Excited-State Intermolecular Proton Transfer of Firefly Luciferin IV

Feb 16, 2011 - the firefly luciferase, undergoes in both acidic aqueous solutions and ice. ... the firefly bioluminescent color pH dependence is the r...
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Excited-State Intermolecular Proton Transfer of Firefly Luciferin IV. Temperature and pH Dependence Yuval Erez, Itay Presiado, Rinat Gepshtein, and Dan Huppert* Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel Aviv University, Tel Aviv 69978, Israel

bS Supporting Information ABSTRACT: Time-resolved emission as well as steady-state UV-vis techniques were employed to study the photoprotolytic processes that D-luciferin, the natural substrate of the firefly luciferase, undergoes in both acidic aqueous solutions and ice. The emission spectrum of D-luciferin in a 20 mM HCl aqueous solution or higher has an additional emission band at 590 nm red-shifted with respect to the strongest emission band positioned at 530 nm of the deprotonated NRO-* form in a pH-neutral aqueous solution. We attribute this emission band to the zwitterion form designated as þHNRO-. The time-resolved emission signals show that the NRO-* emission band at 530 nm and the zwitterion emission band at 590 are strongly quenched by a recombination process with a proton in an acidic solution and in ice. In ice, the quenching rate is 10 times faster than in the liquid state. We attribute the fast quenching rate to the high value of the proton diffusion constant in ice.

’ INTRODUCTION D-Luciferin, shown in Scheme 1, is the active fluorescent substrate of the firefly bioluminescent reaction.1,2 In the firefly, oxidation of D-luciferin is catalyzed by firefly luciferase in the presence of ATP and magnesium ions, to form oxyluciferin in an electronically excited singlet state. The decay of the excited oxyluciferin molecule to its ground state is accompanied by the emission of a photon in the green-red part of the spectrum.3-8 Oxyluciferin is later enzymatically regenerated into D-luciferin.9 Much attention has been drawn to the fact that, although the emitter molecule is identical in different species of firefly, click beetles, and railway worms, the color of the emitted light is not.10,11 This has been attributed to differences in luciferase enzymes between the species,12,13 in ionic concentrations and in pH.14 It has been assumed that emission color differences at different pH levels correspond to different states of the excited oxyluciferin molecule in the luciferase enzyme.15,16 Recently, it has been demonstrated that changes in acidity strongly affect the spectral shape and position of the emission of in vitro bioluminescence. Ando et al.17 showed that below a pH level of 8.5, the quantum yield of the firefly bioluminescent reaction was invariant under changes of reaction conditions except for change in pH. They determined the highest quantum yield of firefly bioluminescence to be 41.0 ( 7.4%, less than onehalf of the previously reported value (88 ( 25%).18 They also found that the pH had a great effect on the reaction rate. Further investigation of quantum yields and kinetics of the firefly bioluminescence followed.19 In the Ando et al. paper, the authors decomposed the spectra into three components, peaking at 560, 620, and 670 nm. The deep-red emitter was observed for the first time. They found that all components showed only small changes in peak energies and peak widths with pH. The 560 nm component peak intensity depended strongly on pH, but the 620 and 670 nm peaks were r 2011 American Chemical Society

insensitive to it. The authors concluded that the assumption that the firefly bioluminescent color pH dependence is the result of a chemical equilibrium between two states of oxyluciferin should be re-examined. In the same issue, Ugarova argued that the new results by Ando et al. support the previous results of other groups, rather than contradict them.20 While debate over the pH dependence of the firefly bioluminescence remains open, in this Article the influence of temperature and pH on the photoluminescence of D-luciferin is discussed, in an attempt to advance the study of its photoacidic properties in aqueous solutions and in ice. In the past decades, intermolecular excited-state proton transfer (ESPT) to a solvent or to a base via a solvent-solute complex in a liquid solution, and more recently in ice, has been extensively studied.21-33 For many years, we used an excitedstate proton transfer (ESPT) model that also accounts for the diffusion-influenced geminate recombination of the transferred proton with the deprotonated form of the photoacid.27,34,35 In a previous study,36 we measured the photoprotolytic processes of excited luciferin in water. D-Luciferin has dual emission bands when excited from its neutral form (the protonated form, NROH). The bands correspond to emission from the NROH* and from the deprotonated species NRO-* of Dluciferin. The decay of the time-resolved emission of the NROH* in neat water is nonexponential and fast, that is, about 25 ps at room temperature. The time-resolved emission signal of the NRO-* is complex because the proton geminate recombination process is irreversible and thus enhances the quenching of the NRO- fluorescence.

Received: November 15, 2010 Revised: January 16, 2011 Published: February 16, 2011 1617

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The Journal of Physical Chemistry A Scheme 1

In a more recent work,37 we studied the reaction of acetate ions (Ac-) in H2O and D2O solutions with the excited NROH form of D-luciferin. We found three time components in the complex decay profile of the excited NROH in 1 M aqueous solution of sodium acetate or higher. We attribute the short-time component of ∼300 and 600 fs for H2O and D2O, respectively, to the PT reaction of a contact ion pair or to a water-bridged complex. We attribute the intermediate time component of 2000 and 3000 fs for H2O and D2O, respectively, to a complex where the bridge is longer by one water molecule. The long-time component we attribute to the diffusion-assisted reaction, in which the proton donor (the NROH*) and the acceptor (Ac-) diffuse to produce either a contact pair or a water bridged complex. In the current study, we focused our attention on the photophysics and photochemistry of d-luciferin in acidic media, aqueous solutions with HCl as well as methanol-doped ice samples. In previous studies of many photoacids, we found that excess protons in liquid water and ice react with the photoexcited molecules in their deprotonated form. Irreversible photoacids such as 1-naphthol analogues were found to recombine with the geminate proton or excess proton with a nearly diffusion-controlled reaction rate. The recombination reaction results in a ground-state protonated photoacid, which leads to fluorescence quenching and a faster signal decay of the deprotonated form. DLuciferin belongs to this class of photoacids, and, consequently, the fluorescence intensity of the 530 nm band is inversely proportional to the acid concentration in water and ice. We also found that additional protonation occurs in D-luciferin in the presence of excess protons in solution, leading to the formation of a new emission band with a peak at 590 nm. We attribute this band to a zwitterion designated as þHNRO-.

’ EXPERIMENTAL SECTION We used the time-correlated single-photon counting (TCSPC) technique to measure the time-resolved emission of D-luciferin in aqueous solutions. For sample excitations, we used a cavity dumped Ti:Sapphire femtosecond laser, Mira, Coherent, which provides short, 80 fs, pulses. The laser’s second harmonics (SHG), operating over the spectral range of 380-420 nm, was used to excite both photoacid ice samples. The cavity dumper operated with a relatively low repetition rate of 800 kHz. The TCSPC detection system is based on a Hamamatsu 3809U photomultiplier and an Edinburgh Instruments TCC 900 computer module for TCSPC. The overall instrumental response was about 35 ps (fwhm). The excitation pulse energy was reduced to about 10 pJ by neutral density filters. Steady-state experiments were conducted with a miniature diode array spectrometer, MS-240 (CVI), with a spectral resolution of 2 nm. This spectrometer allows the measurement of the fluorescence at the front surface that is used for ice samples, in conjunction with the TCSPC measurements. The overall sensitivity of the spectrometer and the optical system that collects the

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emission from the ice sample is roughly 3 orders of magnitude less efficient than a standard commercial fluorometer with photon counting capabilities. (4S)-2-(6-Hydroxy-benzothiazole-2-yl)-4,5-dihydrothiazole4-carboxylic acid (D-luciferin) 99.5% was purchased from Iris Biotech (Germany). HCl (1 N) was purchased from Aldrich. 1-Naphthol-4-sulfonate (1N4S) was purchased from TCI. 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 approximately (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 250 K. Ice samples were doped with 0.25% mole ratio of methanol to prevent expulsion of D-luciferin molecules from the bulk ice to the grain boundaries of the ice microcrystals. Methanol is known to form clathrates with water, and it probably acts as a mediator between the ice molecules and large organic molecules such as D-luciferin.

’ RESULTS Steady State. Figure 1A shows the absorption spectra of Dluciferin in several aqueous acidic solutions. In neutral pH, the lowest energy band’s peak is at 335 nm. As the acid concentration increases, the 335 nm band’s intensity decreases, and a new band appears on the spectrum with a peak at about 390 nm. Moreover, a 5 nm red-shift (200 cm-1) occurs to the 335 nm band even at a low acid concentration of a few millimolars. The carboxyl group on D-luciferin (see Scheme 1) has a pKa value of ∼3, and the band shift may be associated with its protonation. Figure 1B shows the normalized excitation spectra of Dluciferin in aqueous solutions with various concentrations of HCl of up to 220 mM. The excitation spectra correspond to two emission wavelengths, 520 and 600 nm. The broad structureless band red-shifts with rising acid concentrations. The normalized band’s shape only weakly depends on the acid concentration. In acidic solutions, the excitation band red-shifts when the emission wavelength is set on 600 nm. The band shift is in accord with the formation of a new absorption band in highly acidic solutions. Figure 2A shows the normalized emission spectra of D-luciferin in several aqueous acidic HCl samples of up to 220 mM. At cHCl e 11 mM, the spectrum consists of two broad structureless bands with peaks at ∼452 and ∼533 nm for the protonated and deprotonated forms, respectively, and the value of the relative intensities ratio, INRO-*/INROH*, is roughly 35. The intensity ratio of the two bands, INROH*/INRO-*, of a photoacid in a pH-neutral solution is approximately given by: 0

INRO-  =INROH ¼ 1618

kPT 3 kr kr

ð1Þ

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Figure 1. (A) Absorption spectrum of D-luciferin in several acidic aqueous solutions. (B) Steady-state excitation spectra of D-luciferin in several acidic aqueous solutions, measured at 520 and 600 nm, which correspond to NRO-* and þHNRO-*, respectively.

Figure 2. (A) Normalized steady-state emission spectra of D-luciferin in several acidic aqueous solutions. The sample was excited at 390 nm. The narrow peak at 650 nm arises from the scattered light from the excitation at 325 nm. (B) Steady-state emission spectra of D-luciferin in several aqueous HCl solutions on a semilogarithmic scale. Note the large fluorescence quenching of both the NRO-* (520 nm) and the þHNRO-* (600 nm) bands at high acid concentrations.

Scheme 2

where kPT is the proton transfer (PT) rate constant, and kr and 0 k r are the radiative rates of the protonated and deprotonated forms, respectively. The higher the acid concentration gets, the weaker the emission intensity of the NRO-* band becomes, whereas that of the NROH* band remains mostly unchanged as long as kPT > kr. Scheme 2 describes the photoprotolytic cycle processes that D-luciferin in a pH-neutral aqueous solution undergoes after excitation of the ground-state protonated NROH* form. At cHCl g 20 mM, a new emission band is observed at ∼590 nm. We assign this new emission band to the formation of a zwitterion þHNRO-*. At 220 mM HCl, the intensity of the zwitterion emission band is the highest of all three species of D-luciferin. Figure 2B shows the steady-state emission spectra of Dluciferin in the acidic aqueous solutions shown in Figure 2A

but on a semilogarithmic scale. The spectra are not normalized, and it is thus easy to notice the sharp drop in the emission intensity of the NRO-* band as well as the zwitterion band that is formed at high concentrations of acid. The intensity of the NROH* emission band is only weakly affected by the acid concentration. The weakening of the NRO-* band’s intensity with the rise in the HCl concentration is explained by the reaction of the excess proton with the NRO-* form of D-luciferin, which leads to quenching: NRO-* þ Hþ f þHNRO-(g). The reaction rate depends on both the intrinsic proton recombination rate, ka, and the diffusion-controlled rate, kD. As will later be shown, when ka > kD, kD becomes the limiting overall rate constant for the proton recombination reaction, and for a reaction with a proton with a large diffusion constant, that is, DHþ = 1  10-4 cm2/s, kD = 5  1010 M-1 s-1.0 In a 10 mM solution of HCl, the pseudofirst-order reaction rate k D = cHþ 3 kD = 5  108 s-1. This value is higher than that of the radiative decay rate of the NRO-*, which is ∼2  108 s-1. Therefore, weakening of the fluorescence of the NRO-* band with the increase of the acid concentration is observed. Scheme 3 describes the photoprotolytic cycle, which the doubly protonated þHNROH form of D-luciferin undergoes in aqueous solutions with a pH level less than 1.5. At about 50 mM HCl, around 10% of the ground-state population of D-luciferin is in a new protonation state. This is based on the absorption, 1619

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excitation, and emission spectra (see Figures 1A,B, 2A,B). We assume that the protonation in the ground state occurs on the nitrogen in the thiazole ring, and therefore we designated it in Scheme 3 as þHNROH. In the excited state, the heterocyclic nitrogen atom is a stronger base and the hydroxyl group is a stronger acid, so we expect that þHNROH undergoes an ESPT process at the hydroxyl site to form the excited-state zwitterion þ HNRO-* that emits at 590 nm. The excited zwitterion of D-luciferin undergoes a recombination with the geminate and excess protons in both the liquid and ice the phases that leads to fluorescence quenching. The intrinsic recombination reaction rate constant is large; that is why the overall reaction rate is nearly diffusion-controlled. The fluorescence quenching reaction by a proton is represented in Scheme 3 by the diagonal arrow. We were unable to determine the position in which the protonation takes place. Figure 3 shows the steady-state (time-integrated) emission spectra of several samples of D-luciferin in methanol-doped H2O and D2O at several temperatures in both the liquid and the ice phases. The spectra were measured by a miniature diode array spectrometer (see Experimental Section). Therefore, the signalto-noise ratio of the spectra shown in Figure 3 is not good. The spectrum is not corrected for sensitivity variation of the spectrometer as a function of the wavelength. Panel A shows the spectra of D-luciferin in a neutral pH D2O solution also containing 0.25% mole ratio of methanol-d. The methanol doping is used to prevent the expulsion of the D-luciferin molecules from the ice microcrystal bulk and aggregation at the grain boundaries. Previously, we found that methanol serves as a cosolvent with excellent amphiphilic properties that afford the incorporation of photoacids and similar compounds into the ice. The noticeable phenomenon here is the large variation of the spectrum of liquid phase from that of the ice. In the liquid state, the emission spectrum consists of two bands with maxima at ∼465 and 557 nm. We assign the ∼465 nm band to the protonated NROH* form, and the 557 nm band to the deprotonated NRO-* form. The NRO-* band in D2O is red-shifted by ∼7 nm with respect to its position in H2O (see panel C). In D2O ice, the relative intensity of the protonated band increases with respect to the intensity of the band of the deprotonated form. In ice, the band further red-shifts with respect to its position in the liquid solution. Panel B shows the emission spectra of D-luciferin in 2 mM samples of DCl doped with methanol-d. In the liquid phase, the spectrum consists of the bands of the protonated and deprotonated forms, whose relative intensities are similar, as it is for the neutral solutions shown in panel A. In ice doped with 2 mM DCl, the relative intensity of the emission band of the protonated form at 465 nm is weaker than that of the pH-neutral sample and is temperature independent. The emission at long wavelengths splits into two bands at roughly 550 and 600 nm. We suggest that the 550 nm band is that of the NRO-* and the band at 600 nm we assign to the zwitterion þHNRO-*. The 600 nm band we

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assigned to the zwitterion is also visible in room-temperature steady-state emission of D-luciferin at cHCl g 47 mM as shown in Figure 2. In acidic ice samples of D-luciferin, the relative intensity of the 600 nm band is larger than that of the 550 nm band, and this is what led us to suggest these assignments. The time-resolved emission of the NRO-* from a sample with a strong acid measured at 530 nm shows a large increase in the decay rate, which is why the NRO-* band intensity decreases considerably. The decay rate of the NROH* band at 450 nm in ice decreases, and thus its time-integrated emission intensity increases, which obviously enlarges the INROH*/INRO-* ratio. Panel C shows the steady-state emission spectra of D-luciferin in methanol-doped liquid and solid H2O. In the liquid state, the NRO-* band intensity is high, whereas that of the NROH* band is much weaker. This is explained by a fast PT rate and the relatively long average decay time of the NRO-* band. Equation 1 shows how the fluorescence intensities ratio of the two bands is 0 related to kPT, kr, and k r. In ice, the relative emission intensity of the protonated NROH* form increases, whereas that of the NRO-* decreases. Panel D shows the steady-state emission of D-luciferin in liquid and solid 2 mM aqueous solution of HCl to which 0.25% mole ratio of methanol was added. As in the case of the acidic D2O sample, there are two bands at 550 and 600 nm. The relative intensity of the 600 nm band in H2O is stronger than the relative intensity of the respective band in D2O shown in panel B. Time-Resolved Emission. Figure 4 shows the time-resolved emission of D-luciferin in H2O at several temperatures in both the liquid and the ice phases measured by the TCSPC technique. The sample was excited at 390 nm and monitored at 440 nm with a 10 nm spectral bandwidth. Each panel shows the signals of three samples at a given temperature. The three samples contain 0, 2, or 4 mM of HCl as well as 0.25% mole ratio of methanol. The emission signal at 440 nm is assigned to the protonated form, designated as NROH* in Scheme 2. The time resolution of the TCSPC technique is roughly 10 ps, and the 35 ps long full width half-maximum (fwhm) of the instrument response function (IRF) prevents accurate monitoring of the decay of the signal in the first 40 ps. The decay of the signal in a pH-neutral solution is fast and nonexponential. We previously reported36 the decay of the D-luciferin signal in H2O and D2O at room temperature (296 K). The short-time decay was measured by the up-conversion technique with a time resolution of ∼100 fs. For H2O and D2O, the PT time constant was estimated to be 28 and 65 ps, respectively. The nonexponential origin of the decay at 440 nm was attributed to two sources. The first is the reversible geminate recombination with the proton that reforms the excited NROH*. The second source is a nonradiative process unrelated to the reaction with the proton, which may arise from rotation about the C-C bond connecting the two ring systems. In a preliminary experiment, we measured the time-resolved emission of the NROH* band of D-luciferin in propanol at 440 nm and at several hydrostatic pressures of up to 1 GPa using a diamond anvil cell (DAC). At atmospheric pressure, the steady-state emission spectrum mainly consists of the NROH band with a peak at ∼440 nm. The decay is nonexponential, and the decay time is roughly 100 ps. We attribute this short decay time to a nonexponential nonradiative decay process, arising from the relative rotation of the two ring systems about the C2-C20 bond. We found that at 0.15 GPa the decay rate slows considerably. The decay time at pressures higher than 0.15 GPa is nearly pressureindependent, and the average decay time is ∼2 ns, that is, 1620

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Figure 3. Steady-state emission spectra of D-luciferin in both H2O and D2O liquid and ice.

Figure 4. Time-resolved emission of the NROH* form of D-luciferin in ice doped with 0.25% mole ratio of methanol in several acid concentrations.

20 times longer than at atmospheric pressure. This is probably due to the large increase in the viscosity of the solvent at elevated pressures, inhibiting the rotation of the molecule around the C-C bond. As seen in Figure 4, the decay profile of the NROH* signal is almost independent of the acid concentration in both liquid and ice in the temperature range of 242-296 K. This observation is typical of an irreversible geminate recombination

process with a proton, in which the ground-state NROH is reformed. Figure 5 shows the TCSPC time-resolved emission signal of D-luciferin, measured at 530 nm in both liquid and ice at four temperatures. Each panel shows four signals: a pH-neutral sample, and samples with 2, 3, and 4 mM of HCl. We attribute the signal measured at 530 nm to the NRO-* form. In the liquid 1621

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Figure 5. Time-resolved emission of the NRO-* form of D-luciferin, measured at 530 nm, in methanol-doped water and ice in several acid concentrations.

phase at 296 K, the signal of the neutral sample consists of a fast nonexponential decay with an average decay time of ∼300 ps and followed by a nearly exponential long time tail with a time constant of 5.4 ns and a 5-fold increase in the fluorescence quantum yield. We attribute the fast nonexponential decay at short time to a geminate recombination of the proton with the nearest nitrogen atom probably, which leads to fluorescence quenching. The long decay time is attributed to the radiative decay of the NRO-*. When the pH of the solution is 10 or higher, the ground-state population is in the deprotonated form. Excitation of the deprotonated form leads to an exponential decay with a decay time of 5.4 ns. In previous studies,38,39 we found in the presence of mineral acids at concentrations of 1 mM or higher a very sharp increase in the decay rate of the deprotonated form of several 1-naphthol sulfonate photoacids in ice at T > 248 K. We explain this unspecific phenomenon by a large proton diffusion constant in ice. The proton-driven fluorescence quenching of irreversible photoacids in the presence of excess protons is given by the simple reaction below: kðtÞ

RO-  þ Hþ sf ROHðgÞ

ð2Þ

where RO-* and ROH(g) are the excited deprotonated form and the ground-state protonated form, respectively. The Smoluchowski model of irreversible binary collision predicts that if one of the reactants is in excess the reaction rate depends on both the proton diffusion-controlled rate constant, kD, and the intrinsic rate constant, ka, which is the rate constant of the reaction of a proton situated next to the molecule at the socalled contact radius. Usually, the value of the contact radius is considered for such a reaction to lie between 6 and 7 Å, slightly longer than the actual molecular size. In the Supporting Information, we describe the Smoluchowski model for irreversible binary reaction, A þ B f AB. When the intrinsic reaction rate constant ka > kD, then at long times the reaction rate constant k(¥) = kD. kD is linearly dependent on the diffusion constant, D. In ice, the

decay rate measured at 530 nm is faster than that in liquid water, and therefore we attribute this finding to the proton diffusion constant of ice, the value of which is higher than that of liquid water.38-40 Figure 6 shows the TCSPC signals of the same D-luciferin samples shown in Figures 4 and 5, but measured at 595 nm. In the acidic solutions, the TCSPC signals in ice show a distinctive risetime followed by decay with an average decay time constant similar to the one found in the signals detected at 530 nm at long times. In liquid water at 296 K, we find that the signals measured at 595 nm are qualitatively similar to those measured at 530 nm but different in the fine details. The fast initial decay rate is much slower at 595 nm than at 530 nm, and its relative amplitude is larger, that is, ∼0.7 at 595 nm as compared to ∼0.4 at 530 nm. The steady-state emission of the various D-luciferin samples at high and low temperatures may provide further pertinent information that could help resolve the complex decay profiles of the TCSPC signals measured at 595 nm, especially in ice. The steady-state results clearly show that the orange emission band, whose peak is positioned at 572 nm in the liquid state, splits into two emission bands with peaks at ∼530 and 590 nm in ice. The 590 nm band in acidic solution is stronger than the band at 530 nm, whereas in neutral solutions their respective intensities are quite the same. In ice, the excess proton quenches the fluorescence of the zwitterion, measured at 595 nm, as in the case of the NRO-* form at 530 nm. The kinetics of the proton quenching of the zwitterion form is similar to that of the deprotonated form. According to the Smoluchowski model, when kD , ka, it is impossible to derive the absolute or relative values of the intrinsic proton quenching rate constants. Hence, it can be deduced neither that the quenching occurs at a molecular site that is available for both the NRO-* and the þHNRO-* nor that two different sites exist in D-luciferin. Comparison between D-Luciferin and 1N4S. A much simpler photoprotolytic system that we extensively studied in the past is that of 1-naphthol-4-sulfonate (1N4S).39 It is an 1622

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Figure 6. Time-resolved emission of the zwitterion form of D-luciferin measured at 600 nm in liquid and in ice doped with 0.25% mole ratio of methanol in several HCl concentrations.

irreversible photoacid with only two forms: protonated (ROH*) and deprotonated (RO-*). In this subsection, we wish to discuss the similarities and differences between the time-resolved emission of 1N4S and the more complicated system of D-luciferin. Figure 7A shows the TCSPC signals of the protonated ROH* form of 1N4S measured at 360 nm at several temperatures in liquid water and ice. The sample was doped with 0.25% mole ratio of methanol (the same concentration as that of the Dluciferin samples). In the previous 1N4S study, we used a lower methanol concentration of 0.1% mole ratio. The sample also contains 2 mM of HCl, which is a strong acid in liquid H2O. We previously found that HCl dissociates to Hþ and Cl- to a large extent in the ice phase as well. In ice, the average decay rate of the ROH* form decreases with the temperature. In previous studies,39,40 we found that the PT rate of many photoacids including 1N4S strongly decreases when the temperature of the ice drops. In general, the decay of the signal is nonexponential at all temperatures in the range of 237-296 K in both the liquid and the ice phases. Figure 7B shows the time-resolved emission of the NROH* form of D-luciferin measured at 450 nm at several temperatures in ice doped with 0.25% mole ratio of methanol. The temperature dependence of the decay rate of the protonated form of Dluciferin is weaker than that of 1N4S. Careful examination of the results is described in the Discussion. Figure 8 shows the TCSPC signal of the deprotonated RO-* form of 1N4S in liquid H2O and ice doped with 0.25% mole ratio of methanol measured at 470 nm at several temperatures. This sample also contained 2 mM of HCl. In the liquid state, the decay of the RO-* form is long, ∼12.5 ns, whereas in ice the decay times are much shorter, that is, ∼2 ns. So we conclude that the decay rate in ice is 6 times faster than that in liquid water.

We previously found that in methanol-doped ice excess protons exhibit a high degree of reactivity with reversible and irreversible photoacids. 1-Naphthol, 1N4S, and other 1-naphthol sulfonate analogues are irreversible photoacids. These photoacids in their first electronically excited state react irreversibly with the protons to re-form the protonated ROH(g) in the electronic ground state. The rate of these types of reactions can be quantitatively determined by the Smoluchowski model. In the Supporting Information, we briefly describe the rationale behind the model and the results it produces. Figures 5 and 6 show that the zwitterion and deprotonated forms of D-luciferin have similar acid and temperature dependencies. A concentration of 2 mM of acid in ice decreases the long-time component of the NRO-* decay by a factor of 4, much like it does for 1N4S, despite the latter being a simple irreversible photoacid with only two forms, that is, ROH* and RO-*. The RO-* signal of 1N4S lacks the initial fast component seen in the NRO-* decay of D-luciferin measured at 520 nm. We attributed this component to the geminate proton reacting with the nitrogen on the benzothiazole/dihydrothiazole ring. The simpler molecular structure of 1N4S does not offer this additional reaction, so that the TCSPC signals are much simpler to model and analyze quantitatively. Main Findings. The primary findings of this Article are as follows: (1) The distinctive change in the absorption spectrum of D-luciferin in liquid aqueous solutions at a pH level of ∼1 indicates that protonation occurs. We suggest that the protonation occurs on the nitrogen atom of the thiazole ring, thus creating the doubly protonated ground-state þ HNROH form. 1623

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aqueous solution or higher with a quenching rate that is concentration dependent. (4) Time-resolved emission from aqueous acidic solutions shows that both the lifetimes of the deprotonated NRO-* and the zwitterion þHNRO-* forms decreased as the acid concentration was raised. For ice: (5) In the temperature range of 242-296 K, the decay rate of the TCSPC signal measured at 450 nm is nearly temperature independent. The decay of the time-resolved emission of the protonated form consists of two components: The fast component decays with a nearly temperature-independent time constant of ∼30 ps and a relative amplitude of 0.5. The longer time component is temperature dependent with an activation energy of 15 kJ/ mol. (6) In methanol-doped ice containing 2 mM HCl, we found that D-luciferin undergoes a very efficient proton recombination process leading to enhanced fluorescence quenching of both emission bands at 550 and 600 nm. Furthermore, the decay rate of both the NRO-* and the þ HNRO-* bands of D-luciferin in ice is much faster than in the liquid state. We attribute the fluorescence quenching in both liquid and ice to a diffusion-controlled reaction rate of these species with excess proton in ice as follows: NRO-* þ Hþ f þNROH(g) and þHNRO-* þ Hþ f þ HNROH(g). Previously, we found that the proton diffusion constant in ice is 10 times larger than that in water, and this explains the faster decay rates in ice.38-40 Figure 7. (A) Time-resolved emission of the ROH* form of 1N4S in ice doped with 0.25% mole ratio of methanol and 2 mM HCl at several temperatures, measured at 350 nm. (B) Time-resolved emission of the NROH* form of D-luciferin in acid-free ice doped with 0.25% mole ratio of methanol at several temperatures, measured at 440 nm.

Figure 8. Time-resolved emission of the RO-* form of 1N4S in ice doped with 0.25% mole ratio of methanol and 2 mM HCl at several temperatures, measured at 470 nm.

(2) Inspection of the steady-state emission spectra in both liquid water and ice shows that there is a new broadband emission with a peak at ∼590 nm in liquid at acid concentration of g20 mM. We attribute this band to the þHNRO-* zwitterion. (3) Steady-state fluorescence quenching of both the deprotonated form and the zwitterion occurs in a 10 mM acidic

’ DISCUSSION Excess Proton Reaction with Excited Photoacids and in the Liquid State. As seen in Figures 1 and 2,

D-Luciferin

the absorption, steady-state excitation, and steady-state emission spectra of D-luciferin in liquid water are affected by the presence of a few tens of millimolars of HCl more than expected from a simple photoacid such as an irreversible analogue of the 1-naphthol sulfonate photoacids. Scheme 2 describes most of the chemical and photochemical processes involving simple photoacids like naphthol derivatives. Basically, simple reversible and irreversible photoacids exist in either two forms, the protonated, ROH, and the deprotonated, RO-. For reversible photoacids such as 2-naphthol and its derivatives, the photoprotolytic cycle is accurately described by the diffusion-influenced geminate recombination model.34,35 After excitation of the photoacid, the proton is transferred to the solvent. For reversible photoacids, the geminate recombination with the proton is governed by the proton diffusion and the attractive potential, which is usually a Coulomb potential, between the RO-* and the Hþ to re-form the ROH* in its excited state. The reformed ROH* can undergo a second cycle, etc. To detect the photocycle, the photoacid is excited as ROH, and the reactions in the excited state are usually monitored by observing the decay of the ROH* and the formation of the RO-* forms. When excess protons are introduced to the sample, the population of ROH* is strongly affected. The time-resolved fluorescence signal of the ROH* form in acidic solution consists of two contributions. The first is the fast decay due to the ESPT process, and the second is the long-time tail whose decay rate depends on the excited-state lifetimes of both the ROH* and the RO-* forms. The relative amplitude of the 1624

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The Journal of Physical Chemistry A long-time tail increases approximately linearly with the rise in the acid concentration. In the case of irreversible photoacids such as 1N4S, the excess proton in the solution recombines with the RO-* and re-forms the ground-state ROH(g) form (see Figure 8). Consequently, the excited-state lifetime of the RO-* shortens, and it becomes increasingly shorter the more acid is introduced into the solution. The time-resolved emission of the protonated ROH* form of irreversible photoacids, such as 1N4S, is almost invariant under change of the acid concentration (see Figure 7A). The decay of the protonated NROH* form of D-luciferin in the aqueous acidic solutions shows that the long time tail amplitude is not strongly affected when the acid concentration increases (see Figure 7B). This is typical of irreversible photoacids, for which a recombination with a proton generates a ground-state protonated form, thus terminating the photoprotolytic cycle. Careful observation of the long-time fluorescence tail shows that the decay time shortens as the acid concentration is increased. This is expected, because the decay rate of the NRO-* depends on the proton’s concentration. The NRO-* emission of Dluciferin in the liquid state also depends on the acid concentration. Below ∼10 mM HCl, the steady-state emission spectra (see Figure 2A) show only a slight decrease in the NRO-* band intensity at 530 nm, whereas that of the NROH* band at 450 nm is nearly the same. The NRO-* band intensity decreases by a factor of 6 in a 47 mM aqueous solution of HCl, and a new band with a peak at approximately 590 nm is growing. As aforementioned, the band at 590 nm is attributed to the formation of the doubly protonated ground-state þHNROH form, which upon excitation undergoes an ESPT process and forms a zwitterion emitting at 590 nm: þH NROH* a þH NRO-* þ Hþ. D-Luciferin Photochemistry in the Ice Phase. In ice, the fluorescence properties of D-luciferin in neutral pH and even more so in acidic aqueous solutions are considerably different than in the liquid phase. In recent publications, we reported that the proton diffusion constant in ice at temperatures above 240 K is 10 times larger than its value in liquid water at room temperature.40 Eigen and DeMayer41 found by electrical measurements that proton mobility in ice is 10-100 times larger than in water. Later, electrical measurements by other research groups42,43 consistently showed that proton mobility in ice is lower than that in water at room temperature. We expect that the fluorescence of both the NRO-* and the þHNRO-* of Dluciferin in ice containing a few millimolars of HCl in the temperature range of 240-270 K will show that its proton quenching reaction is much more efficient than in liquid water, in agreement with what we found in our previous studies on irreversible photoacids.38,39 Figures 5 and 6 show that the fluorescence quenching rate of NRO-* and þHNRO-* in ice is indeed 4 times higher than in the liquid phase.k If one assumes D that the rate of the reaction: NRO-  þ Hþ sf NROHðgÞ is approximately diffusion-controlled (see the Smoluchowski model in the Supporting Information), then data analysis yields a value of ∼4  10-4 cm2/s for the proton’s diffusion constant in ice doped with 0.25% mole ratio of methanol. We previously found that DHþ in ice strongly depends on the methanol concentration. A similar value of ∼5  10-4 cm2/s is also found for ice doped with 0.2% mole ratio of methanol using the ionic 1N4S and 1N3S photoacids.38,39 When the methanol concentration is ∼0.05% mole ratio, then the analysis of the time-resolved RO-* emission of 1N4S provided a proton diffusion constant that maintains a high value of ∼10-3 cm2/s. Because D-luciferin

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is an uncharged molecule with limited miscibility, we limited ourselves to using high concentrations of methanol. The decay time of the deprotonated form, emitting at 530 nm, depends on the acid concentration in the same way that 1N4S, which is considered a textbook example of an irreversible photoacid, does. The emission decay times of both the NRO-* and the þ HNRO-* forms are approximately linearly dependent on the excess proton concentration. Table 1 in the Supporting Information shows the decay rates of the long-time component of the NRO-* form, measured at 530 nm, and the proton diffusion constants derived from those decay rates. In our previous studies on methanol-doped ice,38-40 we found that the emission decay time of the RO-* form of 1N4S is linearly dependent on the excess proton concentration. The fluorescence quenching rate of the deprotonated form is diffusion-controlled for both D-luciferin and 1-naphthol derivatives in liquid and ice. Figure 8 shows the time-resolved emission of the RO-* of 1N4S, measured at 470 nm, in water and in ice doped with 0.25% mole ratio of methanol and 2 mM of HCl. As seen in the figure, the maximal quenching rate is achieved at ∼260 K. At higher or lower temperatures, the quenching rate slightly decreases. These data corroborate our previous finding regarding the temperature dependence of DHþ in methanol-doped ice. Careful examination of the time-resolved emission of the protonated NROH* form of D-luciferin in ice, emitting at 450 nm shown in Figure 7B, reveals that the decay profile consists of short- and long-time components. In the ice phase, the shorttime component amplitude is about 0.5 and the decay time is short (30 ps or shorter). The fast-time component of the decay is independent of temperature, but because the decay time could not be resolved by the TCSPC technique at all temperatures we cannot be definitely sure. The long-time component shows relatively strong temperature dependence, easily resolved by the TCSPC technique, with an activation energy of 15 kJ/mol in the ice phase. The temperature dependence of the timeresolved signal of the protonated form is not simple to explain, and it may arise from the nonradiative process or the multiple proton accepting sites of the D-luciferin molecule as explained above. A plot of ln(1/τ) versus 1/T of the long-time component of the TCSPC signal of D-luciferin in ice is given in the Supporting Information. We suspect that the longer time component arises from proton transfer to the solvent from a protonated group that accepted a proton first released by ESPT from the hydroxyl group of the phenol (part of the benzothiazole moiety).

’ SUMMARY We employed steady-state and time-resolved emission techniques to study the photoprotolytic processes that the D-luciferin, the natural substrate of the firefly luciferase, undergoes in both acidic aqueous solutions and slightly acidic ice. We found that at a pH level of about 1 (100 mM HCl), the ground-state D-luciferin undergoes a protonation process. The emission spectrum of Dluciferin in a 20 mM acidic aqueous solution has a new emission band at 590 nm red-shifted with respect to the strongest emission band at 530 nm in a neutral aqueous solution. We attribute the new emission band to the zwitterion form designated as þ HNRO-. The time-resolved emission signals show that the NRO-* emission band at 530 nm and the zwitterion emission band at 590 are strongly quenched by a recombination process with a proton in an acidic solution. The recombination process is 1625

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The Journal of Physical Chemistry A nearly diffusion-controlled, where DHþ = 10-4 cm2/s and kD = 5  1010 M-1 s-1. In methanol-doped ice, D-luciferin undergoes the same photoprotolytic processes as in the liquid phase. The proton quenching rate in ice of both the NRO-* and the þHNRO-* forms is much faster than that in water. This result is in accord with our previous findings, when we studied regular hydroxyarene photoacids such as 1-naphthol sulfonate analogs. In ice doped with 0.1% mole ratio of methanol or less, we found that the proton mobility is about 10 times larger than in water.39,40 When the methanol concentration in ice is raised to 0.25%, the proton mobility at 260 K is roughly 4 times as large as that of liquid water, and thus the quenching rate of D-luciferin and other photoacids is only 4 times greater as well.

’ ASSOCIATED CONTENT

bS

Supporting Information. The Smoluchowski model, kinetic analysis of the ESPT of D-luciferin, and kinetic analysis of the excess proton reaction. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Phone: 972-3-6407012. Fax: 972-3-6407491. E-mail: huppert@ tulip.tau.ac.il.

’ ACKNOWLEDGMENT This work was supported by grants from the Israel Science Foundation and from the James-Franck German-Israeli Program in Laser-Matter Interaction. ’ REFERENCES (1) Strehler, B. L.; McElroy, W. D. J. Cell. Physiol. 1949, 34, 457. (2) White, E. H.; McCapra, F.; Field, G. F.; McElroy, W. D. J. Am. Chem. Soc. 1961, 83, 2402. (3) Wilson, T.; Hastings, J. W. Annu. Rev. Cell Dev. Biol. 1998, 14, 197. (4) White, E. H.; Rapaport, E.; Seliger, H. H.; Hopkins, T. A. Bioorg. Chem. 1971, 1, 92. (5) McCapra, F. Acc. Chem. Res. 1976, 9, 201. (6) Wood, K. V. Photochem. Photobiol. 1995, 62, 662. (7) Naumov, P.; Ozawa, Y.; Ohkubo, K.; Fukuzumi, S. J. Am. Chem. Soc. 2009, 131, 11590. (8) Suzuki, M.; Goto, T. Agric. Biol. Chem. 1972, 36, 2213. (9) Gomi, K.; Kajiyama, N. J. Biol. Chem. 2001, 276, 36508. (10) Seliger, H. H.; Buck, J. B.; Fastie, W. G.; McElroy, W. D. J. Gen. Physiol. 1964, 48, 95. (11) Viviani, V. R. Cell. Mol. Life Sci. 2002, 59, 1833. (12) Nakatsu, T.; Ichiyama, J.; Hiratake, J.; Saldanha, A.; Kobashi, N.; Sakata, K.; Kato, H. Nature 2006, 440, 372. (13) Seliger, H. H.; McElroy, W. D. Proc. Natl. Acad. Sci. U.S.A. 1964, 52, 75. (14) Viviani, V. R.; Arnoldi, F. G. C.; Neto, A. J. S.; Oehlmeyer, T. L.; Bechara, E. J. H.; Ohmiya, Y. Photochem. Photobiol. Sci. 2008, 7, 159. (15) Gandelman, O. A.; Brovko, L. Yu.; Ugarova, N. N.; Chikishev, A. Yu.; Shkurimov, A. P. J. Photochem. Photobiol., B 1993, 19, 187. (16) Ugarova, N. N.; Maloshenok, L. G.; Uporov, I .V.; Koksharov, M. I. Biochemistry (Moscow) 2005, 70, 1262. (17) Ando, Y.; Niwa, K.; Yamada, N.; Enomoto, T.; Irie, T.; Kubota, H.; Ohmiya, Y.; Akiyama, H. Nat. Photon. 2008, 2, 44.

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(18) Seliger, H. H.; McElroy, W. D. Biochem. Biophys. Res. Commun. 1959, 1, 21. (19) Niwa, K.; Ichino, Y.; Kumata, S.; Nakajima, Y.; Hiraishi, Y.; Kato, D.; Viviani, V. R.; Ohmiya, Y. Photochem. Photobiol. 2010, 86, 1046. (20) Ugarova, N. N. Nat. Photon. 2008, 2, 8. (21) Ireland, J. E.; Wyatt, P. A. Adv. Phys. Org. Chem. 1976, 12, 131. (22) (a) Gutman, M.; Nachliel, E. Biochem. Biophys. Acta 1990, 391, 1015. (b) Pines, E.; Huppert, D. J. Phys. Chem. 1983, 87, 4471. (23) Tolbert, L. M.; Solntsev, K. M. Acc. Chem. Res. 2002, 35, 19. (24) Rini, M.; Magnes, B. Z.; Pines, E.; Nibbering, E. T. J. Science 2003, 301, 349. (25) Mohammed, O. F.; Pines, D.; Dreyer, J.; Pines, E.; Nibbering, E. T. J. Science 2005, 310, 5745. (26) Tran-Thi, T. H.; Gustavsson, T.; Prayer, C.; Pommeret, S.; Hynes, J. T. Chem. Phys. Lett. 2000, 329, 421. (27) Agmon, N. J. Phys. Chem. A 2005, 109, 13. (28) Spry, D. B.; Fayer, M. D. J. Chem. Phys. 2008, 128, 084508. (29) Siwick, B. J.; Cox, M. J.; Bakker, H. J. J. Phys. Chem. B 2008, 112, 378. (30) Mohammed, O. F.; Pines, D.; Nibbering, E. T. J.; Pines, E. Agnew. Chem., Int. Ed. 2007, 46, 1458. (31) Mondal, S. K.; Sahu, K.; Sen, P.; Roy, D.; Ghosh, S.; Bhattacharyya, K. Chem. Phys. Lett. 2005, 412, 228. (32) Prasun, M. K.; Samanta, A. J. Phys. Chem. A 2003, 107, 6334. (33) Bhattacharya, B.; Samanta, A. J. Phys. Chem. B 2008, 112, 10101. (34) Pines, E.; Huppert, D.; Agmon, N. J. Chem. Phys. 1988, 88, 5620. (35) Agmon, N.; Pines, E.; Huppert, D. J. Chem. Phys. 1988, 88, 5631. (36) Erez, Y.; Huppert, D. J. Phys. Chem. A 2010, 114, 8075. (37) Presiado, I.; Erez, Y.; Huppert, D. J. Phys. Chem. A 2010, 114, 9471. (38) Uritski, A.; Presiado, I.; Huppert, D. J. Phys. Chem. C 2008, 112, 18189. (39) Uritski, A.; Presiado, I.; Huppert, D. J. Phys. Chem. A 2009, 113, 959. (40) Uritski, A.; Presiado, I.; Erez, Y.; Gepshtein, R.; Huppert, D. J. Phys. Chem. C 2009, 113, 10285. (41) Eigen, M.; de Maeyer, L. The Structure of Electrolytic Solutions; Wiley: New York, 1959; p 64ff. (42) Young, I. G.; Salomon, R. E. J. Chem. Phys. 1968, 48, 1635. (43) Camplin, G. C.; Glen, J. W. In Physics and Chemistry of Ice; Walley, E., Jones, S. J., Gold, L. W., Eds.; Royal Society of Canada: Ottawa, 1973; pp 256-261.

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