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Highlights: [•] Two very strong photoacids QCy7, , and QCy9, , are studied in three protic solvents, water, MeOH and EtOH. [•] Weak photoacids do not transfer a proton to alcohols within the excited state lifetime, whereas strong photoacids do. [•] In QCy7 the ESPT rate constant, k PT in water, methanol and ethanol is 1.2×1012, 8×1011 and 7×1011 respectively. [•] The ESPT rate of QCy9 in these solvents is the same, k PT ∼1013 s−1. [•] Non-adiabatic and adiabatic PT theories explain the k PT solvent dependence of weak and very strong photoacids respectively. [ABSTRACT FROM AUTHOR]

Abstract: Steady-state and time-resolved emission spectra were measured to study the relaxation of all-trans retinoic acid (ATRA) from its excited-states in a number of solvents. We found that the time-resolved emission signal is composed of three decay components arising from three different excited-states. The ultrafast component that is shorter than 80fs is dominant at λ ≤500nm, whereas the intermediate component is dominant at longer wavelengths and its lifetime is solvent dependent. For acetonitrile, methanol and 1-pentanol the average decay times of the intermediate component are 0.9, 1.3 and 4ps respectively, and the overall decay is non-exponential. The third decay component has a very small amplitude of ∼0.05 in polar solvents and a longer lifetime of ∼10ps. We assign the fast decaying emission component to the allowed transition from the upper 1 B u + state to the ground 1 A g state. The intermediate lifetime component is assigned to the forbidden transition from the excited 1 A g ππ* state to the ground-state. In n-octane, a non-polar solvent, the amplitude of the intermediate time component is smaller than in polar solvents, and its lifetime is 600fs. This lifetime is shorter than in acetonitrile, whose viscosity is half that of n-octane. Moreover, the amplitude of the long time component in this solvent at long wavelengths is dominant. We therefore propose that the excited state ordering may be different in non-polar solvents. We also conducted a temperature dependence study on ATRA emission in 1-propanol and 1-pentanol, both of which are glass forming liquids. We found that the average emission lifetime increases as the temperature decreases. The value of the activation energy of the non-radiative process is half that of the dielectric relaxation process of the solvent. [Copyright &y& Elsevier]

Abstract: The photoprotolytic properties of a recently synthesized photoacid, hydroxycoumarin-dipicolinium salt (HCD) were studied by means of time-resolved emission techniques. We found that HCD is a super-photoacid with and with an excited-state proton transfer to water with a rate constant of . We also found that HCD undergoes ESPT in a large number of protic solvents. The kinetic isotope effect is about two for water, methanol and ethanol. [Copyright &y& Elsevier]

Abstract: Time-resolved emission techniques were employed to study the pressure effect on curcumin''s luminescence in several aliphatic alcohols. We found that the hydrostatic pressure of up to 2GPa affects the decay rate of curcumin emission in the same way it affects the protonated form of many photoacids. We therefore propose that curcumin, which has two phenol groups, is a strong photoacid. Strong photoacids with pK a*<0 can transfer a proton not only to water but also to alcohols, as curcumin does in these solvents. [Copyright &y& Elsevier]

The fluorescence spectra of the wild-type green fluorescence protein (wt-GFP) and the anionic form of p-hydroxybenzylidenedimethylimidazolone (p-HBDI), which models the protein chromophore, were obtained in the 80-300 K temperature range in glycerol/water solvent. The protein spectra have pronounced and well-resolved vibronic structure, at least at lower temperatures. In contrast, the chromophore spectra are very broad and structureless even at the lowest temperatures. Analysis of the spectra shows that the experimentally observed red-shift of the protein spectrum upon heating is apparently caused by quadratic vibronic coupling of the torsional deformation (TD) of the phenyl single bond of the chromophore to the electronic transition. The broad spectra of the chromophore manifest the contribution of different conformations in the glycerol/water solvent. In particular, the lowest-temperature spectrum reflects the distribution over the same TD coordinate in the excited electronic state, which essentially contributes to the asymmetry of the spectrum. Upon heating, motion along this coordinate leads to a configuration from which the radiationless transition takes place. This narrows the distribution along the TD coordinate, causing a more symmetric fluorescence spectrum. We were able to reconstruct the broad, structureless fluorescence spectra of p-HBDI in glycerol/water solutions at various temperatures by convoluting the original wt-GFP spectra with the function describing the distribution of the transition energies of the p-HBDI chromophore. Thus, both the fluorescence broadening and increase in radiationless transition upon removal of the protein chromophore to bulk solvent are consistent with decay by a barrierless TD of the phenyl single bond. [ABSTRACT FROM AUTHOR]

N-Oxidation of hydroxyquinolines leads to a dramatic increase in their excited-state acidity. Time-resolved and steady-state emission characterization of 6-hydroxyquinoline-N-oxide and 2-methyl-6-hydroxyquinoline-N-oxide reveals a rich but less complex proton-transfer behavior than that of its parent hydroxyquinoline. The electronic effect of the oxidized heterocyclic nitrogen atom makes the excited state both less basic and more acidic than the parent and adds hydroxyquinoline N-oxides to the class of high-acidity excited-state proton donors in photochemistry and photobiology. Adiabatic photoinduced proton transfer is accompanied by the efficient nonreversible deoxygenation and 1-2 oxygen migration. [ABSTRACT FROM AUTHOR]

Time-resolved and steady-state emission characterization of 10-hydroxycamptothecin reveals a rich but less complex proton-transfer' behavior than its parent hydroxyquinoline. The electronic effect of the additional electron-withdrawing ring makes the excited-state both less basic and more acidic than the parent and adds to the class of high-acidity excited-state proton donors in photochemistry and photobiology. [ABSTRACT FROM AUTHOR]

The article discusses the rate of excited state proton transfer (ESPT) in the mKeima, a red fluorescent protein. It states that Green Fluorescent Protein's (GFP's) presence in low temperature emissions affects the efficiency of proton transfer which is characterized by the effect of deuterium isotope. It concludes that mixed population of anionic and protonated states was shown in excitation spectra of GFP and mKeima. It adds that mKeima is the only protein to utilize ESPT for green emission.

The article presents the result of the study which examines the X-ray crystalographic structures and mechanism of photoactivatable green fluorescent protein (PA-GFP). The study shows that the structure is accountable for the changes in the excitation and emission spectra of PA-GFP relative to wild type GFP. It also reveals that in PA-GFP, the dramatic change in the chromophore protanation state results to a large emission contrast between native and photoactivated states.

Steady-state and time-resolved fluorescence techniques were used to study the excited-state proton transfer (ESPT) from an irreversible photoacid, 1-naphthol-3,6-disulfonate (1NP36DS), to methanol-water mixtures. We found that at χ water = 0.3 the ESPT rate constant is higher by a factor of 10 that in neat methanol. TD-DFT calculations show that a mixed molecular bridge of two methanol molecules and one water molecule enables the ESPT from the 1-OH to the 3-sulfonate. The RO - (S 1 ) state is stable by -2.5 kcal/mol in comparison to the ROH(S 1 ) state. We compare the ESPT rate constants of a reversible photoacid, 8-hydroxy-1,3,6-pyrenetrisulfonate (HPTS), in the same methanol-water mixtures. At χ water ≈ 0.3 the ESPT rate constant of HPTS increased by only 15%. We explain the large difference of the ESPT rate of 1NP36DS by the formation of a water bridge or a mixed methanol-water bridge from 1-OH to one of the sulfonates and the absence of such a bridge in HPTS. The water or mixed methanol-water bridge of 1NP36DS enhances the ESPT rate in methanol-water mixtures of low water mole ratio.

Steady-state and time-resolved fluorescence techniques were employed to study a superphotoacid with a p K a * of ∼-7, the chlorobenzoate phenol cyanine picolinium salt (CBCyP) in acetonitrile-water mixtures. We found that the time-resolved fluorescence is bimodal. The amplitude of the short-time component depends on χ water ; the larger χ water , the greater the amplitude. We found that the excited-state proton-transfer (ESPT) rate constant, k PT , is ≥5 × 10 12 s -1 in mixtures of χ water ≥ 0.08, whereas in neat water, k PT = 6 × 10 12 s -1 . The long-time component has a lifetime of 50 ps at χ water = 0.75. We attribute this time component to the CBCyP molecules that are not hydrogen-bonded to H 2 O clusters. The results suggest that the ESPT rate constant to water in acetonitrile-water mixtures depends only slightly on the water cluster size and structure surrounding the CBCyP molecule. We attribute the independence of the ESPT rate on the average water-cluster size to the large photoacidity of CBCyP. QM TD-DFT calculations found that in the excited-state the RO - (S 1 ) species that is formed by the ESPT process is more stable than the ROH(S 1 ) species by -5 kcal/mol when four water molecules accept the proton, and when six water molecules accept the proton, the RO - (S 1 ) drops to -10 kcal/mol. The calculations show that energy stabilities are kept constant in implicit CH 3 CN-H 2 O solvent mixtures of dielectric constant of ε ≥ 45.

Steady-state and time-resolved fluorescence techniques were used to study excited-state proton transfer (ESPT) to water of the reversible photoacid 2-naphthol-8-sulfonate (2N8S) in acetonitrile/water mixtures. In acetonitrile-rich mixtures, up to χ water ≤ 0.12, we found a slow ESPT process on the order of nanoseconds. At χ water ≈ 0.15, the RO - fluorescence band intensity is at the minimum, whereas at χ water ≈ 0.030, it is at the maximum. The steady-state fluorescence spectra of these mixtures show that the intensity of the RO - fluorescence band at χ water ≈ 0.030 is about 0.24 of that of the ROH band. We explain this unusual phenomenon by the presence of water clusters that exist in the acetonitrile-rich CH 3 CN/H 2 O mixtures. We propose that a water bridge forms between the 2-OH and 8-sulfonate by preferential solvation of 2N8S, and this enables the ESPT process between the two sites of the molecular structure of 2N8S. In mixtures of χ water ≥ 0.25, the ESPT process takes place to water clusters in the bulk mixture. The higher the χ water in the mixture, the greater the ESPT rate constant. In neat water, the rate constant is rather small, 4.5 × 10 9 s -1 . TD-DFT calculations show that a single water molecule can bridge between 2-OH and 8-sulfonate in the excited state. The activation energy for the ESPT reaction is about 9 kcal/mol, and the RO - (S 1 ) species is energetically above the ROH(S 1 ) species by about 1.6 kcal/mol.

Steady-state and time-resolved fluorescence techniques as well as quantum-mechanical calculations were used to study the photophysics and photochemistry of a newly synthesized photoacid-the phenol cyanine picolinium salt. We found that the nonradiative rate constant k nr of the excited protonated form of the photoacid is larger than that of the excited-state proton transfer (ESPT) to the solvent, k ESPT . We estimate that the quantum efficiency of the ESPT process is about 0.16. The nonradiative process is explained by a partial trans-cis isomerization reaction, which leads to the formation of a "dark" excited state that can cross to the ground state by nonadiabatic coupling. Moreover, the ESPT process is coupled to the photo-isomerization reaction, as this latter reaction enhances the photoacidity of the studied compound, as a result of photoinduced charge transfer. To prevent trans-cis isomerization of the cyanine bridge, we conducted experiments of PCyP adsorbed on cellulose in the presence of water. We found that the steady-state fluorescence intensity increased by about a factor of 50 and the lifetime of the ROH band increased by the same factor. The fluorescence intensity of the RO - band with respect to that of the ROH band was the same as in aqueous solution. This explains why inhibiting the photo-isomerization reaction by adsorbing the PCyP on cellulose does not lead to a higher ESPT rate., Competing Interests: The authors declare no competing financial interest.

Replacement of the hydroxyl group of a hydrophilic sidechain by an H atom in the proton wire of GFP induces formation of a water-chain proton wire. Surprisingly, this "non-native" water chain functions as a proton wire with response times within 10 ps of the wild type protein. This remarkable rate retention is understood as a natural consequence of the well-known Grotthuss mechanism of proton transfer in water.

Steady-state and time-resolved fluorescence techniques were employed to study the photophysics and photochemistry of trans-resveratrol. trans-Resveratrol is found in large quantities in fungi-infected grapevine-leaf tissue and plays a direct role in the resistance to plant disease. We found that trans-resveratrol in liquid solution undergoes a trans-cis isomerization process in the excited state at a rate that depends partially on the solvent viscosity, as was found in previous studies on trans-stilbene. The hydroxyl groups of the phenol moieties in resveratrol are weak photoacids. In water and methanol solutions containing weak bases such as acetate, a proton is transferred to the base within the lifetime of the excited state. When resveratrol is adsorbed on cellulose (also a component of the plant's cell wall), the cis-trans process is slow and the lifetime of the excited state increases from several tens of picoseconds in ethanol to about 1.5 ns. Excited-state proton transfer occurs when resveratrol is adsorbed on cellulose and acetate ions are in close proximity to the phenol moieties. We propose that proton transfer from excited resveratrol to the fungus acid-sensing chemoreceptor is one of the plant's resistance mechanisms to fungal infection.

Intramolecular proton-transfer dynamics in the lowest excited state (ESIHT) were studied in the natural product quercetin. We found that in all seven solvents used in this study, the ESIHT rate is ultrafast. We estimate that the ESIHT rate is about 70 fs or less. We found that in deuterated protic solvents, such as methanol-d or ethanol-d, the ESIHT rate is slower and the proton-transfer time constant is about 110 fs. The tautomeric form fluorescence quantum yield of quercetin is very low, of the order of the normal form.

Steady-state and time-resolved fluorescence techniques were employed to study the excited-state proton transfer (ESPT) from a photoacid adsorbed on starch to a nearby water molecule. Starch is composed of ∼30% amylose and ∼70% amylopectin. We found that the ESPT rate of adsorbed 8-hydroxy-1,3,6-pyrenetrisulfonate (HPTS) on starch arises from two time constants of 300 ps and ∼3 ns. We explain these results by assigning the two different ESPT rates to HPTS adsorbed on amylose and on amylopectin. When adsorbed on amylose, the ESPT rate is ∼3 × 10(9 )s(-1), whereas on amylopectin, it is only ∼3 × 10(8) s(-1).

Steady-state and time-resolved optical techniques were employed to study the photoprotolytic mechanism of a general photoacid. Previously, a general scheme was suggested that includes an intermediate product that, up until now, had not been clearly observed experimentally. For our study, we used quinone cyanine 7 (QCy7) and QCy9, the strongest photoacids synthesized so far, to look for the missing intermediate product of an excited-state proton transfer to the solvent. Low-temperature steady-state emission spectra of both QCy7 and QCy9 clearly show an emission band at T < 165 K in H2O ice that could be assigned to ion-pair RO(-)*···H3O(+), the missing intermediate. Room-temperature femtosecond pump-probe spectroscopy transient spectra at short times (t < 4 ps) also shows the existence of transient absorption and emission bands that we assigned to the RO(-)*···H3O(+) ion pair. The intermediate dissociates on a time scale of 1 ps and about 1.5 ps in H2O and D2O samples, respectively.

Mutations near the fluorescing chromophore of the green fluorescent protein (GFP) have direct effects on the absorption and emission spectra. Some mutants have significant band shifts and most of the mutants exhibit a loss of fluorescence intensity. In this study we continue our investigation of the factors controlling the excited state proton transfer (PT) process of GFP, in particular to study the effects of modifications to the key side chain Ser205 in wt-GFP, proposed to participate in the proton wire. To this aim we combined mutagenesis, X-ray crystallography, steady-state spectroscopy, time-resolved emission spectroscopy and all-atom explicit molecular dynamics (MD) simulations to study the double mutant T203V/S205A. Our results show that while in the previously described GFP double mutant T203V/S205V the PT process does not occur, in the T203V/S205A mutant the PT process does occur, but with a 350 times slower rate than in wild-type GFP (wt-GFP). Furthermore, the kinetic isotope effect in the GFP double mutant T203V/S205A is twice smaller than in the wt-GFP and in the GFP single mutant S205V, which forms a novel PT pathway. On the other hand, the crystal structure of GFP T203V/S205A does not reveal a viable proton transfer pathway. To explain PT in GFP T203V/S205A, we argue on the basis of the MD simulations for an alternative, novel proton-wire pathway which involves the phenol group of the chromophore and water molecules infrequently entering from the bulk. This alternative pathway may explain the dramatically slow PT in the GFP double mutant T203V/S205A compared to wt-GFP.

Steady-state and time-resolved techniques were employed to study the excited-state proton-transfer (ESPT) rate of two newly synthesized 8-hydroxy-1,3,6-pyrenetrisulfonate (pyranine, HPTS) derived photoacids in three protic solvents, water, methanol and ethanol. The ESPT rate constant k(PT) of tris(1,1,1,3,3,3-hexafluoropropan-2-yl)-8-hydroxypyrene-1,3,6-trisulfonate, 1a, whose pK(a)* ~ -4, in water, methanol and ethanol is 3 × 10(11) s(-1), 8 × 10(9) s(-1) and 5 × 10(9) s(-1) respectively. (8-Hydroxy-N1,N3,N6-tris(2-hydroxyethyl)-N1,N3,N6-trimethylpyrene-1,3,6 trisulfonamide, 1b) is a weaker acid than 1a but still a strong photoacid with pK(a)* ~ -1 and the ESPT rate in water, methanol and ethanol is 7 × 10(10) s(-1), 4 × 10(8) s(-1) and 2 × 10(8) s(-1). We qualitatively explain our kinetic results by a Marcus-like free-energy correlation which was found to have a general form suitable for describing proton transfer reactions in both the proton-adiabatic and the proton-non-adiabatic limits.

The oxyluciferin family of fluorophores has been receiving much attention from the research community and several systematic studies have been performed in order to gain more insight regarding their photophysical properties and photoprotolytic cycles. In this minireview, we summarize the knowledge obtained so far and define several possible lines for future research. More importantly, we analyze the impact of the discoveries on the firefly bioluminescence phenomenon made so far and explain how they re-open again the discussion regarding the identity (keto or enol species) of the bioluminophore., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

To study the dynamics and mechanisms of the proton wire of wild type green fluorescence protein (wt-GFP) and its S205V mutant, we applied molecular dynamics (MD) simulations and compared the results with the X-ray structures of both proteins and with the proton transfer kinetics of these proteins studied by the time-resolved emission technique. The MD simulations for the wt-GFP show that, in the proton wire, the Ser205 exists in two conformations with similar probabilities. One conformation supports the proton transfer, and the other does not. The fluctuation between the two conformers is relatively slow. This result may explain the time-resolved emission spectrum's long-time fluorescence tail of the wt-GFP chromophore's protonated form. The MD simulations of the S205V mutant show that the water molecule in the proton wire is replaced by other bulk water molecules along the simulations of 60 ns. Furthermore, as in the wt-GFP, the Thr203 also exists in two conformations in which only one conformation supports the proton transfer. These two findings give an insight into the relatively slow proton transfer rate in the S205V mutant in comparison to the wt-GFP.

Firefly oxyluciferin presents a pH-sensitive fluorescence in aqueous solutions. Its fluorescence spectra are composed of two green peaks at different pH values, despite the enolate anion being the only emitter. A computational approach was used to further elucidate the photoprotolytic cycle of oxyluciferin and investigate its pH sensitivity. It was found that oxyluciferin forms π-π stacking complexes both in the ground and excited states, at basic and acidic/neutral pH. However, at different pH values, these complexes adopt a different conformation, which explains the lower energy of the emission at acidic/neutral pH, in comparison with the emission at basic pH., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Time-resolved emission of aloesaponarin I was studied with the fluorescence up-conversion and time-correlated single-photon-counting techniques. The rates of the excited-state intramolecular proton transfer, of the solvent and molecular rearrangements, and of the decay from the excited proton-transferred species were determined and interpreted in the light of time-dependent density functional calculations. These results were discussed in conjunction with UV protection and singlet-oxygen quenching activity of aloe.

Steady-state and time-resolved techniques were employed to study the excited-state proton transfer (ESPT) from d-luciferin, the natural substrate of the firefly luciferase, to the mild acetate base in aqueous solutions. We found that in 1 M aqueous solutions of acetate or higher, a proton transfer (PT) process to the acetate takes place within 30 ps in both H(2)O and D(2)O solutions. The time-resolved emission signal is composed of three components. We found that the short-time component decay time is 300 and 600 fs in H(2)O and D(2)O, respectively. This component is attributed either to a PT process via the shortest water bridged complex available, ROH··H(2)O··Ac(-), or to PT taking place within a contact ion pair. The second time component of 2000 and 3000 fs for H(2)O and D(2)O, respectively, is attributed to ROH* acetate complex, whose proton wire is longer by one water molecule. The decay rate of the third, long-time component is proportional to the acetate concentration. We attribute it to the diffusion-assisted reaction as well as to PT process to the solvent.

The excited state of flavin adenine dinucleotide (FAD), dissolved in water, is subjected to intensive quenching due to electron transfer from the adenine moiety to the excited isoalloxazine ring. Increasing the methanol concentration in the solution enhances the quantum yield of the fluorescence. In the present study, we carried out molecular dynamics simulations of FAD in explicit water and water-methanol mixtures over time frames of hundreds of nanoseconds. The simulations record rapid structural fluctuations of the molecule, where the distance between the centers of mass (COMs) of the two nucleotides varied from contact distance (folded) up to fully extended (open) structure. The methanol affected the dynamics of the FAD by enhancing the frequency of unfolding events without any effect on the lifetime of the open state. The correlation of the molecular dynamics simulations with fluorescence titration of the FAD in water/methanol mixtures indicates that the internal quenching takes place when the distance between COMs is <5.5 +/- 0.5 A.

The effects of excess protons on the fluorescence quenching process of 1-naphthol-4-sulfonate (1N4S) and 1-naphthol-3-sulfonate (1N3S) in methanol-doped ice samples were studied by employing a time-resolved emission technique. We found that the fluorescence quenching of the deprotonated form RO(-)* of both photoacids by protonation is very efficient in ice, whereas in liquid water the proton fluorescence quenching is rather small. Using the Smoluchowski diffusion-assisted binary collision model under certain assumptions and approximations, we found that the calculated proton diffusion constant in ice in the temperature range of 240-260 K was 10 times greater than that of water at 295 K.

We used an inhomogeneous excited-state proton-transfer kinetics model to explain the origin of the non-exponential time-resolved emission of the A-band of wt-green fluorescence protein. The calculated fit is rather good for both H 2O and D 2O samples in a wide temperature range of 80-229 K. We attribute the inhomogeneous kinetics to the distance dependence of the excited-state proton-transfer rate between the proton donor (the hydroxyl group of the chromophore) and the oxygen of a nearby water molecule.

Steady-state and time-resolved emission techniques were employed to study the photoprotolytic cycle of an excited photoacid in ice in the presence of a low concentration of a weak base-like F(-). In previous studies we found that the photoprotolytic cycle in methanol-doped ice (1% mol fraction) is too slow to be observed at temperatures below 190 K. In this study we found that at temperatures below 240 K an additional proton-transfer process occurs in ice doped with 10 mM KF. We attributed this reaction to the creation of a mobile L-defect by F(-) ions. We used a diffusion-assisted reaction model, based on the Debye-Smoluchowski equation, to account for the direct reaction of the L-defect with the excited photoacid at temperatures below T < 240 K. Below 160 K the spectroscopic properties as well as the photoprotolytic cycle change dramatically. We propose that below 160 K the sample enters a new phase. The excited-state proton-transfer (ESPT) process was observed and followed down to a liquid nitrogen temperature of approximately 78 K. In the low-temperature phase the ESPT rate is almost twice as much as at 180 K and the temperature dependence of the rate is very small. The kinetic isotope effect of the ESPT at the low-temperature phase is small of about 1.3.

A time-resolved emission technique was employed to study the photoprotolytic cycle of two photoacids 2-naphtol-6-sulfonate (2N6S) and 2-naphtol-6,8-disulfonate (2N68DS) in ice in the presence of a low concentration of a weak base fluoride ion. We found that an additional proton-transfer process occurs in ice doped with F- ions. This reaction takes place between a mobile L-defect (created by static F- ions) and the photoacid. We used a diffusion assisted reaction model, based on the Debye-Smoluchowski equation, to account for the direct reaction of the L-defect with the excited photoacid.

Steady-state emission and time-correlated single-photon counting (TCSPC) are used to measure the temperature dependence of the proton-transfer rate of wt-GFP in H2O and D2O. As the temperature decreases, the proton-transfer rate from the protonated form slows down. At about 80 K, the rate is about 10-fold slower than the rate at room temperature. At lower temperatures of 70 K down to 13 K (the lowest temperature studied), the rate of proton transfer is almost temperature independent. We explain the temperature dependence of the proton-transfer rate by an intermolecular vibration assisted tunneling mechanism. We attribute the specific intermolecular vibration to the oscillation of two oxygen atoms: the chromophore's phenol ring and the nearby water molecule. The kinetic isotope effect is about 5 and is almost temperature independent.

Wild-type green fluorescent protein (wt-GFP) has a prominent absorbance band centered at approximately 395 nm, attributed to the neutral chromophore form. The green emission arising upon excitation of this band results from excited-state proton transfer (ESPT) from the chromophore hydroxyl, through a hydrogen-bond network proposed to consist of a water molecule and Ser205, to Glu222. Although evidence for Glu222 as a terminal proton acceptor has already been obtained, no evidence for the participation of Ser205 in the proton transfer process exists. To examine the role of Ser205 in the proton transfer, we mutated Ser205 to valine. However, the derived GFP variant S205V, upon excitation at 400 nm, still produces green fluorescence. Time-resolved emission spectroscopy suggests that ESPT contributes to the green fluorescence, and that the proton transfer takes place approximately 30 times more slowly than in wt-GFP. The crystal structure of S205V reveals rearrangement of Glu222 and Thr203, forming a new hydrogen-bonding network. We propose this network to be an alternative ESPT pathway with distinctive features that explain the significantly slowed rate of proton transfer. In support of this proposal, the double mutant S205V/T203V is shown to be a novel blue fluorescent protein containing a tyrosine-based chromophore, yet is incapable of ESPT. The results have implications for the detailed mechanism of ESPT and the photocycle of wt-GFP, in particular for the structures of spectroscopically identified intermediates in the cycle.

We have studied the solvation statics and dynamics of coumarin 343 and a strong photoacid (pK* approximately 0.7) 2-naphthol-6, 8-disulfonate (2N68DS) in methanol-doped ice (1% molar concentration of methanol) and in cold liquid ethanol in the temperature range of 160-270 K. Both probe molecules show a relatively fast solvation dynamics in ice, ranging from a few tens of picoseconds at about 240 K to nanoseconds at about 160 K. At about 160 K in doped ice, we observe a sharp decrease of the dynamic Stokes shift of both coumarin 343 and 2N68DS. Its value is approximately only 200 cm-1 at approximately 160 K compared to about 1100 cm-1 at T >/= 200 K (at times longer than t > 10 ps). We find a good correlation between the inefficient and slow excited-state proton-transfer rate at low-temperature ice, T < 180 K, and the dramatic decrease of the solvation energy, as measured by the dynamic band shift, at these low temperatures. We find that the average solvation rate in ice is similar to its value in liquid ethanol at all given temperatures in the range of 200-250 K. The surprisingly fast solvation rate in ice is explained by the relatively large freedom of the water hydrogen rotation in ice Ih.

We have studied the excited-state proton-transfer rate of four photoacids in ice as a function of temperature. For all four photoacids, we have found a non Arrhenius behavior of the proton-transfer rate constant, k(PT). d(ln k(PT))/d(1/T) decreases as the temperature decreases. The average slope of ln(k(PT))versus 1/T depends on the photoacid strength (pK*). The stronger the photoacid is, the smaller the slope. For the strongest photoacid 2-naphthol-6,8-disulfonate (2N68DS) the largest slope is 35 kJ/mol at about 270 K, and the smallest measured slope is about 8 kJ/mol at about 215 K. We propose that the temperature dependence of k(PT) in ice at the temperature range 270 > T > 200 K can be explained as arising from contributions of two proton-transfer mechanisms over the barrier and tunneling under the barrier. At very low temperatures T < 200 K, the slope of ln(k(PT)) versus 1/T increases again. At about 170 K, the proton-transfer rate is much slower than the radiative rate, and the deprotonated form of the photoacid cannot be detected in the steady-state emission spectrum. At lower temperatures, T < 200 K, the rate further decreases because of a limitation on the reaction caused by the restrictions on the H2O hydrogen reorientations.

Time-resolved emission was used to measure the photoprotolytic cycle of an excited photoacid as a function of temperature, both in liquid water and in ice, in the presence of an inert salt. The inert salt affects the geminate recombination between the transferred proton with the conjugate base of the photoacid. We used the Debye-Hückel theory to express the screening of the Coulomb electrical potential by the inert salt. We find that in the liquid phase the measured screening effect is small and the Debye-Hückel expression slightly overestimates the experimental effect. In ice, the screening effect is rather large and the Debye-Hückel expression under estimates the measured effect. We explain the large screening in ice by the "salting-out" effect in ice that tends to concentrate the impurities to confined volumes to minimize the ice crystal energy.

The reversible proton dissociation and geminate recombination of the common photoacid, 8-hydroxypyrene-1,3,6-trisulfonate (pyranine), either in dilute aqueous solution or when forming a complex with gamma-cyclodextrin (gamma-CD), has been studied by time-resolved fluorescence spectroscopy and supplemented by molecular modeling and dynamics simulations. We find that the dissociation rate of the proton from the excited molecule was decreased to about approximately 50% of its value in water, while the rate of recombination was doubled. These observations were evaluated by molecular modeling of the reactants at atomic resolution. The combination of the two methodologies indicates that the pyranine in the complex can assume more than one level of interaction with the solvent. The polysugar torus surrounding the pyranine perturbs the hydrogen bond in the dye's immediate vicinity and deforms the electrostatic potential inside the Coulomb cage, causing major deviations from a simple spheric symmetry. These observations can account for the special kinetic features measured for the complex. We suggest that this system can be used as a basic model for evaluating the mechanism of proton transfer in non-homogeneous systems, such as the surface of proteins or biomembranes.

The reversible proton dissociation and geminate recombination of a photoacid is studied as a function of temperature in water electrolyte solutions and binary water-methanol mixtures, containing 0.1 and 0.2 mole fractions of methanol. 8-Hydroxypyrene-1,3,6-trisulfonate trisodium salt (HPTS) is used as the photoacid. The experimental data are analyzed by the reversible geminate recombination model. We found that the slope of the logarithm of the proton-transfer rate constant as a function of the inverse of temperature (Arrhenius plot) in the liquid phase of these samples are temperature-dependent, while in the solid phase, the slope is nearly constant. The slope of the Arrhenius plot in frozen electrolyte solution is larger than that of the water-methanol mixtures, which is about the same as in pure water. Careful examination of the time-resolved emission in ice samples shows that the fit quality using the geminate recombination model is rather poor at relatively short times. We were able to get a better fit using an inhomogeneous kinetics model assuming the proton-transfer rate consists of a distribution of rates. The model is consistent with an inhomogeneous frozen water distribution next to the photoacid.

Time-resolved emission and steady-state fluorescence techniques are used to study the excited-state intermolecular proton transfer from 8-hydroxypyrene-1,3,6-trisulfonate (HPTS or pyranine) to water in the presence of inert salts, NaCl and MgCl(2). At low salt concentrations, up to about 0.5 M MgCl(2) or about 0.8 M NaCl, the time-resolved emission of both the photoacid and conjugate base can be quantitatively fitted by our diffusion-assisted geminate recombination model. In this concentration range, the proton transfer and geminate recombination rate constants are almost independent of the salt concentrations whereas the proton diffusion constant decreases as the salt concentration increases. At higher salt concentrations, the proton-transfer rate constant decreases while the recombination rate constant increases slightly. For the saturated solution of MgCl(2) (about 5 M at room temperature), the steady-state emission consists of only a single band of the protonated photoacid. Careful examination of the time-resolved emission of HPTS in the presence of a large concentration of MgCl(2) shows that the quality of the fit to the geminate recombination model is rather poor and we fail to find adjustable parameters for a good quality fitting. For this large concentration range of MgCl(2) we were able to get a good fit of the experimental data with a model based on a distribution of proton-transfer rates. The model is consistent with an inhomogeneous water environment next to the excited HPTS molecule in such concentrated solutions.

In a previous work, we proposed an extended model for intermolecular excited-state proton transfer to the solvent. The model invoked an intermediate species, the contact ion-pair RO(-)...H(3)O(+), where a proton is strongly hydrogen bonded to the conjugated photabase RO(-). In this study we tested the extended model by measuring the transient absorption and emission of 8-hydroxypyrene-1,3,6-trisulfonate (HPTS) in an aqueous solution in the presence of a large concentration of mineral acids. In a neutral pH solution, the pump-probe signal consists of three time components, <1, 4, and 100 ps. The 4 ps time component, with a relative amplitude of about 0.3, was attributed to the formation of the contact ion-pair and the long 100 ps component to the dissociation of the ion-pair to a free proton and RO(-). In the presence of acid, the recombination of an excess proton competes with the geminate recombination. At a high acid concentration, the recombination process alters the time-dependent concentrations of the reactant, product and intermediate contact ion-pair. We observed that when the acid concentration increases, the amplitude of both the long and intermediate time components decreases. At about 3 M of acid, both components almost disappear. Model calculations of the acid effect on the transient HPTS signal indeed showed that the amplitude of the intermediate time component decreases as the excess proton concentration increases.

The preparation and characterization of the stable human serum albumin (HSA)-C3 isomer of tris-malonic acid [C60]fullerene complex is reported. Other than the anti-fullerene antibody, a stable protein-fullerene complex with a native protein has never been observed. This study may provide valuable answers to the growing concern regarding the effects of carbonaceous nanomaterials on human health on one hand and, on the other, may lead to the development of novel antioxidant therapeutic agents, radiopharmaceuticals, and components for bioelectronic devices.

The pressure dependence of the excited-state proton dissociation rate constant of four photoacids, 2-naphthol-6,8-disulfonate (2N68DS), 10-hydroxycamptothecin (10-CPT), 5-cyano-2-naphthol (5CN2), and 5,8-dicyano-2-naphthol (DCN2), are studied in methanol. The results are compared with the results of the pressure dependence study we recently conducted for several photoacids in water, ethanol, and propanol. The pressure dependence is explained using an approximate stepwise two-coordinate proton transfer model. The increase in rate, as a function of pressure, manifests a strong dependence of proton tunneling on the distance which decreases with an increase of pressure between the two oxygen atoms involved in the process. The decrease in the proton transfer rate with increasing pressure reflects the dependence of the reaction on the solvent relaxation rate. We found that, for the relatively weak photoacids 2N68DS, 10-CPT, and 5CN2, the proton transfer rate constant increases by a factor of about 5-8 at a pressure of about 1.5 GPa. For a strong photoacid like DCN2, the rate increase was only by a factor of 2.

Picosecond time-correlated single-photon counting was used to measure the proton-transfer rate of green fluorescent protein (GFP) excited by several wavelengths between 266 and 405 nm. When samples of GFP in water and D2O are excited at short wavelengths, lambda(ex) < 295 nm, the fluorescence properties are largely modified with respect to excitation at a wavelength around 400 nm, the peak of the absorption band of the S0 --> S1 transition of the ROH form of the chromophore. The shorter the excitation wavelength, the longer the decay time of the ROH emission band at 450 nm and the longer the rise time of the RO- emission band at 512 nm. The proton transfer is slower by an order of magnitude and about a factor of 3 when GFP in water and D2O are excited by 266 nm, respectively.