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In the present work, we discuss about the relationship between the energy gap law and extended Dutton law in flavoproteins. The extend Dutton law is defined herein as the dependence of logarithmic rates (ln Rate) of photoinduced electron transfer (ET) from aromatic amino acids to excited isoalloxazine (Iso*) on donor–acceptor distances (Rcs). Both functions of ln Rate vs. negative values of the standard free energy gap and ln Rate vs. Rc display a parabolic behavior, when the ET rates are ultrafast. The negative values of the standard free energy gap at peaks of ln Rate [Xm(ES)] were obtained for FMN-binding protein, wild-type pyranose 2-oxidase, T169S (Thr169 is replaced by Ser) pyranose 2-oxidase, and medium-chain acyl-CoA dehydrogenase. The values of Rc at peaks of ln Rate [Xm(Rc)] were also obtained for these flavoproteins. The negative values of the standard free energy gap decreased with approximate linear functions of Rc. The negative values of standard free energy gap [Xm(ESRc)] at Rc = Xm(Rc) were evaluated using the linear functions of the negative standard free energy gap with Rc. The values of Xm(ESRc) were mostly in very good agreement with the values of Xm(ES). This implies that the energy gap law and the extend Dutton law are equivalent. Xm(ES) values in ET donors displaying the linear extend Dutton law with Rc were obtained by energy gap law, and then Xm(Rc) values were evaluated with the negative standard free energy gap. Thus, the obtained Xm(Rc) values were much smaller than the Rc range obtained by the method of molecular dynamics simulation. This suggests that ET processes with linear profiles of the extend Dutton law could be parabolic when Rc becomes much shorter than the Rc range obtained by the method of molecular dynamics simulation.

Development, characterization, and control of large diameter (10-cm) deformable mirrors for multi kW-class CW lasers, capable of operating at a 10-kHz sampling rate, have been described, and their operation has been verified experimentally.

Dynamics of protein structures of five FMN binding isomers, WT, E13K, E13R, E13T and E13Q, were studied through time-dependent residue electrostatic energies (RESTs) of ionic residues and their correlation coefficients. The Arg86 and Glu119 have high positive correlation coefficients which are higher than 0.7 in most of FBP isomers. The Glu23 and FMN, Asp24 and FMN, and Asp61 and Arg63 pairs displayed relatively high negative coefficients. In these residues, the distributions of RESTs showed a few peaks. REST pairs have high positive coefficients when distribution of REST of one residue overlapped with that of REST of another residue, while the REST pairs displayed high negative coefficients when a peak distribution of REST of one residue overlapped with a minimum distribution of another residue. Correlations of REST pairs were not simple and displayed a few groups. Relationships were examined among the correlation coefficients, inter-residue distance (R) and signs of the charge. In the case of R ≤ 1 nm, the total number of the pairs with high negative coefficients in all FBP isomers was much greater when the pairs possess opposite signs of the charge, compared to that when the pairs possess same signs of the charge.

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 (...

Absorption spectrum of ortho-amino benzoate (oAB) – D-amino acid oxidase complex (ODC) displays a charge transfer (CT) band around 570 nm. The fluorescence decays of the second lowest electronic state of ODC were measured using a fluorescence up-conversion technique. The lifetimes were 100 fs and 130 fs at 500 nm and 514 nm fluorescence emission wavelengths, respectively. These ultrafast lifetimes are ascribed to the photoinduced electron transfer (PET) from the oAB to the excited isoalloxazine (Iso*) in the ODC. The PET mechanism in ODC was studied with Marcus-Kakitani-Mataga (MKM) theory. The protein structures were obtained by molecular dynamics simulation (MDS). Mean distances between Iso and oAB over 12,500 MDS snapshots were 0.60 in subunit A (Sub A) and 0.58 nm in Sub B. The calculated lifetimes at 514 nm-emission were 97 and 179 fs in Sub A and Sub B, while at 500 nm there were 97 and 101 fs, respectively. Solvent reorganization energy was quite different between Sub A and Sub B, which is main reason for difference in the calculated lifetimes. Electrostatic (ES) energies between the photoproducts of donor -acceptor, and net ES energies between the photoproducts and ionic groups in the protein were calculated with MDS structures. These ES energies were evaluated with charge densities of neutral radical of oAB produced after PET and anion radical of Iso obtained by semi-empirical molecular orbital (MO) method. The logarithmic PET rate did not display any clear dependence on Rc, despite the ultrafast scale of the PET rates. Most of the logarithmic PET rates in Sub A displayed in the inverted region of the standard free energy gap in the energy gap law. The transition dynamics among local excited states of Iso, CT state and the ion-pair state were discussed.

Molecular interactions between o-aminobenzoate (oAB) and isoallxazine (Iso) in o-aminobenzoate - d -amino acid oxidase complex (ODC) were studied by means of molecular dynamics simulation (MDS) and molecular orbital (MO) method. The distances between oAB and Iso were 0.60 in subunit A (Sub A) and 0.58 nm in Sub B. Four of six heteroatoms of Iso ring formed hydrogen bonding (HB) with nearby amino acids. The oxygen atom and nitrogen atom of oAB also formed HB with amino acids. The ODC displays a broad absorption band around 570 nm, which is considered to be charge transfer (CT) band between oAB and Iso in the complex. The ODC displayed fluorescence at around 520 nm, which is different from that of DAAO (emission peak at 535 nm) without oAB. The emission is from a local excited state (LE) without CT interaction. The CT interaction was studied by a semi-empirical molecular orbital (MO) method, using the structures of oAB and Iso obtained by MDS. The CT state did not form in the ground state of Iso, but formed in the excited state of Iso (Iso*), when the both cannot move appreciably from the MDS structures. The CT states formed both in the oAB – Iso and oAB – Iso* (excited state of Iso) systems when the both molecules can move so as to attain the minimum energy state. It is concluded that a part of oAB could move to form CT complex with Iso in the ground state, which displays the CT absorption band, but others could not move in the protein without CT interaction, which displays the fluorescence.

Pyranose 2-oxidase (P2O) from Trametes multicolor contains FAD as cofactor, and forms a tetramer. The protein structure of a mutated P2O, T169S (Thr169 is replaced by Ser), in solution was studied by means of molecular dynamics simulation and analyses of photoinduced electron transfer (ET) from Trp168 to excited isoalloxazine (Iso*), and was compared with wild type (WT) P2O. Hydrogen bonding between Iso and nearby amino acids was very similar as between T169S and WT protein. Distances between Iso and Tyr456 were extremely heterogeneous among the subunits, 1.7 (1.5 in WT) in subunit A (Sub A), 0.97 (2.2 in WT) in Sub B, 1.3 (2.1 in WT) in Sub C, 1.3 nm (2.0 in WT) in Sub D. Mean values of root of mean square fluctuation over all residues were greater by four times than those in WT. This suggests that the protein structure of T169S is much more flexible than that of WT. Electrostatic (ES) energies between Iso anion in one subunit and ionic groups in the entire protein were evaluated. It was found that more than 50% of the total ES energy in each subunit is contributed from other subunits. Reported fluorescence decays were analyzed by a method as WT, previously reported. Electron affinities of Iso* in T169S were appreciably higher than those in WT. Static dielectric constants near Iso and Trp168 were also quite higher in T169S than those in WT. This article is protected by copyright. All rights reserved.

Conformational changes in the single mutated flavin mononucleotide binding proteins (FBP), E13K (Glu13 of wild type FBP is replaced by Lys), E13R (Glu13 replaced by Arg), E13T (Glu13 replaced by Thr) and E13Q (Glu13 replaced by Gln) upon dimer formations were studied through the phenomena of photoinduced electron transfer (ET) from tryptophan 32 (Trp32) and tryptophan 106 (Trp106) to the excited isoalloxazine (Iso∗). The ET rates were obtained with atomic coordinates of the proteins determined by a method of molecular dynamics simulation (MDS) and an ET theory using the reported experimental fluorescence decays of the FBPs. The value of the net electrostatic energy (NetES) between the photo-products and ionic groups inside the proteins increased in magnitude upon the dimer formation in every mutated FBP. Dependencies in the logarithmic ET rate (ln Rate) on the donor-acceptor distances (Rc) could not be approximated with simple linear nor parabolic functions in most ET donors, which are ascribed to the relatively greater values of the NetES in the dimers compared to the energy terms of the standard free energy gap (SFEG), solvent reorganization energy (SROE) and the electrostatic energy between the photo-products (ESDA) in total free energy gap (GT) in the ET rates. The NetES vs Rc relations displayed two distinct groups in Trp32KB, Trp32QA and Trp32QB. The ln Rate vs Rc relations in these donors displayed different behaviors between the two groups.

Fluorescence dynamics of human d -amino acid oxidase (hDAAO) and its five inhibitors have been studied in the picoseconds time domain, and compared with one in d -amino acid oxidase from porcine kidney (pkDAAO) reported. The fluorescence lifetimes were identified as 47 ps in the dimer, 235 ps in the monomer, which are compared with those of pkDAAO (45 ps–185 ps). The fluorescence lifetimes of the hDAAO did not change upon the inhibitor bindings despite of modifications in the absorption spectra. This indicates that the lifetimes of the complexes are too short to detect with the picosecond lifetime instrument. Numbers of the aromatic amino acids are similar between the both DAAOs. The fluorescence lifetimes of hDAAO were analysed with an ET theory using the crystal structure. The difference in the lifetimes of the dimer and monomer was well described in terms of difference in the electron affinity of the excited isoalloxazine (Iso*) between the two forms of the protein, though it is not known whether the structure of the monomer is different from the dimer. Three fastest ET donors were Tyr314, Trp52 and Tyr224 in the dimer, while Tyr314, Tyr224 and Tyr55 in the monomer, which are compared to those in pkDAAO, Tyr314, Tyr224 and Tyr228 in the dimer, and Tyr224, Tyr314 and Tyr228 in the monomer. The ET rate from Trp55 in hDAAO dimer was much faster compared to the rate in pkDAAO dimer. A rise component with negative pre-exponential factor was not observed in hDAAO, which are found in pkDAAO.

Rates of photoinduced electron transfer (ET) from tryptophan residues (Trps) to excited isoalloxazine (Iso*) of flavin adenine dinucleotide (FAD) in medium-chain acyl Co A dehydrogenase from porcine kidney (MCAD) have been studied using its entire tetrameric structures obtained by molecular dynamics simulation (MDS) and experimental ultrafast fluorescence decays reported. The ET rates were fastest from Trp166 to Iso* among the four Trps. The emission-wavelength dependent decays were elucidated by introducing emission-wavelength dependent electron affinity of Iso*. The ET rates in Sub C were much slower than those in other subunits, which was ascribed to non-equivalent electrostatic interactions among the subunits. Effect of the protein association on the ET rates and related physical quantities were examined comparing those in the tetramer and monomer reported. The donor-acceptor distances in the tetramer were shorter than those in the monomer. The ET rates from Trp166 in the tetramer were slower than that in the monomer. The ET rates and related physical quantities were examined at seven emission wavelengths. The values of standard free energy gap between the photo-products and reactants displayed to increase as the emission wavelength becomes longer at all subunits. The ET rates displayed to decrease in Sub A, Sub C and Sub D, as the emission wavelength becomes longer.

Ultrafast transient absorption (TA) spectroscopy has been one of the most powerful experimental tools to study the mechanism of photoinduced electron transfer (ET) as in photosynthetic and flavin photoreceptor systems in plants. However, no work has been reported on their quantitative mechanisms. Apparent rates of charge separation (CS) from tryptophans (Trps) to the excited isoalloxazine (Iso*) and charge recombination (CR) from the produced ion pairs to Trps and Iso in the ground states are reported to be 0.25 ps and 3.2 ps in medium-chain acyl-CoA dehydrogenase (MCAD), and 0.15 ps and 6.6 ps in flavin mononucleotide binding protein (FMN-bp), obtained by an ultrafast TA method. The decays of the CS and CR processes were for the first time simultaneously analyzed with an ET theory and structures obtained by molecular dynamics simulation. MCAD and FMN-bp form a tetramer and dimer, respectively. The CS and CR rates of an individual donor and various related physical quantities were numerically obtained. It was found that both CS and CR rates were fastest from Trp166 among four Trps in MCAD and those from Trp106 among two Trps in FMN-bp. Logarithmic CS rates in MCAD were dependent on the donor–acceptor distance (Rc) with parabolic functions, while those of CR rates linearly decreased with Rc. Reasons why CS rates were faster than CR rates in both MCAD and FMN-bp were elucidated in terms of pre-exponential factors in the theory. The present method could be useful to understand the precise mechanisms of initial steps of biological functions of photoreceptors in plants.

Mechanisms of photoinduced electron transfer (ET) from tryptophanes 32 and 106 in subunits A and B (Trp32A, Trp32B, Trp106A and Trp106B) of wild type flavin mononucleotide binding protein (FBP) dimer were studied through relations of the logarithmic ET rate (ln Rate) vs the donor-acceptor distance (Rc). The sum (GT) of standard free energy gap (SFEG) between the products and reactants, electrostatic energy (ESDA) between the photo-products and solvation reorganization energy (SROE) and electrostatic energy (NetES) between the photo-products and ionic groups inside the protein were numerically determined for the all donors with atomic coordinates obtained by molecular dynamic simulation. The GT values of Trp32A and Trp32B displayed always negative in the entire Rc range, which predicts that ET rate becomes slower as the Rc shorter. The reason of negative GT values in Trp32A and Trp32B were numerically elucidated with the mean values of SFEG, ESDA, SROE, and NetES.

A CO 2 laser can be used to melt ice. Here we use a CO 2 laser at 10.6 μm, a wavelength at which ice strongly absorbs, to drill (via melting) through ice. The resulting drilling speed is measured at several irradiation intensities, ice-snow densities, and beam angles relative to the horizontal axis. The speed increases nearly in proportion to the laser intensity. For an intensity of about 50 W/cm 2 , for instance, the melting speed is 4 mm/s for snow of density 153 kg/m 3 and 0.8 mm/s for solid ice. Results also show that for downward beam angles, melt-water accumulates in the hole, reducing the drilling speed. Nevertheless, we also consider other laser mediums and argue that an optical-fiber-coupled laser drilling system could be used for drilling on glaciers and ice sheets.

Pyranose 2-oxidase (P2O) from Trametes multicolor forms a homotetramer in which each of the subunits contains flavin adenine dinucleotide (FAD). The fluorescence of P2O decays with two lifetime components; a slow (358 ps) and a fast (∼90 fs) decay. The lifetime of the fast component is emission-wavelength dependent and is ascribed to fast photoinduced electron transfer (ET) from Trp168 to the excited isoalloxazine (Iso*) in FAD. The donor–acceptor distances were ∼0.7 nm. The extraordinary heterogeneous decays were analyzed with atomic coordinates obtained by a molecular dynamics simulation and the ET rate by Kakitani and Mataga. The emission-wavelength dependent decays in the fast component were elucidated by introducing emission-wavelength dependent standard free energy related to electron affinity of Iso*. Examination of all possible combinations of the four subunits revealed that the slow component was from subunit A and the fast component was from the other three subunits. Agreements between the observed and calculated decays were all excellent. The large difference in the fast and slow fluorescent lifetimes is ascribed to the difference in the standard free energy gap related to electron affinity of Iso*. The dependence of the logarithmic ET rates on the center-to-center distance displayed approximate linear functions (Dutton rule) when the rate was relatively slow and parabolic functions when the rate was ultrafast. The Dutton rule originated from the exponential term of the ET rate, not from the electronic coupling term.

The basic principles and main characteristics of the ultrafast time-resolved fluorescence upconversion technique (conventional and space-resolved), including requirements for nonlinear crystals, mixing spectral bandwidth, acceptance angle, etc., are presented. Applications to flavoproteins [wild-type (WT) FMN-binding protein and its W32Y, W32A, E13R, E13K, E13Q and E13T mutants] and photoresponsive proteins [WT photoactive yellow protein and its R52Q mutant in solution and as single crystals] are demonstrated. For flavoproteins, investigations elucidating the effects of ionic charges on ultrafast electron transfer (ET) dynamics are summarized. It is shown that replacement of the ionic amino acid Glu13 and the resulting modification of the electrostatic charge distribution in the protein chromphore-binding pocket substantially alters the ultrafast fluorescence quenching dynamics and ET rate in FMN-binding protein. It is concluded that, together with donor-acceptor distances, electrostatic interactions between ionic photoproducts and other ionic groups in the proteins are important factors influencing the ET rates. In WT photoactive yellow protein and the R52Q mutant, ultrafast photoisomerization dynamics of the chromophore (deprotonated trans-p-coumaric acid) in liquid and crystal phases are investigated. It is shown that the primary dynamics in solution and single-crystal phases are quite similar; hence, the photocycle dynamics and structural differences observed at longer time scales arise mostly from the structural restraints imposed by the crystal lattice rigidity versus the flexibility in solution.

Pyranose 2-oxidase (P2O) from Trametes multicolor contains FAD as cofactor, and forms a tetramer. The protein structure of a mutated P2O, T169S (Thr169 is replaced by Ser), in solution was studied by means of molecular dynamics simulation and analyses of photoinduced electron transfer (ET) from Trp168 to excited isoalloxazine (Iso*), and was compared with wild type (WT) P2O. Hydrogen bonding between Iso and nearby amino acids was very similar as between T169S and WT protein. Distances between Iso and Tyr456 were extremely heterogeneous among the subunits, 1.7 (1.5 in WT) in subunit A (Sub A), 0.97 (2.2 in WT) in Sub B, 1.3 (2.1 in WT) in Sub C, 1.3 nm (2.0 in WT) in Sub D. Mean values of root of mean square fluctuation over all residues were greater by four times than those in WT. This suggests that the protein structure of T169S is much more flexible than that of WT. Electrostatic (ES) energies between Iso anion in one subunit and ionic groups in the entire protein were evaluated. It was found that more than 50% of the total ES energy in each subunit is contributed from other subunits. Reported fluorescence decays were analyzed by a method as WT, previously reported. Electron affinities of Iso* in T169S were appreciably higher than those in WT. Static dielectric constants near Iso and Trp168 were also quite higher in T169S than those in WT.

Flavin mononucleotide (FMN)-binding proteins (FBP) from Desulfovibrio vulgaris , Miyazaki F, contain FMN as a cofactor. Upon photo-excitation of the FBP, photoinduced electron transfer (ET) takes place from tryptophan (Trp)32, tyrosine (Tyr)35 and Trp105 residues to the excited isoalloxazine ring (Iso ∗ ) of FMN. The ultrafast fluorescence dynamics of the wild type, and the E13K, E13R, E13T and E13Q substitution isoforms of FBP were simultaneously analyzed with molecular dynamics simulation structures and Kakitani-Mataga ET theory, to obtain the time-dependent ET rates. A bell-shaped behavior of the logarithmic ET rates (In k ET ) from Trp32 and Trp106 to Iso ∗ versus the centre-to-centre donor–acceptor distances ( Rc ) was obtained in the time domain of sub-ps to ps in all five FBP isoforms. The In k ET from Tyr35 to Iso ∗ linearly decreased with the Rc distance, which were much slower than those of Trp32 and Trp105. These findings suggest that the nuclear term in the ET theory is important in the ultrafast time domain, whereas the electronic coupling term may be dominant in slower ET rates.

The mechanism of photoinduced electron transfer (ET) from benzoate (Bz) and aromatic amino acids to the excited isoalloxazine (Iso*) in the D-amino acid oxidase–benzoate complex (DAOB) dimer from porcine kidney was studied using molecular dynamics simulation (MDS) and an electron transfer theory, and compared with that in the DAOB monomer. The DAOB dimer displayed two fluorescent lifetime components of 0.85 ps and 4.8 ps, as reported. The ET parameters contained in the Kakitani and Mataga (KM) model were determined so as to reproduce these lifetimes with MDS atomic coordinates. The Bz–isoalloxazine (Iso) distances were 0.66 nm in subunit A (Sub A), 0.68 nm in subunit B (Sub B) and 0.61 nm in the monomer. The fluorescent lifetimes of 4.8 ps and 0.85 ps were found to originate from Sub A and Sub B, respectively. In Sub A, Tyr228 was the fastest ET donor followed by Bz and Tyr55, while Bz was followed by Tyr228 and Tyr314 in Sub B. The ET rate from Bz was fastest in Sub B, followed by that in Sub A and the DAOB monomer. The static dielectric constants obtained near Iso were 2.4–2.6 in the DAOB dimer and monomer and 5.8–5.9 in holo D-amino oxidase (DAAO). The different dielectric constants could account for the experimental fluorescence peak observed for DAOB (524 nm) and DAAO (530 nm). Logarithmic ET rates decreased linearly with the donor–acceptor distance expressed by both center to center distance (Rc) and edge to edge distance (Re) in Sub A and Sub B of DAOB dimer and monomer, which reveals that the conventional Dutton rule holds in the ET processes in DAOB. The logarithmic ET rates were decomposed into the electronic coupling (EC), square root (SQ) and exponential (GTRAM) terms. It was found that both the EC term and the GTRAM term also decreased linearly with Rc. The sum of the slopes in the EC and GTRAM vs. Rc plots coincided with the slopes in the logarithmic ET rate vs. Rc functions, suggesting that the GTRAM term makes a significant contribution to the linear relations between logarithmic ET rate and Rc.

Photoinduced electron transfer in the Helicobacter pylori flavodoxin (HPFD) was investigated using both experimental and theoretical techniques. Recombinant (r)HPFD was prepared by expression in Escherichia coli and the ultrafast fluorescence dynamics of the purified rHPFD was measured by means of fluorescence up-conversion. The fluorescence was found to decay with a lifetime of 173 fs (amplitude, 0.963) and 2.08 ps (0.037). These data were used in the Kakitani and Mataga theory to calculate the electron transfer (ET) rate inside the native HPFD. Molecular dynamics simulations of HPFD were performed over a 50 ns period to enable an inclusion of the protein dynamics effect on the ET process. The ET rate from Tyr91 to an excited isoalloxazine (Iso*) ranged from 6 to 23 ps −1 , depending on the analysis method used, while the ET rates from Trp63 and Tyr120 to Iso* were negligibly slow. The logarithmic ET rate and net electrostatic (ES) energy between the photo-products and ionic groups inside HPFD both displayed a parabolic relationship with the total free energy gap, indicating that the net ES energy is one of the most influential factors upon the ultrafast ET rate. Although the logarithmic ET rate should theoretically be decreased with increasing center-to-center distances according to Dutton's law, this relationship was not observed in this system. One possible reason for this was elucidated by comparing our results with those from the flavin mononucleotide binding protein.

Crystal structures of E13K [Glu13 with negative charge in wild type (WT) was replaced by Lys with positive charge] and E13R (Glu13 was replaced by Arg with positive charge) of flavin mononucleotide...

The photochemistry of fac-[Re(bpy)(CO)(3)Cl] (1 a; bpy=2,2'-bipyridine) initiated by irradiation using330 nm light has been investigated. Isomerization proceeded in THF to give the corresponding mer-isomer 1 b. However, in the presence of a small amount of MeCN, the main product was the CO-ligand-substituted complex (OC-6-24)-[Re(bpy)(CO)(2) Cl(MeCN)] (2 c; bpy=2,2'-bipyridine). In MeCN, two isomers, 2 c and its (OC-6-34) form (2 a), were produced. Only 2 c thermally isomerized to produce the (OC-6-44) form 2 b. A detailed investigation led to the conclusion that both 1 b and 2 c are produced by a dissociative mechanism, whereas 2 a forms by an associative mechanism. A comparison of the ultrafast transient UV-visible absorption, emission, and IR spectra of 1 a acquired by excitation using higher-energy light (e.g., 270 nm) and lower-energy light (e.g., 400 nm) gave detailed information about the excited states, intermediates, and kinetics of the photochemical reactions and photophysical processes of 1 a. Irradiation of 1 a using the higher-energy light resulted in the generation of the higher singlet excited state with τ≤25 fs, from which intersystem crossing proceeded to give the higher triplet state ((3)HES(1)). In THF, (3)HES(1) was competitively converted to both the triplet ligand field ((3)LF) and metal-to-ligand charge transfer ((3)MLCT) with lifetimes of 200 fs, in which the former is a reactive state that converts to [Re(bpy)(CO)(2)Cl(thf)](+) (1 c) within 10 ps by means of a dissociative mechanism. Re-coordination of CO to 1 c gives both 1 a and 1 b. In MeCN, irradiation of 1 a by using high-energy light gives the coordinatively unsaturated complex, which rapidly converted to 2 c. A seven-coordinate complex is also produced within several hundred femtoseconds, which is converted to 2 a within several hundred picoseconds.

Pyranose 2-oxidase from Trametes multicolor (P2O) catalyzes oxidations of aldopyranoses by using molecular oxygen. The enzyme is composed of four identical subunits with a native molecular weight of 270 kDa and each of which contains flavin adenine dinucleotide (FAD) as a cofactor. P2O contains the Trp168 residue near the flavin isoalloxazine ring (Iso), which can potentially act as an electron donor to the excited Iso (Iso*). Iso is covalently linked to His167 in WT P2O, but not in H167A (His167 replaced by Ala). Ultrafast fluorescence dynamics of H167A, T169S (Thr169 replaced by Ser) and acetate complexes with WT and T169S were studied by the fluorescence up-conversion method. The fluorescence decays were well described with two-exponential functions. The shorter fluorescence lifetimes ( τ 1 ) were 87–184 fs depending on an emission wavelength, and the longer lifetimes ( τ 2 ) was 120 ps without the emission wavelength-dependence in H167A. The shorter lifetimes of T169S were 92–240 fs, and the longer lifetime 15 ps. In WT P2O–acetate complex τ 1 was 110 fs and τ 2 50 ps, and in T169S P2O–acetate complex τ 1 was 250 fs and τ 2 300 ps at 530 nm. The ratio of the lifetime ( τ 2 / τ 1 ) was extraordinary high compared to the other flavoproteins. This result was interpreted as two conformers of P2O, Conformers 1 and 2 (Conf 1 and Conf 2), in which each was associated with τ 1 and τ 2 , respectively. The ultra-short lifetimes were ascribed to photoinduced electron transfer (ET) mainly from Trp168 to the excited Iso (Iso*). ET rates of Confs 1 and 2 were calculated based on structural parameters reported. According to the crystal structures, distances between Iso and Trp168 were not different among the four subunits. It was found that static dielectric constants of the Conf 1 with τ 1 ( ɛ 1 = 3.51) was different from that of the Conf 2 with τ 2 ( ɛ 2 = 5.95). Fluorescence spectra with τ 1 and τ 2 were constructed based on the emission-wavelength dependent-decay functions. Emission peak with τ 1 was time-dependent from the wavelengths shorter than 480 to 540 nm within 1 ps after excitation, whereas that with τ 2 was steady at around 530 nm.

Ultrafast fluorescence dynamics of flavin adenine dinucleotide (FAD) in wild type pyranose 2-oxidase (P2O) has been investigated in solution by means of fluorescence up-conversion method. Fluorescence decays were well described by two-exponential model function. Fluorescence lifetimes were τ1 ∼ 110 fs and τ2 ∼ 360 ps, respectively. The (τ2/τ1) ratio (∼3200) was extraordinary high compared to other flavoproteins without subunit structure. The heterogeneous distribution of emission lifetimes were elucidated in terms of two different conformers of P2O; conformer 1 with τ1 and conformer 2 with τ2. Emission peaks of conformer 1 and conformer 2 were determined to be at ∼540 nm and 510 nm, respectively, using transient spectral reconstruction procedure. Using dynamics analysis by Kakitani and Mataga (KM) theory, both quenching processes were ascribed to photoinduced electron transfer (ET) reactions mainly from Trp168 to the excited isoalloxazine (Iso*) in different protein tetramers having different static dielectric constants (ɛ1 ∼ 3.25 for conformer 1 and ɛ2 ∼ 5.93 for conformer 2). The quaternary structure seems to be responsible for the observed conformational heterogeneity.

Medium chain acyl-CoA dehydrogenase (MCAD) binds flavin adenine dinucleotide (FAD) as cofactor, and catalyze α,β-dehydrogenation of fatty acid acyl-CoA conjugates as the first step of β-oxidation of fatty acids in mitochondria. The dynamic properties of geometrical factors as distances between isoalloxazine (Iso) and the aromatic amino acids of Trp and Tyr were studied by molecular dynamic (MD) simulation. The center-to-center distances ( R c ) between Iso and aromatic amino acids were shortest in Tyr365 (0.99 nm) and then in Trp156 (1.00 nm), Tyr123 (1.29 nm), Tyr362 (1.42 nm). The Iso moiety was buried inside the protein, and surrounded by rigid and hydrophobic amino acids. The motions of the aromatic amino acids were suggested to be cooperative and synchronized with each other at some places in the protein. H-bonds were formed between Iso and Tyr123, Val125, Thr126 and Thr158. Photoinduced electron transfer (ET) from aromatic amino acids to the excited Iso (Iso*) was analysed from the reported ultrafast fluorescence dynamics of MCAD with Kakitani and Mataga (KM) theory. The ET rate was fastest from Trp156, and then from Tyr365, Trp47, Tyr302 and Trp165 in this order. Physical constants contained in KM theory were determined by a best-fit procedure between the observed and calculated fluorescence decays. Most of those quantities were similar to those derived from other flavoproteins. The free energy related to electron affinity of Iso* ( G Iso 0 ) and dielectric constant ( ɛ 0 ) were, however, quite low compared to their mean values among four other flavoproteins, which was elucidated by the low polarity around Iso. The energy gap law of ET was examined and revealed that ET in MCAD takes place in the normal region.

Time–dependent changes in the geometrical factors near the isoalloxazine (Iso) residue of FMN in three mutant isoforms [Y97F, W59F and W59F–Y97F (DM, double mutation)] of the flavodoxin (FD) from Desulfovibrio vulgaris, strain Miyazaki F., were obtained by molecular dynamic (MD) simulation. The center to center distances from Iso to Trp59 in Y97F and to Tyr97 in W59F were 0.78 nm and 0.55 nm, respectively. The remarkable fluorescence quenching in these proteins has been explained in terms of photoinduced electron transfer (ET) from the Trp59 and/or Tyr97 residues to the excited isoalloxazine (Iso*). The ultrafast fluorescence dynamics of the wild type (WT) and the Y97F, W59F and DM variant FDs reported by Mataga et al. (J. Phys. Chem. B 106 (2002) 8917–8920), were simultaneously analyzed by the electron transfer theory of Kakitani and Mataga (KM theory) and the atomic coordinates determined by MD, according to a non-linear least squares method. Agreements between the observed and calculated decays were all very good. The obtained physical constants contained in the KM theory were, for Trp and Tyr, respectively, a frequency factor (ν0) of 3.09 × 103 ps−1 and 2.46 × 103 ps−1, an ET process coefficient (β) of 55.6 nm−1 and 9.64 nm−1, a critical transfer distance (R0) of 0.772 nm and 0.676 nm, plus a free energy related to the electron affinity of Iso* ( G Iso 0 ) of 7.67 eV. These constants were common to all three mutant FD systems. In contrast, the static dielectric constant depended on the FD systems, being 4.78, 4.04 and 2.28 in the Y97F, W59F and DM variant FDs, respectively. The mean ET rate to Iso* was fastest from Trp59 in Y97F among the three systems. The total free energy gap in the FD systems was obtained as a sum of the net electrostatic (ES) energy between ion pairs and the standard free energy gap. A plot of ln kET/λS vs. − Δ G T 0 / λ S in all ET donors, where kET is ET rate, λS is the reorganization energy and Δ G T 0 is the total free energy gap, revealed that ln kET/λS can be expressed by a parabolic function of − Δ G T 0 / λ S and the ET process in FD took place mostly in the normal region.

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.

The ultrafast non-exponential fluorescence decay of FMN binding protein (FBP) was analyzed with three electron transfer (ET) theories, Marcus theory, Bixon and Jortner theory and Kakitani and Mataga theory. Center to center distances between electron acceptor, the excited isoalloxazine, and donors, Trp-32, Tyr-35 and Trp-106, in FBP were determined by molecular dynamic simulation. Electron transfer parameters containing in these theories were determined so as to fit the calculated decay with the observed decay, according to a non-linear least squares method. Introduction of electrostatic energies between isoalloxazine anion and other ionic groups and between the donor cations and other ionic groups in the protein into any ET theories improved the fitting. The non-exponential behavior in the fluorescence decay is considered to be ascribed to a fluctuation of the protein structure with long period.

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 dynamics of FMN in FMN-binding protein (FMN-bp), and its mutated proteins, W32Y and W32A, were investigated by the fluorescence up-conversion method. Fluorescence lifetimes were 167 fs (96%) and 1.5 ps (4%) in wild-type FMN-bp (WT), and 3.4 ps (23%), 18.2 ps (74%), and 96 ps (3%) at 530 nm in W32Y, and 30.1 ps in W32A. The fluorescence lifetime of W32A, in which Trp-32 was absent, was about 140 times longer than that of WT. Tyr-32 in W32Y was not so effective quencher as Trp-32 in WT. This was explained in terms of different ionization potentials of quenchers and average donor–acceptor distances in the protein.

Reported donor–acceptor distance-dependent rates of photo-induced electron transfer from tryptophan (Trp), tyrosine (Tyr), and benzoate (Bz) to the excited isoalloxazine in ten flavoprotein systems were analyzed with three kinds of electron transfer theories by Marcus, by Bixon and Jortner, and also by Kakitani, Yoshimori, and Mataga. Average donor–acceptor distances that were obtained from X-ray structures of flavoproteins were used for the analysis, rather than the edge-to-edge distance. The observed photo-induced electron transfer rates were best reproduced by the Kakitani, Yoshimori, and Mataga theory.

Dynamics of the photodissociation process of a hexaarylbiimidazole derivative, 2,2′-di(ortho-chlorophenyl)-4,4′,5,5′-tetraphenylbiimidazole (o-Cl-HABI), in benzene solution was investigated by means of the femtosecond–nanosecond laser spectroscopy. The time evolution of the transient absorption of the lophyl radical, the product of the photodissociation, was biphasic with the faster and the slower time constants of 80 fs and 1.9 ps, respectively. The faster time constant was ascribable to the formation of the lophyl radical through the bond fission after the excitation, and the slower time constant may be attributed to the conformational change and/or vibrational cooling of nascent radicals.

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.

International audience; We investigated the earliest steps of the photocycle of photoactive yellow protein (PYP) and its R52Q mutant in aqueous solution, by subpicosecond transient absorption spectroscopy. Our aim was to address the role of the positively charged Arg52 residue. We found that the relaxation mechanism of R52Q is similar to that of wt-PYP and discarded that Arg52 plays a key role in the chromophore photoreactivity. The excited-state decay of R52Q is however slower, confirming previous fluorescence up-conversion data. The experiments also reveal a slower stabilization of the cis isomer product. The loosening of the protein pocket and structural heterogeneities are discussed.

In many flavoproteins photoinduced electron transfer (ET) efficiently takes place from aromatic amino acids such as tryptophan or tyrosine to the excited isoalloxazine, so that the fluorescence lifetimes of isoalloxazine in some flavoproteins become ultrashort. The mechanism of ET in the flavoproteins was classified into four classes from the relationship between logarithmic ET rates (ln Rate) and the donor-acceptor distances (Rc), using reported data. The physical quantity, GT, is defined as the sum of solvent reorganization energy, electrostatic energy between a donor cation and an Iso anion, the standard free energy gap between the photoproducts and reactants, and net electrostatic energy between the photoproducts and other ionic groups in the flavoproteins (NetES). When GT fluctuates around zero with Rc, the ET rate becomes fastest (faster than 1 ps(-1)) in Kakitani and Mataga rates. In the ultrafast ET processes, the ln Rate becomes a parabolic function (category 1) of Rc as in FMN binding proteins and pyranose 2-oxidase at the shorter emission wavelengths, when NetES is negligible compared to the other quantities in the GT function. In the ultrafast ET processes, the ln Rate does not display any clear function of Rc (category 2) when NetES is dominant in the GT function, because of no direct relation between NetES and Rc. ET in flavodoxin from Helicobacter pylori may be classified into category 2. When GT linearly varies with Rc around a certain positive value, the ET rates become much slower (1 ps(-1)). In this case the ln Rate linearly decreases with Rc (category 3), as Tyr224 in d-amino acid oxidase dimers. It is also conceivable that the ln Rate decreases with much scattered function of Rc (category 4), when NetES is dominant in the GT function, as Tyr314 in d-amino acid oxidase dimers. In ET processes of category 1, ET rates decrease as Rc becomes shorter than the distance at the maximum values of ln Rates, where GT is negative. Conditions and physical meanings were discussed for the GT-negative region.

We developed a Yb:YAG Zig-Zag Active-Mirror laser amplifier with jet impingement cooling system for high average power laser systems. The output power of laser was beyond 1kW which was stable nearly 30 minutes.