Flavin Adenine Dinucleotide Photophysics in Ice - American Chemical

Feb 11, 2009 - Flavin Adenine Dinucleotide Photophysics in Ice. Itay Presiado and Dan Huppert*. Raymond and BeVerly Sackler Faculty of Exact Sciences,...
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J. Phys. Chem. C 2009, 113, 3835–3843

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Flavin Adenine Dinucleotide Photophysics in Ice Itay Presiado and Dan Huppert* Raymond and BeVerly Sackler Faculty of Exact Sciences, School of Chemistry, Tel AViV UniVersity, Tel AViV 69978, Israel ReceiVed: September 7, 2008; ReVised Manuscript ReceiVed: December 16, 2008

Steady-state, time-resolved emission and pump-probe techniques were employed to study the nonradiative process of flavin adenine dinucleotide (FAD) in methanol-doped ice and in a deuterated solvent mixture of methanol-d-D2O in a large range of temperatures of 79-268 K. We found that the nonradiative process depends on the temperature, i.e., the lower the ice temperature the smaller the nonradiative rate. The timeresolved emission is nonexponential, and a large portion of the decay curve could be reasonably fitted to a stretched exponent with R ) 0.55 in a large temperature range. We found a relatively large and unexpected isotope effect, KIE ) 1.8, on the nonradiative rate. The large nonradiative rate in FAD (compared to flavin mononucleotide (FMN)) possibly arises from a coupled electron-proton transfer from adenine to flavin in a “closed” conformation existing predominantly in an ice crystal. Introduction Flavoproteins containing the flavin chromophore can be found in many biological systems, where they undergo important redox reactions. The physical and chemical properties of the isoalloxazine ring and the two most common flavin cofactors, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), have been extensively studied for several decades.1-3 Weber4 found that the fluorescence intensity of FAD with respect to free riboflavin is smaller. An intramolecular ground-state complex between the flavin and the adenine was proposed to explain the small fluorescence of FAD in aqueous solutions. Furthermore, it was proposed that FAD exists in two conformations: a nonfluorescent “closed” conformation, in which the flavin and adenine rings interact through π-π interactions in a stacked conformation, and an extended, “open” conformation that is responsible for the remaining weak fluorescence of FAD. In solution, the FAD molecule is considered to predominantly assume the stacked conformation, which explains the low fluorescence intensity. From the comparison of the fluorescence intensity of FMN and FAD it is estimated that about 80% of the FAD molecules are in the “closed” conformation.5 Femtosecond fluorescence quenching was observed in flavoproteins such as flavodoxin,6,7 riboflavin-binding protein,8,9 and the D-amino acid oxidase/benzoate complex.9 The ultrafast fluorescence decay is explained as arising from an electron transfer process in a coplanarly stacked complex between the isoalloxazine ring and an aromatic amino acid such as tryptophan or tyrosine and also a benzoate. Visser and co-workers10 have investigated the structural dynamics of FAD using a combination of time-resolved fluorescence and molecular dynamics (MD) simulations. Polarized subnanosecond time-resolved-fluorescence experiments under various temperature and solvent conditions yielded experimental data on the dynamic behavior of the flavin cofactor. Nanosecond molecular dynamics simulations in water provided an additional insight into the dynamic behavior of the FAD molecule. Special attention was given to the interrelation * Corresponding author. Telephone: 972-3-6407012. Fax: 972-3-6407491. E-mail: [email protected].

between the MD and fluorescence data in terms of fluorescence quenching and rotational behavior. In a subsequent paper,11 Mataga, Visser, and their co-workers used the fluorescence upconversion technique to examine the femtosecond-picosecond fluorescence decay kinetics of FAD in aqueous solution. In the observation range of 30 ps three fluorescent lifetimes can be distinguished. The shortest-lived component (∼1 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 a 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-statefluorescence spectrum. Fluorescence anisotropy of 0.4 is instantaneously observed. In this study we researched the photophysics and photochemistry of FAD in water and in methanol-doped ice in a large temperature range of 79-295 K by looking at its time-resolved emission (measured by the time-correlated single photon counting technique, TCSPC, as well as the pump-probe technique with ∼200 fs time resolution (for the liquid state)). A small methanol concentration (0.1% mole ratio) is added to prevent the FAD from being excluded from the bulk of a microcrystal of the polycrystalline ice. A major fraction of the fluorescence quenching is attributed to the stacked flavin adenine “closed” conformation. The fluorescence decay rate of this component strongly depends on the temperature. At high temperatures, T g 160 K, the nonradiative rate constant approximately follows the solvent dynamics of the water molecules in ice. Below 160 K, the temperature dependence of the nonradiative decay rate is rather small. Experimental Section We used the time-correlated single photon counting (TCSPC) technique to measure the time-resolved emission of flavin adeneine dinucleotide (FAD) in this study. For sample excitations we used a cavity dumped Ti:sapphire femtosecond laser, Mira, Coherent, which provides short, 80 fs, pulses. The laser’s

10.1021/jp8079364 CCC: $40.75  2009 American Chemical Society Published on Web 02/11/2009

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Figure 1. Steady-state emission spectra of FAD at several temperatures in an aqueous solution containing 0.1% mole ratio of methanol. Broken line: liquid state.

second harmonic, operating over the spectral range 380-420 nm, was used to excite the FAD in the ice samples. The cavity dumper operated with a relatively low repetition rate of 500 kHz. The TCSPC detection system is based on a Hamamatsu 3809U, photomultiplier, and Edinburgh Instruments TCC 900 computer module for TCSPC. The overall instrumental response was about 35 ps (full width at half-maximum (fwhm)). The excitation pulse energy was reduced to about 10 pJ by neutral density filters. For the pump-probe experiments reported, we used an amplified femtosecond Ti:sapphire laser system. In brief, laser pulses (50 fs duration, centered near 800 nm with pulse energy of 600 µJ) at a 1 kHz repetition rate were generated by a Ti:sapphire-based oscillator (Coherent Mira seed) and amplified by a multipass Ti:sapphire amplifier (Odin Quantronix). Samples were excited by the second harmonic of the amplified laser (400 nm). To obtain probe pulses, we generated a supercontinuum by focusing 1 µJ of 800 nm pulse onto a 2 mm thick sapphire window. The continuum provided a probe pulse in the region of 480-750 nm. The probe beam signal was measured by a combination of a chopper/lock-in amplifier and computer averaging. Interference filters of 8 nm fwhm bandwidth at the proper wavelength were used in front of the probe beam detector, a silicon photodiode. Samples were placed in a rotating optical cell to avoid degradation. FAD and FMN were purchased from Sigma, and riboflavin was purchased from TCI Japan. For transient measurements the sample concentrations were between 2 × 10-4 and 2 × 10-5 M. Deionized water had a resistance of >10 MΩ. Methanol of analytical grade was purchased from Fluka. All chemicals were used without further purification. The temperature of the irradiated sample was controlled by placing the sample in a liquid N2 cryostat with a thermal stability of (1.5 K. Ice samples were prepared by first placing the cryogenic sample cell for about 20 min at a supercooled liquid temperature of about 265 K. The second step involved a relatively rapid cooling (5 min) to a temperature of about 240 K. Subsequently, the sample froze within a few minutes. To ensure ice equilibration prior to the time-resolved measurements, the sample temperature was kept for another 10 min at about 240 K. Results Figure 1 shows the steady-state emission spectrum of FAD in an aqueous solution containing 0.1% mole fraction of

Figure 2. Time-resolved emission of FAD in methanol-doped ice at several temperatures in the range of T g 197 K. (a) Linear scale; (b) semi-log scale. The IRF of the TCSPC system is also included (broken line).

methanol in water and in ice. The addition of a small fraction of methanol ensures the presence of the FAD molecules in the bulk of the microcrystal of polycrystalline ice sample rather than in the grain boundaries. Figure 1 shows the spectra at several temperatures in the temperature range 80-291 K. In the liquid phase the spectra are broad and the vibrionic substructure is blurry. The intensities of the FAD spectra in solid ice at high temperatures are about 20 times smaller than in the liquid phase. A large reduction in the steady-state emission upon sample freezing was also observed in FMN ice samples. Part of the emission intensity reduction we attribute to the exclusion of FAD molecules from the bulk ice upon sample freezing. Another reduction in the emission intensity arises from a large increase in the percentage of molecules in the nonradiative “closed” conformation: from 80% in liquid water to about 95% in ice. As the temperature of the FAD ice sample decreases, the emission intensity increases, and two vibrionic structures are observed. The large increase in the intensity supports the time-resolved emission observation that the nonradiative rate decreases as the temperature decreases. The spacing between the two vibrionic peaks is about 1100 cm-1, and it may be assigned to the flavin skeleton stretching vibration. Another important point that is clearly seen in Figure 1 is the large red shift of the emission spectra of the ice sample compared to the liquid phase. The shift is roughly 400 cm-1. The intensities of the emission spectra increase as the temperature decreases. At about 80 K the intensity almost equals that of the liquid at 290 K. Figure 2a shows on a linear scale the time-resolved emission of FAD in 0.1% methanol-doped ice sample at several temper-

FAD Photophysics in Ice

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Figure 3. Time-resolved emission of FMN and FAD in ice doped with 0.1% mole ratio of methanol.

atures in the high-temperature range of 197-260 K. The decay curves were measured by the time-correlated single photon counting technique with a limited time resolution determined by the instrument response function (IRF) shown in the figure of about 35 ps at full width at half-maximum. The samples were excited at 430 nm by 200 fs pulses at a rate of 500 kHz, and the emission was detected at 550 nm. The rate of the major part of the decay is nonexponential and rather short. At 260 K the initial decay time is ∼45 ps, whereas at 222 K the decay time is ∼150 ps. The fast decay component at temperatures below 230 K covers about 97% of the signal’s initial amplitude. Figure 2b shows, on a semilogarithmic scale, the timeresolved emission of FAD of the same sample in the same temperature range. The time scale is longer than that of Figure 2a by a factor of 10 and extends to cover the first 10 ns, and a dynamic range of two decades. As seen in Figure 2b, the signal is nonexponential at all temperatures. At high temperatures, the signal seems to be composed of short- and long-time components. The two lower temperature emission curves measured at 197 and 210 K seem to miss (at this time and dynamic ranges) the long-time fluorescence component, since its amplitude reduces to only 0.025. Figure 3 shows the time-resolved emission of FAD and FMN at several temperatures. Each panel shows the fluorescence signal of both FMN and FAD in methanol-doped ice (0.1% mole ratio) at a particular temperature. At temperatures above 240 K, the FMN decay is almost exponential with a lifetime of 5.4 ns, whereas the FAD decay is nonexponential with a very short decay time. At low temperatures, the FMN fluorescence decay signal shows a distinct short-time component of a relatively small amplitude. Both the decay time and its amplitude are independent of the temperature in the range 79-230 K. Figure 4a shows on a semilog plot the time-resolved emission of FAD in 0.1% mole fraction of methanol-doped ice sample at several temperatures in the low-temperature range of 79-197 K. The decay curves shown in the time range 0-5 ns are

Figure 4. Time-resolved emission of FAD in methanol-doped ice at several temperatures in the range of 79 e T < 197 K. (a) Stretched semilog scale; (b) semi-log scale.

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Figure 5. Time-resolved emission of FAD in methanol-doped ice and methanol-d and D2O ice at several temperatures. Each frame contains the data of both D2O and H2O ice samples.

nonexponential at all temperatures, including the lowest temperature, 79 K, near the liquid nitrogen temperature. Figure 4b shows the same signals shown in Figure 4a, only stretched to cover the fluorescence decay up to about 50 ns. In general, the signal can be fitted to a stretched exponent exp[-(t/τ)R], where R ) 0.55 ( 0.05, and is nearly independent of the temperature. Figure 5 shows the time-resolved emission of two FAD samples that differ in their isotopic constituents, at several temperatures in the range 79-291 K. In addition to the 0.1% mole fraction methanol in H2O, Figure 5 also shows the signal of a second sample, which is deuterated with 0.1% mole ratio of methanol-d in D2O. In general, the isotope effect has a moderate value of roughly 1.8 ( 0.1 at all temperatures. The decay curves of the D2O sample are also nonexponential and could be fitted by a stretched exponential function with the same stretched exponent factor R but with a somewhat larger decay time, τ. The relatively large isotope effect indicates that a proton transfer may be involved, to some extent, in the nonradiative process. Sobolewski and Domcke23,24 proposed that the nonradiative process of heterocyclic compounds containing NH2 and OH may proceed via a coupled electron-proton transfer. We further discuss this possibility in the next section. As seen in Figure 5, the amplitude of the long-time fluorescence tail, assigned to the “open” conformation, is larger in D2O samples than in H2O samples at all temperatures. This may indicate that the “open”-“closed” conformation equilibrium constant depends on the isotopic constituent. In D2O the “closed” conformation is less favored compared to H2O. Figure 6 shows the time-correlated single photon counting emission signal of FAD in a methanol-rich water mixture (5% mole fraction of methanol) at several temperatures in the range 210-248 K. For comparison we also plot in each panel in Figure 6 the signal of an FAD sample with 0.1% mole fraction of methanol. In the liquid state the amplitude of the short-time

component in 5% methanol mixture is somewhat larger than in the low methanol concentration sample at all studied temperatures. In ice the situation reverses, and the amplitude of the short component of the time-resolved emission of the sample containing 0.1% mole fraction of methanol is greater by a factor of 5 than that of the 5% sample. This observation indicates either that in the ice phase a high methanol concentration prevents the nonradiative process of the “closed” conformation, or that the ground-state equilibrium constant between the “open” and “closed” conformations strongly depends on the methanol concentration. A water-methanol mixture of 50% mole fraction freezes at -75 °C. The time-resolved emission of FAD in such a sample in the liquid phase at low temperatures of 200-260 K shows distinctive solvation dynamics components, especially at wavelengths of 480-510 nm, in a time frame ranging from a few tens of picoseconds to several hundreds of picoseconds depending on the temperature. The nonradiative rate of the methanol-rich samples (50% mole fraction) is rather small. Therefore, we conclude that the “closed” conformation population in 50% mole fraction of methanol mixtures is rather small. Another plausible explanation for the small nonradiative rate in this mixture is that that a coupled electron-proton transfer occurs in the excited “closed” conformation rather than an electron transfer reaction. In such a case, the solvent mixture composition strongly influences the proton transfer reaction rate. In general, electron transfer reaction rates are less sensitive than proton transfer reactions to the mole fraction of methanol in water-methanol mixtures. The rate of proton transfer in photoacids in pure methanol is rather small.12 For strong photoacids the rate decreases by about 2 orders of magnitude, whereas for weak photoacids the rate decreases by 3 orders of magnitude or more.12 In water-methanol mixtures, the excitedstate proton transfer decreases exponentially with an increase in the mole fraction of methanol.13 Therefore, we propose that

FAD Photophysics in Ice

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Figure 6. Time-resolved emission of FAD in methanol-doped samples of 0.1% and 5% mole fraction of methanol at several temperatures.

a large mole fraction of methanol may prevent the coupled proton-electron transfer process from taking place. Pump-Probe Spectroscopy. Samples of FAD, FMN, and riboflavin were excited at 400 nm by 1 kHz, ∼100 fs Ti:sapphire laser pulses, and probed by a white light supercontinuum at several wavelengths in the spectral range 520-640 nm. Figure 7 shows the probe signals of the three different samples measured at 560 nm close to the maximum of the emission band of the three compounds. All three samples show a negative signal, which signifies the larger contribution of the stimulated emission than absorption of S1 to higher excited states at the wavelength region of the emission spectra of the three similar compounds. The signal of FAD consists of a short-lived component of about 7 ps with an amplitude of about 0.4 and a long-lived decay component of τ > 100 ps. The FMN and riboflavin signals lack this short component. We attribute the short-lived time component of the FAD sample to an electron transfer between the adenine and the flavin in the closed conformation. The long lifetime component (τ > 100 ps) we attribute to the open conformation, in which the electron transfer process is much less effective (if at all). Figure 8 shows the probe signal at 580 nm of the three samples (FAD, FMN, and riboflavin). The signal is positive, and thus shows that the transient absorption component is larger than the stimulated emission. We assign the absorption component to the absorption from the first electronically excited singlet state to a higher excited state. The FAD signal contains a short-lived signal of ∼7 ps on top of a long-lived signal with a lifetime longer than 100 ps. The probe signals of both FMN and riboflavin do not contain a short-lived component. We found that at wavelengths shorter than 530 nm the signal is positive, whereas the signal in the range 540-560 nm is negative. The analysis of the overall transient signals in the range 520-640 nm indicates the existence of an absorption band (or bands) overlapping the emission band of these compounds in the range 520-580 nm. The short-time component of the probe signal of

Figure 7. Pump-probe signal of FAD, FMN, and riboflavin in water. The pump pulse is the SHG of an amplified Ti:sapphire system operating at 1 kHz. The probing beam measures the transients at 560 nm.

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Figure 8. Pump-probe signal of FAD, FMN, and riboflavin, excited at 400 nm, probed at 580 nm. Note that the signal denotes absorption.

FAD at 560 nm (negative) and 580 nm (positive) of about 7 ps may be compared with the fluorescence up-conversion signal observed in the experiment of Mataga, Visser, and co-workers.11 The up-conversion signal analysis of FAD consisted of three components: τ1 = 1 ps, τ2 = 9 ps, and a longer component of ∼2.4 ns. The 9 ps component of the up-conversion signal is attributed by the authors to the electron transfer in the closed conformation of the FAD. This value is similar to the 7 ps value we find in the pump-probe signal at 580 nm. Figure 9 shows the pump-probe signal of the FAD sample in H2O and D2O measured at a probe wavelength of 580 nm. The signal of both H2O and D2O is positive and consists of two components, the same as the signal as shown in Figure 8. The relative amplitude of the short-time component in the D2O sample, a ) 0.6, is larger than that of the H2O sample, a ) 0.4. The fitting analysis of the signals of both H2O and D2O shows that the lifetime of the short component for the D2O sample is 8.8 ps, compared to 6.8 ps for the H2O sample. Thus, a kinetic isotope effect (KIE) is clearly measured, but it is rather small in the liquid state; i.e., KIE ≈ 1.3, whereas in ice we found for the time-resolved emission an isotope effect of 1.8 (see Figure 5). Analysis of Time-Resolved Emission Signals. The TCSPC signals in methanol-doped ice at all temperatures are nonexponential. The signals can be fitted reasonably well with four exponents. We chose instead to use two stretched exponents to fit the data. From the analysis of all the data at a wide range of temperatures, 80-263 K, it seems that the data fit nicely to

Presiado and Huppert

Figure 9. Pump-probe signal of FAD in H2O and D2O samples probed at 580 nm.

well-separated short- and long-lived components. The shortlived component strongly depends on the temperature, whereas the long-lived component’s dependence on it is much weaker. We attribute the long-time decay component to the open FAD conformations, in which the electron transfer process is inefficient, and their emission lifetime is close to a radiative lifetime of ∼5 ns. In addition, the samples purchased from Sigma (>95% pure) probably contained a few percent FMN. FMN fluorescence in liquid H2O is long-lived and the steady-state emission is roughly 10 times more intense than that of FAD.5 Thus, in timeresolved experiments we expect an additional contribution to the amplitude fraction of the long-time component of about 0.01-0.04, depending on the FMN content in the FAD sample. We find an amplitude of 0.01-0.04 in the TCSPC signal in ice at temperatures below 250 K and above 150 K. This long-time component may arise from both FMN contamination and FAD in the open conformation. In the steady-state emission measurements of the FAD sample from Sigma, we find that the fluorescence intensity of the sample is 8.0 times smaller than that of a FMN sample with the same absorbance. This intensity ratio indeed shows that the sample contains about 2.5% FMN. We fit the signal using the following equation:

f(t) ) [a1 exp[-(t ⁄ τ1)R1] + (1 - a1) exp[-(t ⁄ τ2)R2]] × exp(-t ⁄ τf)R3 (1) There are five fitting parameters of the TCSPC signal: the two lifetimes τ1 (the short lifetime component) and τ2, the stretched exponent factors R1 and R2, and the amplitude of

FAD Photophysics in Ice

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TABLE 1: Fitting Parameters of the Nonradiative Decay of FAD in Ice to a Stretched Exponenta

a

T [K]

τ [ns]

79 88 100 112 124 148 160 173 197 210 235 248 260 268

0.800 0.700 0.600 0.550 0.460 0.320 0.280 0.210 0.125 0.090 0.047 0.030 0.018 0.008

R ) 0.55 (see text).

TABLE 2: Fitting Parameters of the Nonradiative Decay of FAD in D2O Ice to a Stretched Exponenta

a

T [K]

τ [ns]

79 100 124 136 148 160 173 185 197 210 222 227 232 237 242 248 253 258 263

2.200 1.600 1.250 0.950 0.800 0.600 0.480 0.300 0.210 0.160 0.110 0.090 0.085 0.075 0.070 0.060 0.050 0.040 0.025

R ) 0.55 (see text).

the short-time component, a1.The radiative lifetime of the FAD sample, τf, was to be that of the FMN sample; it was observed that the decay signal of the time-resolved emission of the FMN sample in ice at temperatures below 240 K is nonexponential (see Figure 3). We assume that the time-resolved emission of the open conformation of FAD exhibits the same decay profile as that of FMN. We used a stretched exponent (τf, R3) to fit the FMN data. We apply the same decay pattern also for the FAD data. The stretched exponent factor R1 was found to be a constant value of 0.55 in a wide temperature range below 210 K. At higher temperatures its value slightly increases with temperature from R ) 0.55 to about R ) 0.6. The value of R2 weakly depends on the small mole fraction of methanol added to some of the samples in order to increase the solubility of FAD in bulk ice. In pure water samples R2 = 0.55, whereas for 0.1% mole fraction of methanol R2 = 0.80. τ2, the long lifetime component, varies from 3.5 ns to about 6.6 ns at the lowest temperature. In Tables 1 and 2, we present the values of the short-time component, τ1, in a wide range of temperatures in H2O and D2O samples, respectively. The value of τ1 in the H2O sample at about 248 K is 30 ps, whereas at 80 K it is 0.8 ns. We attribute this decay time component, τ1, to the electron transfer process. We found that

the values of τ1 are independent of the small amount of mole fraction of methanol we added to the sample (0.1% and 0.2% mole fraction of methanol). In previous experiments we found that a small fraction of methanol in H2O prevents probe molecules, such as 2-naphtholsulfonate derivatives, from being expelled from the bulk ice. We also noticed that in pure water ice samples with FMN the total fluorescence intensity is reduced by 2 orders of magnitude upon freezing of the sample. The timeresolved emission of FMN in pure water ice and a pure FMN powder sample at room temperature showed a short-lived nonexponential decay of ∼0.2 ns. This observation indicates that in a pure water ice sample the FMN indeed tends to aggregate at the grain boundaries. The aggregation leads to the drastic reduction in the fluorescence intensity. In pure water samples with FAD the signal intensity is reduced by a factor of 20 upon freezing, which is ∼5 times more intense than for FMN in pure ice. The main component of the time-resolved emission is short (τ ) 30 ps at 260 K), and the signal in ice lacks the relatively large amplitude (0.05) of the long lifetime (τ ) 5 ns) component that is observed in ice samples with 0.1% mole fraction of methanol. The value of the short-lived component is the same as that of a sample that contains 0.1% or 0.2% mole fraction of methanol, for which the total fluorescence intensity is ∼5 times larger, since it also contains a relatively large amplitude (0.05) of a long-time component. A plausible explanation for the FAD emission in pure ice is as follows: the main portion of the FAD aggregates at the grain boundaries of the ice microcrystals. Each excited FAD molecule is only in the closed conformation or finds a close adenine (or flavin) at a nearby molecule and, thus, to efficiently transfer an electron between the flavin and the adenine. The long-lived emission of the FMN molecules (the impurity) in the sample and that of the open conformation of FAD, which also has a long lifetime emission in the liquid phase, are nonfluorescent at the grain boundaries of the ice. Discussion In a recent study we measured the photophysics and photochemistry of flavin mononucleotide (FMN) in liquid water and in ice in neutral pH samples and in acidic solutions in the range 2.3 < pH < 3.6 (2.5 × 10-4 < cH+ < 5 × 10-3).14 In ice we found that in an acidic solution the nonradiative rate is 10 times larger than in liquid water. We attributed the nonradiative rate in acidic ice and water to reaction of a proton with the excited flavin that leads to a protonation of one of the many basic sites on the flavin rings. The main conclusion of the FMN time-resolved study and similar studies using several photoacids in acidic solution is that the proton diffusion in ice is ∼10 times larger than in liquid water. The time-resolved emission of FAD in neutral pH ice is clearly distinct from that of FMN. FMN in water and ice decays almost exponentially in neutral pH solution with a lifetime of τf = 5.4 ns. Unlike FMN, the time-resolved emission of FAD in ice exhibits a short-time decay component of a few tens of picoseconds at 265 K with a large amplitude of a > 0.95, followed by a long-time component of ∼3.5 ns. The decay profile of the fast component is nonexponential. At about 220 K the emission signal could be fitted by a stretched exponent exp[-(t/τ)R], where R ) 0.54and τ220 K ≈ 150 ps. An additional long-time fluorescence tail with a small amplitude of 0.025 and a lifetime of about 8 ns covers the rest of the signal from about 2% of the initial intensity (at time zero) at about 10 ns to about 50 ns. Penzkofer et al.15 studied the fluorescence quenching of flavins in aqueous solutions by reducing compounds. They used

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knr1(T) ) k01 exp(-Ea1 ⁄ RT)

(2a)

knr2(T) ) k02 exp(-Ea2 ⁄ RT)

(2b)

where k10 . k20 and Ea1 . Ea2. At a certain temperature the total nonradiative rate constant is given by

ktot ) k1 + k2

Figure 10. Arrhenius plot of the nonradiative decay rate of FAD in (a) H2O and (b) D2O ice.

three flavin derivatives, one of which was FAD, and three reducing agents. Flavins are reduced by strong reducing agents such as dithiothreitol (DTT). The fluorescence quantum yields and fluorescence lifetimes are determined as a function of the reducing compound concentration. It was found that the dynamics of the fluorescence quenching is diffusion-controlled, and it is proposed that the fluorescence quenching is governed by an electron transfer process from the donor, the reducing agent, to flavin. Mataga and co-workers7 and Zewail and coworkers8 attributed the very large nonradiative rates of several flavoproteins also to electron transfer from aromatic amino acids such as tryptophan and tyrosine to flavin. Temperature Dependence of the Nonradiative Rate. Figure 10 shows an Arrhenius plot of the short-time component τ1 (see Tables 1 and 2) rate constant, k ) 1/τ1, versus 1/T for the FAD samples in H2O and D2O ice. As seen in Figure 10, the slope of the Arrhenius plot is not constant. At low temperatures, the slope is much lower than at high temperatures. We observed similar non-Arrhenius plots in the nonradiative processes of both dGMP and G-4,16 and also for the proton transfer rate constant in GFP.17 A plausible nonradiative mechanism that might explain the non-Arrhenius temperature dependence of the nonradiative rate in ice in the temperature range 79-270 K may include two coordinates that control the overall nonradiative rate, rather than one. In this case, we can assign a rate constant for each coordinate (k1 and k2). The temperature dependence of each rate constant follows an Arrhenius law. Let us assume that k1 and k2 are substantially different from each other in both their activation energies and their preexponential factors:

(3)

In such a case (by choosing the precise parameters) the main nonradiative channel at high temperatures is that of the rate constant k1, whereas at a low enough temperature we find a switch over, where k2 g k1. Figure 10 also shows the calculated curves of ktot, k1, and k2 (blue, red, and green curves, respectively), and the values of k(T) as obtained from the best fit of the time-resolved emission for H2O and D2O samples. The fit is rather satisfactory at both high and low temperatures. We used for H2O k10 ) 4.1 × 1013 s-1, k20 ) 5.0 × 109 s-1, Ea1 ) 15 kJ/mol, and Ea2 ) 1 kJ/mol. For D2O samples k10 ) 2.8 × 1013 s-1, k20 ) 1.95 × 109 s-1, Ea1 ) 15 kJ/mol, and Ea2 ) 1 kJ/mol. An alternative theoretical explanation to the dynamics of the fluorescence quenching of FAD in water and in ice is based on the role of solvent fluctuations in promoting electron and/or coupled electron-proton transfer reactions. In general, there are two extreme regimes in the formulation of the electron transfer reaction rate constant: the adiabatic and the nonadiabatic limits. In the nonadiabatic limit, the rate is determined by the rate of electron tunneling. The preexponent of the rate constant, kET, is then determined by the coupling matrix element and to a lesser extent by the reorganization energy and the temperature.18 In the adiabatic regime, the solvent’s fluctuation mainly determines the preexponential factor. An expression for solvent-controlled adiabatic electron transfer is given by18



ketAD ) τL-1

ES exp(-Ea ⁄ kBT) 16πkBT

(4)

where τL is the longitudinal dielectric relaxation time, ES is the solvent reorganization energy, and Ea is the activation energy.19 The preexponent in the adiabatic solvent-controlled limit strongly depends on the solvent’s dynamic properties represented in eq 4 by τL. In a previous study20 we examined the solvation statics and the dynamics of Coumarin 343 and the strong photoacid (pK* ∼ 0.7) 2-naphthol-6,8-disulfonate (2N68DS) in methanol-doped ice (1% mole ratio concentration of methanol) in the temperature range 160 - 240 K. Both probe molecules showed a relatively fast solvation dynamics in ice, ranging from a few tens of picoseconds at 240 K to about a nanosecond at 160 K, whereas in doped ice below T < 175 K we observed a sharp decrease in the dynamic Stokes shift of both Coumarin 343 and 2N68DS. Its value is approximately equal to only 200 cm-1 at ∼160 K, far less than the value of about 1100 cm-1 at T g 200 K (at times longer than t > 10 ps). We previously found that at a particular temperature the rate of the solvation dynamics in 1% methanol-doped ice is comparable to the nonradiative rate of deoxyguanosine monophosphate (dGMP) in 1% methanol-doped ice.16 We therefore proposed that the nonradiative rate of dGMP in particular, and perhaps also of other nucleobases in ice in general, is determined by the solvent translational and rotational motions. In the liquid state these solvent motions are rather fast. The solvation dynamics in water is bimodal. The long-time component’s length is ∼1 ps,21 and this is the same time scale as that of the long-time component of the nonradiative rate of dGMP in liquid water at room temperature.22 Based on the nonradiative rate of FAD in liquid and in ice found in this work,

FAD Photophysics in Ice and the formal expression of the adiabatic electron transfer rate constant (eq 4) and the FAD study of Visser and co-workers9,11 in liquid water, we propose that the nonradiative rate of the FAD’s closed conformation may be determined by the solvent dynamics rather than by the electron coupling matrix element. The similarity of the time scale of solvation dynamics in ice to the nonradiative rate of the FAD “closed” conformations in ice as well as the relatively large isotope effect of 1.8 in ice at intermediate temperatures of 173-240 K fits nicely also to Sobolewski23,24 and Domcke’s model on the origin of the large nonradiative rate of nucleobases and similar compounds. Domcke et al.23 and Sobolewski et al.24 carried out ab initio electronic structure calculations on nucleobases and related heterocyclic compounds containing NH2 and OH. From these calculations, they drew a general mechanistic picture of the nonradiative decay of nucleobases and other similarly structured molecules. An important part of their model is an intermediate excited singlet state with πσ* character that has repulsive potential energy functions with respect to the stretching of OH or NH bonds. The 1πσ* potential-energy surfaces intersect not only the bound potential-energy surfaces of the 1ππ* excited states, but also the potential-energy surface of the electronic ground state. An ultrafast internal-conversion process takes place via predissociation of the 1ππ* state to the 1πσ* state, and subsequently, a conical intersection with the ground state ends the photocycle. In protic solvents, the 1πσ* states encourage a hydrogen transfer process from the chromophore to the solvent. Calculations for chromophore-water clusters have shown that a charge-separation process takes place spontaneously in the solvent shell, producing a microsolvated hydronium cation and a microsolvated electron. Their findings show that the 1πσ* state promotes a coupled electron-proton transfer (hydrogen transfer) process from the chromophore to the solvent. Such a model may apply (with some modifications) to the nonradiative process of the “closed” conformation of FAD. Summary We employed steady-state, time-resolved emission and femtosecond pump-probe techniques to study the nonradiative process of flavin adenine dinucleotide (FAD) in methanol-doped ice and in deuterated solvent mixture in the large temperature range of 79-268 K. The time-resolved emission is nonexponential and could reasonably be fitted to a stretched exponent with R ) 0.55 in a large temperature range. We found that the nonradiative process depends on the temperature, i.e., the lower the ice temperature the smaller the nonradiative rate. The decay time constants are in the range 15 ps-1 ns: for high temperature ice samples, as high as 268 K, it would be 15 ps and for low temperature ice samples, as low as 79 K, it would be about 1 ns. We found in ice a relatively large and unexpected isotope effect, KIE ) 1.8, on the nonradiative rate. We therefore propose that the large nonradiative rate in FAD (compared to flavin mononucleotide) could be explained as arising from a coupled electron-proton transfer (rather than an electron transfer) from adenine to flavin of a “closed” conformation existing predominantly in an ice crystal. In a previous study20 we found that solvation dynamics in methanol-doped ice is rather fast in the range of 10 ps at high temperatures to a few nanoseconds at

J. Phys. Chem. C, Vol. 113, No. 9, 2009 3843 low temperature of ∼160 K. Thus, the solvent relaxation rate is in close proximity to the nonradiative rate of FAD. Theories of proton and electron transfer predict that these reactions may also proceed via a rate-limiting step, where the solvent fluctuation determines the reaction rate (the so-called solvent-controlled adiabatic limit).18 Therefore, we propose an alternative explanation for the origin of the rate-determining step in the nonradiative process of FAD in ice. The temperature dependence of the nonradiative rate process in FAD in ice exhibits a non-Arrhenius behavior. The slope of ln knr versus 1/T depends on the temperature: the lower the temperature the smaller the slope (concave shape). Similar dependences of the nonradiative rates were also found for deoxyguanosine monophosphate in ice samples in the same temperature range.16 Acknowledgment. This work was supported by a grant from the James-Franck German-Israel Program in Laser-Matter Interaction. References and Notes (1) Song, P. S. In Quantum Aspects of Heterocyclic Compounds Chemistry and Biochemistry; Bergmann, E. D., Pullman, B., Eds.; Israel Academy of Sciences and Humanities: Jerusalem, 1970; p 358. (2) Leijonmarck, M. Chem. Commun. 1977, 8, 1. (3) Hall, L. H.; Orchard, B. J.; Tripathy, S. K. Int. J. Quantum Chem. 1987, 31, 217. (4) Weber, G. Biochem. J. 1950, 47, 114. (5) Spencer, R. D.; Weber, G. In Structure and Function of Oxidation Reduction Enzymes; Åkeson, Å., Ehrenberg, A., Eds.; Pergamon Press: Oxford, 1972; p 393. (6) Mataga, N.; Chosrowjan, H.; Taniguchi, S.; Tanaka, F.; Kido, N.; Kitamura, M. J. Phys. Chem. B 2002, 106, 8917. (7) Tanaka, F.; Chosrowjan, H.; Taniguchi, S.; Mataga, N.; Sato, K.; Nishina, Y.; Shiga, K. J. Phys. Chem. B 2007, 111, 5694. (8) Zhong, D.; Zewail, A. H. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 11867. (9) Mataga, N.; Chrosrowjan, H.; Shibata, Y.; Tanaka, F.; Nishima, Y.; Shiga, K. J. Phys. Chem. B 2000, 104, 10667. (10) van den Berg, P A. W.; Feenstra, K. A.; Mark, A. E.; Berndsen, H. J. C.; Visser, A. J. W. G. J. Phys. Chem. B 2002, 106, 8858. (11) Chosrowjan, H.; Taniguchi, S.; Mataga, N.; Tanaka, F.; Visser, A. J. W. G. Chem. Phys. Lett. 2003, 354. (12) Carmeli, I.; Huppert, D.; Tolbert, L. M.; Haubrich, J. E. Chem. Phys. Lett. 1996, 260, 109. (13) Solntsev, K.; Huppert, D.; Agmon, N.; Tolbert, L. M. J. Phys. Chem. A 2000, 104, 4658. (14) Uritski, A.; Presiado, I.; Huppert, D. J. Phys. Chem. C, ASAP. (15) Penzkofer, A.; Bansal, A. K.; Song, S.-H.; Dick, B. Chem. Phys. 2007, 336, 14. (16) Gepshtein, R.; Huppert, D.; Lubitz, I.; Amdursky, N.; Kotlyar, A. B. J. Phys. Chem. C 2008, 112, 12249. (17) Leiderman, P.; Gepshtein, R.; Tsimberov, I.; Huppert, D. J. Phys. Chem. B 2008, 112, 1232. (18) Rips, I.; Jortner, J. J. Chem. Phys. 1987, 87, 2090. (19) (a) Marcus, R. A. J. Chem. Phys. 1956, 24, 966. (b) Marcus, R. A. Annu. ReV. Phys. Chem. 1964, 15, 155. (c) Marcus, R. A. Faraday Discuss. Chem. Soc. (London) 1982, 74, 7. (20) Uritski, A.; Huppert, D. J. Phys. Chem. A 2007, 111, 10544. (21) Rosental, S. J.; Xie, X.; Du, M.; Fleming, G. R. J. Chem. Phys. 1991, 95, 4715. (22) Crespo-Herna´ndez, C. E.; Cohen, B.; Hare, M. P.; Kohler, B. Chem. ReV. 2004, 104, 1977. (23) Domcke, W.; Stock, G. AdV. Chem. Phys. 1997, 100, 1. (24) Sobolewski, A. L.; Domcke, W.; Dedonder-Lardeux, C.; Jouvet, C. Phys. Chem. Chem. Phys. 2002, 4, 1093.

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