 17 ps–1 at 480 nm, 14 ps–1 at 500 nm, 9 ps–1 at 530 and 555 nm, and 11 ps–1 at 580 nm, while the rate for the slow component in T169S (...

The photodissociation dynamics of a hexaarylbiimidazole (HABI) derivative with two pyrenyl groups was investigated by time-resolved transient absorption spectroscopy and fluorescence measurements. Transient absorption spectroscopy revealed that photodissociation took place in the wide time region of

International audience; Subpicosecond transient absorption spectroscopy was used to address the role of the local environment on the photoisomerization process of the p-coumaric thioester chromophore in Photoactive Yellow Protein (PYP) by studying two point mutants, T50V and E46Q. These mutations introduce alterations of the hydrogen-bond network involving the amino acids of the active site close to the chromophore and the chromophore phenolate group, respectively. Transient-absorption spectra of T50V and E46Q are found to be qualitatively similar to those of the wild-type PYP (WT) and R52Q, suggesting that the earliest steps of the photoinduced processes in all three mutants remain similar to those of the WT. Target analyses of the transient spectra of T50V, E46Q, R52Q and WT, were successfully performed by using a model based on the one previously published by Larsen et al. (Biophys. J. 2004, 87, 1858), which involves heterogeneous excited-state populations undergoing deactivation along two competitive relaxation pathways. A so-called reactive pathway leads to the sequential formation of the well-characterized cis intermediates, I0 and I1, of the photocycle. The second pathway is non reactive and produces a transient species that restores the initial trans ground state in 3-6 ps. This transient is tentatively attributed to a distorted vibrationally-hot trans ground state. The most prominent effect of mutation is observed for T50V and R52Q which exhibit significantly slower excited-state deactivations, whereas E46Q behaves like the WT protein. This difference is analyzed in terms of a significant decrease, in T50V and R52Q, of the fraction of heterogeneous excited-state population that undergoes isomerization. The quantum yield of isomerization deduced from the target analyses was found to be 0.31 ± 0.08 for WT, 0.22 ± 0.06 for T50V, 0.29 ± 0.08 for E46Q and 0.19 ± 0.05 for R52Q. The decrease of isomerization yield observed in T50V and R52Q is mainly attributed to the loss of rigidity of the protein active site, induced by these mutations, rather than to the deletion of the positive charge of Arg52 in R52Q.

CT interactions between Iso* and nearby aromatic amino acids in FBP were investigated by a semiempirical MO method. Atomic coordinates of lumiflavin as Iso, 3-methylindole as Trp, and 4-methylphenol as Tyr, used for MO calculations, were obtained from crystal, 20 NMR structures and 40 MD structures (20 ps time intervals). Geometries of Iso-Trp32, Iso-Trp106 and Iso-Tyr35 systems were optimized by the PM3 method. The interaction energies (kcal/mol) of crystal structure were -16.9 in the Iso-Trp32 system, -7.4 in the Iso-Trp106 system and 1.4 in the Iso-Tyr35 system. The interaction energies (kcal/mol) of NMR structures were -16.5 +/- 0.28 in the Iso-Trp32 system, -10.6 +/- 0.14 in the Iso-Trp106 system, and 0.97 +/- 0.09 in the Iso-Tyr35 system. The interaction energies (kcal/mol) of MD structures were -24.3 +/- 0.19 in the Iso-Trp32 system, -10.2 +/- 0.49 in the Iso-Trp106 system, and 0.285 +/- 0.037 in the Iso-Tyr35 system. CT interaction from the aromatic amino acids to Iso* was judged from negative charge at Iso*. The charge in the Iso-Trp32 system was -0.490 in crystals, -0.439 +/- -0.099 in NMR structures, -0.454 +/- 0.048 in MD structures. The charge in the Iso and Trp106 system was -0.011 +/- 0.004 in MD structures, but negligible in other structures. CT interactions in Iso-Tyr35 system were also negligible. The ET rate obtained with Kakitani and Mataga theory and MD decreased as the magnitude of the interaction energy decreased. Correlation between the ET rate and CT interaction in FBP was examined. The interaction energy (Y) was approximated with ln(ET rate) (X) by a function, Y = 0.0036X(3) + 0.0306X(2) - 1.7822X - 21.177.

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 (nu 0), a coefficient of the ET process (beta), a critical distance of the ET process ( R 0), standard free energy related to the electron affinity of the excited Iso ( G Iso (0)), and the static dielectric constant in FBP species (epsilon 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, nu 0, beta, and R 0 were 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.

Ultrafast fluorescence quenching of flavin in flavodoxin from Megasphaera elsdenii was investigated by means of a fluorescence up-conversion method. Fluorescence lifetimes of flavodoxin from M. elsdenii were estimated to be tau(1) approximately 165 fs (0.97%) and tau(2) approximately 10 ps (0.03%). Correlation of photoinduced electron-transfer rates (k(ET)) with averaged distances (D(av)) between isoalloxazine and nearby tryptophan or tyrosine was examined and obtained an empirical equation of ln k(ET) vs D(av) by means of a nonlinear least-squares method using reported data together with flavodoxin from M. elsdenii. The values of D(av) were calculated from X-ray structures of the flavoproteins. The ln k(ET) was approximately linear at D(av) shorter than 7 A. The model free empirical equation was expressed as ln k(ET) = 29.7 + (-0.327 D(av) + 2.84 x 10(-5))/(0.698 - D(av)(2)). We also analyzed the observed values of ln k(ET) with 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.

Photoinduced charge separation (CS) due to electron transfer from the S2 state of zinc porphyrin (ZP) to a directly linked imide (I) has been studied by means of femtosecond fluorescence up-conversion and femtosecond−picosecond transient absorption spectral measurements. S2 fluorescence decay curves of ZP−I dyads in various solvents were all single-exponential, from which accurate rate constants for the CS reaction were obtained. The whole bell-shaped energy-gap law (EGL) has been confirmed unambiguously in polar solvents tetrahydrofurane, acetonitrile, and triacetin as the first example for CS, which can be reproduced on the basis of a nonadiabatic mechanism considering solvent reorganization and intrachromophore high-frequency vibrations (hfm) approximated by an averaged single mode. Mainly the top and inverted regions have been observed in nonpolar solvents cyclohexane and toluene. This result may be ascribed to the large decrease of the solvent reorganization energy in nonpolar solvents, which causes ...

We have studied the fluorescence dynamics of “nonfluorescent” flavoproteins including flavodoxin (FD), its mutants W60F, Y98F, and W60F/Y98F, and riboflavin binding protein (RBP) with the femtosecond fluorescence up-conversion method and have observed the fluorescence quenching dynamics of FD and its mutants for the first time. The strong fluorescence quenching in these flavoproteins seems to be caused by ultrafast electron transfer (ET) from aromatic amino acid residues to the excited flavin chromophore in stacked configuration according to previous transient absorption studies. In the present work, we have made comparative studies on the dynamics of fluorescence quenching due to ET to the excited chromophore in RBP and FD. We have observed also fluorescence dynamics of FD mutants where active electron donors Trp·NH and Tyr·OH are partially (either of them) or completely replaced by inactive phenylalanine and directly demonstrated the ET mechanism of the ultrafast fluorescence quenching in PNS of FD.

We have studied excited-state dynamics of “nonfluorescent” flavoproteins including riboflavin binding protein (RBP), d-amino acid oxidase benzoate complex (DAOB), and others by means of femtosecond fluorescence up-conversion method and have observed ultrafast fluorescence quenching dynamics for the first time. We have interpreted the fluorescence quenching mechanisms of these flavoproteins as due to the ultrafast electron transfer (ET) to flavin chromophore (F) in the excited electronic state from nearby tryptophan (Trp.NH) or tyrosine (Tyr.OH) residues placed in the protein nanospace (PNS), on the basis of their X-ray structures. Extremely fast fluorescence quenching in RBP (τf ∼ 90−100 fs) could be attributed to the compact stacked arrangement, Trp.NH.....F.....Tyr.OH, supremely favorable for the ultrafast ET reaction dynamics. Comparisons of fluorescence time profiles and spectral characteristics of F in solution with those in PNS have indicated the existence of extremely fast FC (Franck−Condon) → Fl (...

The effect of the protein environment surrounding a chromophore [protein nanospace (PNS)] on the photoinduced ultrafast reaction of the chromophore (deprotonated p-coumaric acid covalently bonded to the protein via a thioester linkage) has been investigated by comparing the femtosecond to picosecond fluorescence dynamics of wild-type (w-t) photoactive yellow protein (PYP) with those of the chromophore in aqueous solution as well as in various PNSs modified by site-directed mutagenesis. The fluorescence decay dynamics of the chromophore are considerably accelerated in PNSs compared with those in solution. The ultrafast nonexponential fluorescence decay dynamics of the w-t PYP in the 100 fs to 10 ps time regime, which are probably due to the formation of a twisted state by the flipping of the thioester linkage of the chromophore, have been slowed in the mutants, which seem to have a looser PNS structure because of the weakening of the chromophore−amino acid residue hydrogen-bonding interaction and the parti...

The light-induced photoprocesses of phycocyanobilin were studied by femtosecond spectroscopy. After excitation with 100 fs pulses at 610 nm the transient absorption spectra were measured throughout...

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 (τav = 220 fs), and 730 fs (60%) and longer than 10 ps (40%) in crystals (τav = 4.44 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 A in solution, respectively (obtained by NMR spectroscopy), and 7.05, 7.72, and 8.49 A 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 molecule...

We have studied excited-state dynamics of “nonfluorescent” flavoproteins by means of the femtosecond fluorescence up-conversion method. We have interpreted the ultrafast fluorescence quenching mechanisms of these flavoproteins as due to the ultrafast electron transfer or electron transfer followed by proton-transfer interactions between excited flavin chromophore and nearby tryptophan and tyrosine residues placed in the protein nanospace, on the basis of their X-ray structures. Comparisons of fluorescence time profiles and spectral characteristics of flavin chromophores in solutions with those in protein environments have suggested the existence of extremely fast Franck−Condon → fluorescent state relaxations specific to the protein environments. These results of the ultrafast fluorescence dynamics studies on the nonfluorescent flavoproteins have many features in common with other photobiologically important proteins.