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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.

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.

Chemical reactions of metal ions in solution were studied using 800 nm, 90 femtosecond laser pulses with a laser intensity range beginning at sub-micro-Joules/pulse, where the intensities were at or just below supercontinuum generation. Products were observed as surface resonance absorptions assignable to Ag, Au nanoparticles (NPs) and light scattering, indicating the formation of Pd NPs. The consumption efficiencies of metal ions from the Au and Pd ions were estimated to be 10−3 per incident photon. The major absorbing species of the laser energy was water, which led to the formation of solvated electrons that acted as a reducing agent of metal ions, while direct multi-photon dissociation to neutral atoms was unlikely. Metal ion Fe3+and Yb3+ systems are also discussed, where Fe3+ to Fe2+ was ascribed to two-photon absorption and Yb3+ to Yb2+ to a reduction followed by solvent ionization.

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.

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.

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.

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.

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 have investigated photoinduced electron transfer and related processes from the higher excited electronic state (S 2 ) of Zn-porphyrin-imide acceptor directly linked supramolecular systems (ZP-I) designed especially for the critical studies of the energy gap law (EGL) of the charge separation (CS) from the S 2 state and solvent effects upon EGL. We have confirmed the modification of the EGL by change of solvent polarity from acetonitrile (ACN), tetrahydrofuran (THF) to toluene (Tol) and methyl-cyclohexane (MCH), from rather typical bell-shaped one in ACN to that with less prominent normal region and prominent inverted region with moderate slope extending over wider range of −Δ G CS values in nonpolar solvent MCH. We have demonstrated that these solvent effects upon EGL affect delicately various radiationless relaxation processes from S 2 state. We have examined also effects of the hydrogen bonding solvent ethanol (EtOH) on the EGL for CS and found very specific effect controlling the CS reaction and related processes.

We have investigated photoinduced electron transfer (ET) and related processes from the higher excited electronic state (S2) of Zn-porphyrin-imide acceptor directly linked supramolecular systems (ZP-I) designed especially for the critical studies of the energy gap law (EGL) of the charge separation (CS) from the S2 state, effects of solvent dynamics and intramolecular vibrations on this CS, and competition or cooperation between this CS and S2--S1 conversion, etc. In this study, we have confirmed the modification of the EGL for the CS from S2 induced by the change of solvent polarity by comparing the EGL in toluene solution with that in THF, i.e. the EGL in toluene extends over a wider range of the energy gap for CS in the inverted region and becomes somewhat similar to the case of the weak coupling limit of an intramolecular radiationless transition. Moreover, we have compared the rate constants (lambda p) of the S1 state formation by the S2 excitation with the decay rate constants (lambda 1) of the S2 state in the ZP-I series and have also examined solvent polarity effects on these rate constants comparing THF and toluene solutions. Our studies have revealed that S1 formation by S2 excitation occurs mainly due to the CS in S2 followed by charge recombination (CR) producing S1, and these processes are affected by the modification of EGL owing to the solvent polarity, resulting in the smaller lambda p in toluene.

We have investigated primary processes of ultrafast photoreactions of various photoresponsive proteins by means of fs fluorescence dynamics measurements. Based on these studies, the effects of the protein nanospace (PNS), containing the chromophore, on the dynamics and mechanisms of the ultrafast and highly efficient reactions of these proteins have been elucidated. In this article, we discuss mainly the results of our studies on ultrafast photoisomerization of photoactive yellow protein (PYP), its mutants and analogues. The chromophore of the PYP, deprotonated coumaric acid thioester (O−-phenyl-CHCH–CO–S–CH2–), fixed in PNS by hydrogen (H) bonding interactions at the head part, O−-phenyl-, and by covalent bonding at the tail part, –CO–S–, undergoes ultrafast twisting by flipping the thioester bond, owing to the intrachromophore head to tail charge transfer caused by the photoexcitation. In the site-directed mutants where the PNS structure is looser and more disordered, the photoinduced twisting reaction becomes slowed compared with the wild-type PYP and moreover, the twisting becomes much slower in the denatured PYP, showing the supreme importance of more regulated PNS for the fast twisting. We found also coherent vibrations in the fluorescence decay curves which might be coupled with the twisting. Furthermore, we found for a PYP analogue with replaced chromophore ultrafast dynamic Stokes shift of fluorescence rather than the quenching due to twisting, indicating the importance of chromophore-PNS fine adjustment for the ultrafast twisting.

We have studied primary processes of ultrafast photoisomerization reactions of photoactive yellow protein (PYP) and visual rhodopsin (Rh) as well as ultrafast photoinduced electron transfer reactions of flavoproteins (FP) by means of the fs fluorescence up-conversion method. On the basis of these studies, we have examined the effects of the protein nanospace (PNS), where the chromophore is held, on the dynamics and mechanisms of the ultrafast and highly efficient photoinduced reactions of these proteins. In this article, we discuss mainly results of our comparative fluorescence dynamics studies on wild type (w. -t.) PYP, it’s site-directed mutants, analogues with replaced chromophore and FP, including the coherent vibrations in the ultrafast fluorescence decays of w. -t. PYP and mutants in the course of the photoisomerization, ultrafast dynamic Stokes shift of fluorescence of PYP analogues, and ultrafast electron transfer of FP.

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.

A series of 1,4-phenylene-bridged ZP-HP hybrid porphyrins (ZP = zinc porphyrin, HP = free-base porphyrin) 1-8 ZH have been prepared in which an electron-donating ZP moiety is kept constant and electron-accepting HP moieties are varied by introducing electron-accepting substituents, so that the energy gap for charge separation, ZP-1HP*--> ZP(+)-HP-, covers a range of about 0.9 eV in DMF. Here selective excitation at the HP moiety was employed to avoid complication in the determination of electron transfer rates derived from energy transfer, 1ZP*-HP --> ZP-1HP*. Definitive evidence for the electron transfer has been obtained in three solvents (benzene, THF, and DMF) through picosecond-femtosecond transient absorption studies, which have allowed the determination of the rates of the photoinduced charge separation, ZP-1HP* --> ZP(+)-HP-, and subsequent thermal charge recombination ZP(+)-HP- --> ZP-HP. Dyad 1ZH in THF exhibits a biphasic fluorescence decay that indicates thermal repopulation of the ZP-1HP* from ZP(+)-HP-; this has been also supported by the transient absorption spectra. On this ground, the energy levels of the ZP(+)-HP- ion pairs have been estimated. Similar biphasic fluorescence decay has been observed for 5 ZH in benzene; this allows furhter estimation of the energy level of the ZP(+)-HP- ion pairs. The free-energy-gap dependence (energy-gap law) has been probed from the normal to the upper limit region for the rate of the charge separation alone, and only the inverted region for the rate of the charge recombination. It was not possible to reproduce both energy-gap dependencies of the charge separation and the charge recombination assuming common parameter values for the reorganization energy and electronic interaction responsible for the electron transfer with the classical Marcus equation. Although both energy-gap dependencies can be approximately reproduced by means of the simplified semiclassical equation, which takes into consideration the effect of the high-frequency vibrations replaced by one mode of averaged frequency, many features, which include the effects of solvent polarity, electron-tunneling matrix element, and so forth on the energy-gap law, are considerably different from those of the previous studied porphyrin-quinone systems with weaker inter-chromophore electronic interactions.

The synthesis is described of fixed distance triads consisting of zinc porphyrin ( ZP ), metal-free porphyrin ( HP ) and pyromellitimide (I) in which the ZP and HP moieties are bridged by a rigid 4,4'-biphenylene spacer and related spacers, being held with dihedral angles defined by the torsional angle (θ) of the 4,4'-biphenylene spacer varying from nearly coplanar (θ = 0°, phenanthrene spacer) to nearly orthogonal (θ = 90°, 2,2',6,6'-tetramethylbiphenylene spacer). Using these models, the effects of the dihedral angle of the two porphyrins have been examined for singlet–singlet excitation energy transfer from ZP to HP , hole transfer from HP + to ZP and charge recombination of ZP +– HP – I – to the ground state. In the three processes examined, the observed rate constants are roughly in the order of 1d (coplanar)≥1c (nearly coplanar) >1a (tilted) >1b (orthogonal).

Self-assembled monolayers (SAMs) technique has been applied to porphyrin-linked fullerene systems to mimic supramolecular photoinduced charge-separation (CS) events in photosynthesis. Porphyrin-linked C60 molecules with a sulfide group were designed to self-assemble on a gold surface using S–Au interaction. The structure of the SAMs formed on gold electrodes was investigated by spectroscopic methods including X-ray photoelectron spectroscopy (XPS), ultraviolet (UV)-visible spectroscopic ellipsometry, UV-visible absorption spectroscopy, and Fourier transform infrared (FTIR) spectroscopy as well as electrochemical studies. These results indicate that the SAMs have loosely packed structure; the porphyrin-C60 molecules are tilted and nearly parallel onto the gold surface. Short-circuit photocurrent with a level of sub-μA cm−2 was observed for the photoelectrochemical cell in the presence of methylviologen (MV) under illumination with ca. 4 mW cm−2. The maximum intensity of the photocurrent in free-base porphy...

Motions of the trans-p-coumaric acid carbonyl group following the photoexcitation of the R52Q mutant of photoactive yellow protein (PYP) are investigated, for the first time, by ultrafast time-resolved circular dichroism (TRCD) spectroscopy. TRCD is monitored in the near-ultraviolet, over a time scale of 10 ps. Immediately after excitation, TRCD is found to exhibit a large negative peak, which decays within a few picoseconds. A quantitative analysis of the signals shows that, upon excitation, the carbonyl group undergoes a fast (≪0.8 ps) and unidirectional flipping motion in the excited state with an angle of ca. 17-53°. For the subset of proteins that do not enter the signaling photocycle, TRCD provides strong evidence that the carbonyl group moves back to its initial position, leading to the formation of a nonreactive ground-state intermediate of trans conformation. The initial ground state is then restored within ca. 3 ps. Comparative study of R52Q and wild-type PYP provides direct evidence that the absence of Arg52 has no effect on the conformational changes of the chromophore during those steps.

Four different kinds of C60-linked zincporphyrins have been prepared by changing systematically the linking position at meso-phenyl ring from ortho to para and their photophysical properties have been investigated. Regardless of the linkage between the two chromophores, photoinduced charge separation (CS) and subsequent charge recombination (CR) were observed in a series of zincporphyrin-C60 dyads by picosecond fluorescence lifetime measurements and time-resolved transient absorption spectroscopy. In THF the CS occurs from both the excited singlet state of the porphyrin and the C60 moieties, implying that the increase of the absorption cross section by both the chromophores results in the efficient formation of the ion pair (IP) state. On the other hand, in benzene the IP state generated by the photoinduced CS from the excited singlet state of the porphyrin to the C60 produces or energetically equilibrates with the locally excited singlet state of the C60. Both the CS and CR rates for the meta isomer are ...

Fucoxanthin-pyropheophorbide dyads, 3c and 4c, and zeaxanthin-pyropheophorbide dyads, 3d and 4d, were prepared as the first example of natural carotenoid-linked pyropheophorbide. Singlet–singlet energy transfer from carotenoid to pyropheophorbide is more efficient in fucoxanthin-pyropheophorbide dyads than the corresponding zeaxanthin-pyropheophorbide dyads, while the singlet excited state of the pyropheophorbide is quenched more strongly in the zeaxanthin-linked molecules. Marked differences in carotenoid-pyropheophorbide singlet excited-state interactions between fucoxanthin and zeaxanthin strongly suggest their different roles in vivo; antenna function for fucoxanthin and energy-dissipation for zeaxanthin. Both carotenoids similarly quench the triplet excited state of the pyropheophorbide through triplet–triplet energy transfer with rates which depend on temperature and the linkage.

The synthesis and excited-state dynamics are described for fixed-distance porphyrin–oxochlorin–pyromellitdimide triads (P–C–Im) and related reference compounds. In zinc-oxochlorin–pyromellitdimide (ZnP–Im), the 1(ZnC)* is quenched by the attached Im through intramolecular charge separation (CS) in benzene, THF, and DMF, while the 1(H2C)* in the corresponding free base is not significantly quenched by the Im even in polar DMF. In the steady-state fluorescence emission spectra, only the emission from the 1(C)* is commonly observed, indicating an efficient intramolecular singlet–singlet excitation energy transfer from P to C. Of these, the fluorescence intensities of the 1(H2C)* in ZnP–H2C and ZnP–H2C–Im are significantly reduced in polar DMF solution and this is attributed to the intramolecular CS that gives (ZnP)+–(H2C)−–Im and (ZnP)+–(H2C)−, respectively. The (ZnP)+–(H2C)−–Im ion pair is clearly shown, by picosecond absorption spectroscopy, to be converted into a secondary, longer-lived charge separated s...

Using pyromellitimide-linked, linear, 1,4-phenylene-bridged di- or tri-porphyrins, the intramolecular electron-transfer reaction from the singlet excited state of the distal doubly strapped free-base porphyrin to the pyromellitimide acceptor was studied by means of picosecond time-resolved fluorescence and transient absorption spectroscopy. The electron transfer is more effective in magnesium-porphyrin bridged models than in zinc-porphyrin bridged ones. Remarkably, the electron transfer over two porphyrins proceeds with rates almost similar to that of electron transfer over one porphyrin regardless of the bridging metal porphyrin.

Upon photoexcitation of fixed-distance triads cosisting of zinc-porphyrin (ZnP) or magnesium-porphyrin (MgP), a doubly strapped metal-free porphyrin (SH 2 P), and pyromellitimide (PIm), (ZnP) + SH 2 P(PIm) − and (MgP) + SH 2 P(PIm) − ion pair states are produced in high yields by two-step electron-transfer reactions: (1) charge separation between 1 (SH 2 P)* and PIm and (2) hole transfer from (SH 2 P) + to the metalloporphyrin. A (MgP) + SH 2 P(PIm) − ion pair state is fairly long-lived, while a (ZnP) + SH 2 P(PIm) − state is only short-lived due to a decay pathway to the ground state through a reverse hole transfer to give ZnP(SH 2 P) + (PIm) − followed by charge recombination.

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.

Photoactive yellow protein (PYP) is a blue-light photoreceptor responsible for the negative phototaxis of the purple sulfur bacterium Ectothiorhodospira halophila . Photoexcitation of PYP with a blue light triggers a photocycle that involves several intermediate states. High-resolution gas-phase fluorescence excitation experiments performed on the neutral form of PYP's p CA chromophore involved excitation wavelength-dependent photochemistry. This is explained by the existence of a low-lying second electronic state ( n → π * origin). Based on steady state and transient absorption measurements, it is concluded that upon excitation, simultaneous population of those states may occur and the complex photophysics of PYP arise due to competing relaxation pathways from those states. This chapter uses femtosecond time-resolved fluorescence up-conversion spectroscopy to elucidate the quasi-degenerate nature of the PYP's excited state. Detailed fluorescence anisotropy measurements are carried out on native PYP, 3,4-dihydroxy cinnamic acid analogue, and several mutant PYP systems.

Low frequency modes of PYP (photoactive yellow protein), its several mutant and analogue systems have been investigated by fluorescence up-conversion and resonance Raman scattering techniques, complemented by DFT and ab initio MO calculations. Assignments of the oscillatory components to particular vibrations are proposed. Their role in photoisomerization reaction is discussed.

The fluorescence upconversion technique has been applied to examine the picosecond fluorescence decay kinetics of flavin adenine dinucleotide (FAD) in aqueous solution. In the observation range of 30 ps three fluorescent lifetimes can be distinguished. The shortest-lived component (similar to1 ps) arises from water relaxation around the excited flavin. The 9-ps component originates from the intramolecular complex between flavin and adenine, whereas the nanosecond decay is attributed to the unstacked form of FAD. The spectra of the three forms are derived from global analysis of decay curves at different emission wavelengths and time regimes using a triple exponential function. It is assumed that the amplitude belonging to the nanosecond fluorescence component reflects the steady-state fluorescence spectrum. Fluorescence anisotropy to its maximum value of 0.4 is instantaneously created. (C) 2003 Elsevier B.V. All rights reserved.

In photosensory or photoactive proteins with chromophores absorbing in the visible region, such as rhodopsin (Rh) for vision, bacteriorhodopsin (bR), photoactive yellow protein (PYP) etc., light absorption leads to ultrafast and highly efficient reactions. Interestingly, despite quite different chromophores of these proteins, their behavior concerning the photoinduced primary processes in the protein nanospace in the course of the relaxation and twisting, including coherent processes seems to be similar. For instance, the fluorescence dynamics in all three proteins is nonexponential, no dynamic Stokes shift but a slight narrowing of the fluorescence spectra within first few picoseconds of the reaction were observed [1-3]. The spectral narrowing effect is most probably due to damping of coherently coupled intrachromophore high frequency modes and/or low frequency vibrations of environmental protein interacting with chromophore along the twisting coordinate. If this is the case, oscillatory behavior in fluorescence dynamics of photosensory proteins should be observed. To our knowledge, no such observation has been reported yet.

One-photon excitation of one of the two porphyrins within porphyrin-tetracyanoanthraquinodimethane-porphyrin triad with picosecond laser pulse leads to single reduction of the acceptor by intramolecular electron transfer, while an additional delayed excitation of the non-excited porphyrin results in double reduction of the acceptor.

Electron-transfer reactions in an H-bonded assembly composed of a diacylaminopyridine bearing zinc porphyrin and 1,8:4,5-naphthalenetetracarboxylic diimide (Ka = 2.8 × 102 dm3 mol–1) in benzene occur with kCS = 4.1 × 1010 s–1 and kCR = 3.7 × 109 s–1, while the corresponding covalently linked model with a comparable distance and energy gap undergoes electron transfer with kCS = 9.9 × 1010 s–1 and kCR = 6.7 × 108 s–1.

Excitation of a hydrogen-bonded complex of zinc porphyrin bearing a 2,6-diacylaminopyridine binding site and N-monosubstituted 1,8:4,5-naphthalene-tetracarboxamide in benzene results in efficient electron transfer to give a charge separated state detectable by picosecond transient absorption spectroscopy.

Low-frequency vibrational modes of the native photoactive yellow protein (PYP) and its several mutant and analogue systems have been investigated in “time” (femtosecond fluorescence up-conversion) ...