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Ultrafast Fluorescence Quenching Dynamics of Atto655 in the Presence of N‑Acetyltyrosine and N‑Acetyltryptophan in Aqueous Solution: Proton-Coupled Electron Transfer versus Electron Transfer Ying Zhang, Shuwei Yuan, Rong Lu, and Anchi Yu* Department of Chemistry, Renmin University of China, Beijing 100872, People’s Republic of China S Supporting Information *

ABSTRACT: We studied the ultrafast fluorescence quenching dynamics of Atto655 in the presence of N-acetyltyrosine (AcTyr) and N-acetyltryptophan (AcTrp) in aqueous solution with femtosecond transient absorption spectroscopy. We found that the charge-transfer rate between Atto655 and AcTyr is about 240 times smaller than that between Atto655 and AcTrp. The pH value and D2O dependences of the excited-state decay kinetics of Atto655 in the presence of AcTyr and AcTrp reveal that the quenching of Atto655 fluorescence by AcTyr in aqueous solution is via a proton-coupled electron-transfer (PCET) process and that the quenching of Atto655 fluorescence by AcTrp in aqueous solution is via an electron-transfer process. With the version of the semiclassical Marcus ET theory, we derived that the electronic coupling constant for the PCET reaction between Atto655 and AcTyr in aqueous solution is 8.3 cm−1, indicating that the PCET reaction between Atto655 and AcTyr in aqueous solution is nonadiabatic.

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INTRODUCTION The development of innovative fluorescence-based techniques to probe biomolecular interaction and dynamics is one of the major interests in biophysical chemistry.1−6 In recent years, it turns out that the photoinduced electron transfer based fluorescence correlation spectroscopy (PET-FCS) is a powerful tool to study the conformational dynamics of biopolymers.5−16 Compared to the fluorescence resonance energy transfer based fluorescence correlation spectroscopy (FRET-FCS),17−21 the PET-FCS has two advantages. First, the PET-FCS does not require site-specific labeling of two extrinsic fluorophores. Second, the PET-FCS can probe subtle spatial changes of biopolymers because PET requires contact formation between labeled fluorophores and quenchers at a van der Waals distance.16 In the traditional two-state PET-FCS model, researchers generally assumed the brightness of the dark species to be zero so that they could extract both the forward and reverse rate constants from a single PET-FCS curve.22 However, our recent studies suggested that the PET-FCS data alone are no longer sufficient to extract the forward and reverse rate constants when more accurate physical interpretation is considered.23−26 Table 1 summarizes the calculated deviation of the PET-FCS amplitude (α) from the equilibrium constant (K) for a K = 1 reaction at several values of the brightness of the dark species (Q) with eq 4 of ref 26. Clearly, for Q ≤ 0.001 PET-FCS system (e.g., Atto655/tryptophan and MR121/ tryptophan systems7,8), the assumption of Q = 0 would not introduce noticeable errors in their equilibrium constant and a single PET-FCS study could obtain the reasonable forward and reverse rate constants of a reaction. However, for Q ≥ 0.01 © XXXX American Chemical Society

Table 1. Calculated Deviation of the PET-FCS Amplitude (α) from the Equilibrium Constant (K) for a K = 1 Reaction at Several Values of the Brightness of the Dark Species (Q) Q

K

α

α−K

100(α − K)/K

0 0.001 0.01 0.1

1 1 1 1

1 0.996 0.961 0.669

0 −0.004 −0.039 −0.331

0 −0.4 −3.9 −33.1

system (e.g., tetramethylrhodamine/guanosine,23,24 MR121/ guanosine,22 and Atto655/guanosine25 systems), the value of Q has to be determined with an additional experiment, i.e., transient absorption,24−26 transient fluorescence,24,26 and/or ensemble steady-state fluorescence measurement,23,24 so as to obtain the correct forward and reverse rate constants. Among amino acids, tryptophan and tyrosine are the two that can cause the most obvious fluorescence quenching of a labeled fluorophore in protein or aqueous solution.1,7,8,12−16,21,27−49 Up to date, the fluorescence quenching dynamics of a fluorophore by tryptophan has been extensively studied and it is generally reported that the selected fluorophore has PET interaction with tryptophan.1,7,8,12−16,21,27−37 However, the mechanism for the fluorescence quenching of a fluorophore by tyrosine is still debated. Some reports suggested that the fluorescence Received: May 6, 2013 Revised: May 30, 2013

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dx.doi.org/10.1021/jp404466f | J. Phys. Chem. B XXXX, XXX, XXX−XXX

The Journal of Physical Chemistry B

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quenching of a fluorophore by tyrosine is via PET interaction,28,30,36,38,39 while some other reports suggested that the fluorescence quenching of a fluorophore by tyrosine is via proton-coupled electron transfer (PCET) interaction.40−48 More importantly, it is unknown what the time scale is for the fluorescence quenching dynamics of a labeled fluorophore by tyrosine in protein or aqueous solution. And the time scale for the fluorescence quenching dynamics of a labeled fluorophore by tyrosine is an important parameter to understand the experimental observation involving tyrosine reaction in biophysical chemistry. On the basis of the unsolved issues related to the fluorescence quenching of a fluorophore by tyrosine, in this work we studied the ultrafast fluorescence quenching dynamics of Atto655 (Scheme 1) in the prescence of different N-

acetyltyrosine (AcTyr) concentration in aqueous solution by using femtosecond transient absorption spectroscopy. Atto655 is a member of the oxazine family,50 and it has been widely used in PET-FCS experiments to explore the conformational dynamics of proteins and DNA-hairpins.2,12,50−53 Comparing with other red absorptive and emissive dyes, such as Cy-5 and Alexa Fluor 647, Atto655 has excellent photostability and brightness. In this work, we first measured the first electronic excited-state decays of Atto655 in the presence of different AcTyr concentrations in aqueous solution with femtosecond transient absorption spectroscopy. Then we measured the first electronic excited-state decays of Atto655 in the presence of 200 mM AcTyr in H2O solution at different pH values and in D2O solution with femtosecond transient absorption spectroscopy. The pH value and D2O dependences of the excited-state decay kinetics of Atto655 in 200 mM AcTyr solution reveal that the fluorescence quenching of Atto655 by AcTyr in aqueous solution is via a PCET process. As a comparative study, we also investigated the ultrafast fluorescence quenching dynamics of Atto655 in the presence of N-acetyltryptophan in aqueous solution with femtosecond transient absorption spectroscopy.

buffer. Ultrapure H2O (18.2 MΩ·cm) was obtained through a Milli-Q water purification system (Millipore, Billerica, MA). Ultra-D D2O (>99.9%) was purchased from Sigma-Aldrich, St. Louis, MO, and used as received. Steady-State Absorption and Fluorescence Measurements. Steady-state absorption spectra were recorded by a Cary 50 UV−vis spectrometer (Varian, Forest Hill, Victoria, Australia). Steady-state fluorescence spectra were recorded on a LS-55 luminescence spectrometer (Perkin-Elmer, Waltham, MA). In the steady-state absorption and fluorescence spectroscopy measurements, the concentrations of Atto655 were kept at about 1 × 10−6 M. Femtosecond Transient Absorption Measurements. The details of our femtosecond transient absorption apparatus have been described elsewhere.24−26 Briefly, we used an amplified Ti:sapphire laser system (Spitfire, Spectra Physics, Mountain View, CA), which generates about 100 fs laser pulses at 800 nm with a repetition rate of 1 kHz and an average power of around 1.0 W. These fundamental pulses were used to pump an optical parametric amplifier (OPA) as well as to generate the white light continuum. The OPA pulses were about 100 fs and could be tuned from 450 to 700 nm. The white light continuum was generated in a spinning fused-silica disk with the 800 nm pump pulse and its spectrum covers the range from 420 to 750 nm. The OPA outputs were used as the pump pulses, and the white light continuum were used as the probe pulses. The timing between the pump and probe pulses was controlled using a motorized translation stage (M-ILS250CC, Newport, Irvine, CA). The pump and probe beams were noncollinearly focused into the sample cell using two achromatic lenses (300 mm focal length for pump and 100 mm focal length for probe, respectively). At the sample position, the average powers were about 0.5 mW for pump beam, and around 10 μW for the white light continuum probe beam. The signals were collected by a large area adjustable gain balanced photoreceiver (2307, Newport, Irvine, CA) which was attached to the output port of a monochromator (SP2358, Princeton Instruments, Acton, MA) and sent to a lock-in amplifier (SR850, Stanford Research Systems, Sunnyvale, CA), where it was synchronized by an optical chopper (75160, Newport, Irvine, CA). The chopped frequency was 160 Hz. The polarization of the pump pulses was set at 54.7° with respect to the polarization of the probe pulses to get rid of the molecular reorientation effect. The time resolution for this femtosecond transient absorption apparatus was estimated to be about 150 fs through the cross correlation between the pump and probe pulses in buffer solution. In the femtosecond transient absorption measurements, the concentrations of Atto655 were about 1 × 10−5 M. To keep the sample solution fresh, a homemade magnet stirring bar was placed inside a 1 mm path length sample cell and rotated by an external magnet motor. The steady-state absorption spectra of the sample were measured before and after each kinetic measurement to check the sample’s quality and stability. The difference between the absorption spectra of the sample before and after each kinetic measurement is less than 1%.

EXPERIMENTAL SECTION Materials. Atto655, N-acetyltyrosine (AcTyr), and Nacetyltryptophan (AcTrp) were purchased from Sigma-Aldrich, St. Louis, MO, and used as received. 100 × TE buffer (1 M Tris-HCl + 100 mM EDTA, pH = 8.0) was purchased from Sigma-Aldrich, St. Louis, MO. One × TE buffer (10 mM TrisHCl + 1 mM EDTA, pH = 8.0) was diluted from 100 × TE

RESULTS AND DISCUSSION Ensemble Steady-State Absorption and Fluorescence Measurements. The formation of a ground-state complex between a fluorophore and a quencher can be revealed through the ensemble steady-state absorption and fluorescence spectra of the fluorophore.27,29,31,54 Figure 1 displays the ensemble steady-state absorption and fluorescence spectra of Atto655

Scheme 1. Chemical Structures of Atto655, NAcetyltyrosine, and N-Acetyltryptophan

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the inset of Figure 2, it is derived that AcTyr quenches the fluorescence of Atto655 with a SV constant (KSV) value of 27 ± 1 M−1. Through the “sphere-of-action” fluorescence quenching model (F0/F = (1 + KSV[Q]) exp(λ[Q])), it is derived that AcTyr quenches the fluorescence of Atto655 with a KSV value of 23 ± 1 M−1 and a λ value of 3.5 ± 0.5 M−1. The obtained KSV value for the fluorescence quenching of Atto655 by AcTyr in aqueous solution is 4−5 times smaller than the KSV value for the fluorescence quenching of Atto655 by tryptophan reported in the literature,29 indicating that the fluorescence quenching efficiency of tyrosine is much weaker than that of tryptophan. Femtosecond Transient Absorption Spectroscopy Measurements. To understand the fluorescence quenching dynamics of Atto655 by AcTyr, we measured the first electronic excited-state decay kinetics of Atto655 in the absence and presence of AcTyr in 1 × TE buffer solution with femtosecond transient absorption spectroscopy through monitoring the transient absorption of the S1 → Sn transition after Atto655 was excited into its first electronic excited state.25 Figure 3

Figure 1. Normalized steady-state absorption (solid line) and fluorescence (dash line) spectra of Atto655 in the absence (black) and presence (red) of 100.0 mM N-acetyltyrosine in 1 × TE pH = 8 buffer.

with and without 100 mM AcTyr in 1 × TE buffer solution. It is clear that the absorption maximum of Atto655 in 100 mM AcTyr solution has an obvious red shift, and that the fluorescence emission maximum of Atto655 in 100 mM AcTyr solution has only a slight red shift. The red shifts in the absorption and fluorescence maxima of Atto655 in 100 mM AcTyr solution indicate that a ground-state complex is formed between Atto655 and AcTyr in aqueous solution. To further explore the interaction between Atto655 and AcTyr, we respectively recorded the fluorescence spectra of Atto655 in the presence of different AcTyr concentrations, as shown in Figure 2. The fluorescence intensity of Atto655 decreases with

Figure 3. Femtosecond magic-angle pump−probe transients of Atto655 in the presence of different N-acetyltyrosine concentration (a, 0 mM; b, 35 mM; c, 100 mM; and d, 500 mM) in 1 × TE pH = 8 buffer. Pump, 665 nm; probe, 570 nm.

shows the first electronic excited-state decay of Atto655 with different AcTyr concentrations in 1 × TE buffer. It is clear that the first electronic excited-state decay kinetics of Atto655 becomes faster with the increase of AcTyr concentration. The decay of the first electronic excited state of Atto655 without AcTyr can be fitted by a single exponential, while the decays of the first electronic excited state of Atto655 with AcTyr need a summation of two exponential functions to be fitted. The fitting parameters for the first electronic excited-state decay of Atto655 in different AcTyr concentrations are summarized in Table 2. From the data listed in Table 2, it is found that the lifetime of the fast component of Atto655 in the presence of AcTyr does not vary with the increase of AcTyr concentration, but its amplitude does. This is a well-known behavior and indicates the formation of a ground-state complex between Atto655 and AcTyr in aqueous solution. To confirm that the obtained decays in the femtosecond transient absorption

Figure 2. Steady-state fluorescence spectra of Atto655 in the presence of different N-acetyltyrosine concentration (from top to bottom: 0, 10, 20, 30, 40, 50, 60, 70, 85, and 100 mM) in 1 × TE pH = 8 buffer. Inset shows the fluorescence intensity Stern−Volmer plot of Atto655 in the presence of N-acetyltyrosine in 1 × TE buffer. Excitation: 630 nm.

the increase of AcTyr concentration, indicating that AcTyr can distinctly quench the fluorescence of Atto655. The inset in Figure 2 shows the fluorescence intensity Stern−Volmer (SV) plot of Atto655 in the presence of AcTyr in 1 × TE buffer. It is clear that the SV plot of Atto655 in AcTyr solution has an upward curvature. The reason for the nonlinearity of a SV plot has been discussed thoroughly in the literature, where it has been suggested that the deviation from linearity with the increase of the quencher concentration in a SV plot is due to different populations of fluorophores.29 With the linear region data of the quencher concentration dependent F0/F shown in C



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Table 2. Excited-State Decay Parameters for Atto655 in the Presence of Different N-Acetyltyrosine (AcTyr) and NAcetyltryptophan (AcTrp) Concentrations in 1 × TE pH = 8 Buffer, Obtained from Femtosecond Transient Absorption Measurements quencher

concn/mM

AcTyr

0 35 100 500 0 60 200

AcTrp

a1

τ1/ps

0.45 0.72 0.94

230 ± 20 220 ± 20 220 ± 20

0.76 0.97

1.2 ± 0.2 1.1 ± 0.2

a2

τ2/psa

1 0.55 0.28 0.06 1 0.24 0.03

1800 1800 1800 1800 1800 1800 1800

a

Fixed to match the lifetime obtained by time correlated singlephoton-counting measurements. Figure 4. Calculated fluorescence quenching ratio (F0/F) of Atto655 in the presence of 200 mM N-acetyltyrosine in 1 × TE pH = 8 buffer at different temperature.

measurement were associated with the initially populated first electronic excited state of Atto655, we further recorded the fluorescence decay profiles of Atto655 in the presence of different AcTyr concentrations in 1 × TE buffer solution with time-correlated single-photon-counting experiment (TCSPC, Figure S1 of the Supporting Information). The TCSPC measurement gave a consistent result (Table S1 of the Supporting Information). Thus, the 220 ps decay component in Table 2 reflects the excited-state lifetime of Atto655/AcTyr complex, and the 1800 ps decay component in Table 2 is the excited-state lifetime of free Atto655. With the data listed in Table 2, we derived that the relative brightness of Atto655/ AcTyr complex (Q = τ1/τ2) is 0.12 ± 0.01, which is an important parameter for the PET-FCS study involving Atto655 and tyrosine in biopolymers. Herein, we used a summation of two exponential decay functions to fit the first electronic excited-state decay of Atto655 in the presence of different AcTyr concentrations in 1 × TE buffer and attributed the short lifetime (220 ps) of Atto655 in the presence of AcTyr entirely to a ground-state complex between Atto655 and AcTyr, indicating that the contribution of the bimolecular fluorescence quenching between Atto655 and AcTyr is minor at the experimental conditions utilized in the current study. To confirm that the contribution of the bimolecular fluorescence quenching between Atto655 and AcTyr is minor, we investigated the fluorescence quenching behavior of Atto655 in the presence of 200 mM AcTyr in 1 × TE buffer with steady-state fluorescence spectroscopy at different temperatures (Figure S2 of the Supporting Information). Figure 4 displays the calculated fluorescence quenching ratio (F0/F) of Atto655 in the presence of 200 mM AcTyr in 1 × TE buffer solution at different temperatures. Clearly, it is found that the fluorescence quenching ratio of Atto655 by 200 mM AcTyr is maintained constant with the increase of the temperature. It is known that the increase of the temperature would increase the diffusion between a fluorophore and a quencher and cause the extent of the bimolecular fluorescence quenching increases. However, from the data shown in Figure 4, we do not observe an obvious temperature effect on the fluorescence quenching behavior of Atto655 by AcTyr, indicating that either the bimolecular fluorescence quenching process is minor or the ground-state complex dissociates with the increase of the temperature. To check the effect of the temperature on the ground-state complex formation between Atto655 and AcTyr, we further recorded the absorption spectrum of Atto655 with 200 mM

AcTyr in 1 × TE buffer at different temperature (Figure S3 of the Supporting Information). The almost identical absorption spectrum of Atto655 with 200 mM AcTyr as a function of the temperature reveals that the ground-state complex between Atto655 and AcTyr does not dissociate in the temperature range of 10−50 °C. Thus, the data shown in Figure 4 indicate that the bimolecular fluorescence quenching process is minor and the ground-state complex formation (or static quenching) process is major in the fluorescence quenching of Atto655 by AcTyr. AcTrp also can dominantly quench the fluorescence of Atto655 in aqueous solution (Figure S4 of the Supporting Information). For a comparative study, we also investigated the fluorescence quenching dynamics of Atto655 in the presence of different AcTrp concentration in 1 × TE buffer solution. Figure 5 shows the first electronic excited-state decay of Atto655 in the presence of different AcTrp concentrations in 1 × TE buffer obtained with femtosecond transient absorption spectroscopy through monitoring the transient absorption of the S1 → Sn

Figure 5. Femtosecond magic-angle pump−probe transients of Atto655 in the presence of different N-acetyltryptophan concentrations (a, 0 mM; b, 60 mM; and c, 200 mM) in 1 × TE pH = 8 buffer. Pump, 665 nm; probe, 570 nm. D



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transition after Atto655 was excited into its first excited electronic state.25 Similarly, the decays of the first electronic excited state of Atto655 in the presence of AcTrp solution also needs a summation of two exponential decay functions to be fitted (Table 2). From the fitting parameters listed in Table 2, it is found that the excited-state lifetime of Atto655/AcTrp complex is 1.1 ps, which is extremely shorter than that of the Atto655/AcTyr complex. Herein, we measured the first electronic excited-state decay of Atto655 in AcTrp solution at a probe wavelength of 570 nm. It is reported that the absorption maximum of the Trp cation radical is around 570 nm.49 To confirm that the absorption of Trp cation radical does not have obvious effect on the measured first electronic excitedstate decay kinetics of Atto655 in AcTrp solution, we measured the stimulated emission decay of Atto655 in 200 mM AcTrp solution with femtosecond transient absorption spectroscopy (Figure S5 of the Supporting Information). The stimulated emission decay of Atto655 in 200 mM AcTrp solution was collected with femtosecond magic-angle pump−probe experiment at the probe wavelength of 710 nm, where Atto655 does not have obvious absorption. From the data shown in Figure S5, it is found that the stimulated emission decay of Atto655 in 200 mM AcTrp solution can be fitted by a summation of two exponential decay functions with the time constant (amplitude) of 1.0 ps (0.95) and 1800 ps (0.05). The almost identical time constant (amplitude) that was extracted from the data shown in Figure S5 and from the data shown in Figure 5c indicates that the absorption of Trp cation radical has no obvious effect on the first electronic excited-state decay kinetics of Atto655 in AcTrp solution. Excited-State Decays of Atto655 in 200 mM AcTyr and AcTrp Solution at Different pH. In this work, we studied the fluorescence quenching dynamics of Atto655 in the presence of AcTyr and AcTrp in 1 × TE buffer solution. We found that the excited-state lifetimes of Atto655/AcTyr and Atto655/AcTrp complexes in 1 × TE buffer are 220 and 1.1 ps, respectively. The excited-state lifetime of Atto655/AcTyr complex is about 200 times longer than that of Atto655/ AcTrp complex. To understand the difference in the excitedstate lifetime between Atto655/AcTrp and Atto655/AcTyr complexes, we first measured the first one-electron oxidation peak potentials of AcTyr and AcTrp in 1 × TE buffer solution with cyclic voltammetry (Figure S6 of the Supporting Information). It is found that the first one-electron oxidation peak potential of AcTyr is only slightly smaller than that of AcTrp. The small difference in the first one-electron oxidation peak potential between AcTrp and AcTyr cannot introduce such larger difference in the excited-state lifetime between Atto655/AcTrp and Atto655/AcTyr complexes. It is reported that tyrosine often undergoes a PCET process in its fluorescence quenching of a fluorophore40−48,55 and that tryptophan often undergoes an ET process in its fluorescence quenching of a fluorophore.28,30,36,38,39 Thus, it is expected that any ET reaction that involves tyrosine will be significantly slower than that of tryptophan, which is in agreement with our current experimental observation. To confirm that the fluorescence quenching of Atto655 by AcTyr is through a PCET process and that the fluorescence quenching of Atto655 by AcTrp is through an ET process, we respectively measured the first electronic excited-state decay kinetics of Atto655 in 200 mM AcTyr and AcTrp solutions at different pH values. Figure 6 shows the first electronic excited-state decay kinetics of Atto655 in 200 mM AcTyr and AcTrp solutions at pH = 6, 8,

Figure 6. (A) Femtosecond magic-angle pump−probe transients of Atto655 in 200 mM N-acetyltyrosine solution at different pH values. (B) Femtosecond magic-angle pump−probe transients of Atto655 in 200 mM N-acetyltryptophan solution at different pH values. Pump, 665 nm; probe, 570 nm.

10 and 12, respectively. It is clear that the pH value dependence of the first electronic excited-state decay kinetics of Atto655 in 200 mM AcTrp solution is different from that of Atto655 in 200 mM AcTyr solution. For Atto655 in 200 mM AcTrp solution, the first electronic excited-state decays of Atto655 at all pH values can be fitted with a summation of two exponential decay functions (Table 3), and the excited-state lifetime of Atto655/AcTrp complex is maintained constant as a function of the solution’s pH value. However, for Atto655 in 200 mM AcTyr solution, the first electronic excited-state decays of Atto655 at pH = 6 and pH = 8 can be fitted with a summation of two exponential decay functions (Table 3), whereas the first electronic excited-state decays of Atto655 at pH = 10 and pH = 12 need a summation of three exponential decay functions to be fitted (Table 3). Comparing with the first electronic excitedstate decays of Atto655 in 200 mM AcTyr solution at pH = 6 and pH = 8, an extra 3.0 ps decay component appears in the first electronic excited-state decays of Atto655 in 200 mM AcTyr solution at pH = 10 and pH = 12. This indicates that an extra process was introduced in the first electronic excited-state decays of Atto655 in 200 mM AcTyr solution at pH = 10 and pH = 12. To understand the pH value dependence of the first electronic excited-state decay kinetics of Atto655 in 200 mM AcTrp solution and in 200 mM AcTyr solution, we have to know the pKa of AcTrp and AcTyr in aqueous solution. It is known that the pKa of AcTrp is around 17 and the pKa of AcTyr is about 10 in aqueous solution.41,42,49,55 Then, the quencher AcTrp only has one existing form (AcTrpNH) in the pH range of 6−12 whereas the quencher AcTyr could have two existing forms (AcTyrOH and AcTyrO−) in the pH range of 6−12. Therefore, we concluded that the 1800 ps decay component in Table 3 is the excited-state lifetime of free Atto655, the 1.1 ps decay component in Table 3 is the excitedstate lifetime of the Atto655/AcTrpNH complex, the 230 ps decay component in Table 3 is the excited-state lifetime of the Atto655/AcTyrOH complex, and the 3.0 ps decay component in Table 3 is the excited-state lifetime of the Atto655/AcTyrO− complex. The first electronic excited-state lifetime of Atto655/ AcTyrOH complex is about 80 times longer than that of the Atto655/AcTyrO− complex, and the first electronic excitedstate lifetime of the Atto655/AcTrpNH complex is similar to E



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Table 3. Excited-State Decay Parameters for Atto655 in 200 mM N-Acetyltyrosine (AcTyr) and N-Acetyltryptophan (AcTrp) Solution at Different pH Values, Obtained from Femtosecond Transient Absorption Measurements quencher

pH

200 mMAcTyr

6 8 10 12 6 8 10 12

200 mM AcTrp

a

a1

0.14 0.32

τ1/ps

a2 0.87 0.86 0.70 0.36 0.96 0.97 0.97 0.97

3.5 ± 0.5 3.0 ± 0.5

τ2/ps

a3

τ3/psa

± ± ± ± ± ± ± ±

0.13 0.14 0.16 0.32 0.04 0.03 0.03 0.03

1800 1800 1800 1800 1800 1800 1800 1800

230 220 230 260 1.1 1.1 1.1 1.1

20 20 20 20 0.2 0.2 0.2 0.2

Fixed to match the lifetime obtained by time-correlated single-photon-counting measurements.

that of the Atto655/AcTyrO− complex. To confirm that the above experimental observations are not from the pH effect on the photophysics of Atto655 alone, we further recorded the steady-state fluorescence intensity and the fluorescence lifetime (Figure S7 of the Supporting Information) of Atto655 in aqueous solution at different pH values. It is clear that the solution’s pH value does not have any obvious effect on the photophysical parameters of Atto655. Thus, the result shown in Figure 6 reveals that the fluorescence quenching of Atto655 by AcTyr in a low pH solution involves a PCET process between Atto655 and AcTyrOH, and that the fluorescence quenching of Atto655 by AcTrp in all pH solution involves an ET process between Atto655 and AcTrp. However in a high pH (pH > 12) solution, the quenching of Atto655 by AcTyr could also involve an ET process between Atto655 and AcTyrO−. To our knowledge, this is the first systematic study on the PCET reaction between a labeled fluorophore and tyrosine with femtosecond transient absorption spectroscopy. Excited-State Decays of Atto655 with 200 mM AcTyr and AcTrp Solution in D2O. To confirm that the fluorescence quenching of Atto655 by AcTyr in 1 × TE pH = 8 buffer undergoes a PCET process and that the fluorescence quenching of Atto655 by AcTrp in 1 × TE pH = 8 buffer undergoes an ET process, we also measured the first electronic excited-state decay of Atto655 in the presence of 200 mM AcTyr and AcTrp in H2O and D2O solution. Figure 7 shows the first electronic excited-state decay of Atto655 in the presence of 200 mM AcTyr and AcTrp in H2O and D2O solution, respectively, obtained with femtosecond transient absorption spectroscopy through monitoring the transient absorption of the S1 → Sn transition after Atto655 was excited into its first excited electronic state.25 Clearly, it is found that

the first electronic excited-state decay of Atto655 in the presence of 200 mM AcTyr in D2O solution is slower than that in H2O solution, and that the first electronic excited-state decay of Atto655 in the presence of 200 mM AcTrp in D2O solution is identical to that in H2O solution. The first electronic excitedstate kinetics of Atto655 in the presence of 200 mM AcTyr in H2O solution can be fitted by a summation of two exponential decay functions with the time constant (amplitude) of 230 ps (0.86) and 1800 ps (0.14), and the first electronic excited-state kinetics of Atto655 in the presence of 200 mM AcTyr in D2O solution can be fitted by a summation of two exponential decay functions with the time constant (amplitude) of 390 ps (0.84) and 3400 ps (0.16). The first electronic excited-state kinetics of Atto655 in the presence of 200 mM AcTrp in H2O solution can be fitted by a summation of two exponential decay functions with the time constant (amplitude) of 1.1 ps (0.96) and 1800 ps (0.04), and the first electronic excited-state kinetics of Atto655 in the presence of 200 mM AcTrp in D2O solution can be fitted by a summation of two exponential decay functions with the time constant (amplitude) of 1.1 ps (0.97) and 3400 ps (0.03). The different lifetimes of Atto655/AcTyr complex in H2O and D2O solution (230 versus 390 ps) suggest that the fluorescence quenching of Atto655 by AcTyr in aqueous solution undergoes a PCET process between Atto655 and AcTyr. The identical lifetimes of Atto655/AcTrp complex in H2O and D2O solution (both are 1.1 ps) confirm that the fluorescence quenching of Atto655 by AcTrp in aqueous solution undergoes an ET process between Atto655 and AcTrp. It is reported that the observation of a kinetic isotope effect (KIE) is the hallmark of a concerted PCET reaction.56,57 Herein, we derived the KIE of the reaction between Atto655 and AcTyr in aqueous solution is 1.70, indicating that the fluorescence quenching of Atto655 by AcTyr in aqueous solution undergoes a concerted PCET between Atto655 and AcTyr. The mechanism of a concerted PCET is defined as the simultaneously transfer of electron and proton. Electronic Coupling Constant between Donor and Acceptor. In this work, we studied the fluorescence quenching dynamics of Atto655 in the presence of AcTyr and AcTrp in aqueous solution at different pH value and found that the excited-state lifetimes of Atto655/AcTyrOH, Atto655/AcTyrO− and Atto655/AcTrpNH complexes are 230 ps, 3.0 and 1.1 ps, respectively. With the kinetic scheme shown in our previous reports,24,25 we derived that the PCET rate of Atto655/AcTyrOH complex is 3.8 × 109 s−1, and that the ET rates of Atto655/AcTyrO− and Atto655/AcTrpNH complexes are 3.3 × 1011 s−1 and 9.1 × 1011 s−1, respectively. The PCET rate of Atto655/AcTyrOH complex is 100 times smaller than the ET rates of Atto655/AcTyrO− and Atto655/AcTrp

Figure 7. (A) Femtosecond magic-angle pump−probe transients of Atto655 in 200 mM N-acetyltyrosine H2O solution and in 200 mM Nacetyltyrosine D2O solution. (B) Femtosecond magic-angle pump− probe transients of Atto655 in 200 mM N-acetyltryptophan H2O solution and in 200 mM N-acetyltryptophan D2O solution. Pump, 665 nm. probe, 570 nm. F



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FluA−Fl complex is 170 cm−1, which is very close to our obtained electronic coupling constant for reaction between Atto655 and AcTrpNH. Gotz et al.30 also reported that the electronic coupling constant for ET reaction between labeled fluorescein and Tyr residue in the FluA−Fl complex is 140 cm−1, which is larger than the obtained electronic coupling constant for the reaction between Atto655 and AcTyrOH or AcTyrO−. However, Mayer and co-workers71 studied the PCET reactions of a series of hydrogen-bonded phenols and found that the electronic coupling constants for the reactions of hydrogen-bonded phenols are in the range of 20−30 cm−1, which is quite similar to the obtained 8.3 cm−1 electronic coupling constant for the reaction between Atto655 and AcTyrOH. The 8.3 cm−1 electronic coupling constant between Atto655 and AcTyrOH reveals that the PCET reaction between Atto655 and AcTyrOH in aqueous solution is nonadiabatic.

complexes. For a pure ET process, the transfer rate can be calculated using the Marcus-type equation58 ket =

⎛ −(ΔG + λ)2 ⎞ exp⎜ ⎟ 4λkT ⎝ ⎠ ℏ 4πλkT 2π |J |2

(1)

where ΔG is the driving force for the electron transfer reaction, J is the electronic coupling matrix element between donor and acceptor, λ is the reorganization energy, ℏ is the Planck constant, k is the Boltzmann constant, and T is the temperature. The driving force ΔG can be estimated by using Weller’s equation59 ΔG = Eox − Ered − ES + C

(2)

where Eox is the first one-electron-oxidation peak potential of the donor, Ered is the first one-electron-reduction peak potential of the acceptor, ES is the energy of the zero−zero transition to the lowest excited singlet state, and C is the solvent-dependent Coulombic interaction energy, which can be neglected in moderately polar environment. However, for a PCET process, various levels of theory have been employed. Researchers have used versions of Hammes−Schiffer’s multistate continuum theory to analyze the rate of a PCET reaction.48,57,60−66 The versions of Hammes−Schiffer’s multistate continuum theory generally require simplifications because it includes many parameters that are not easily accessible.48,64−66 Alternatively, researchers have also used versions of the semiclassical Marcus ET theory (eq 1) to analyze the rate of a PCET reaction.46,67−71 Recently, Mayer and co-workers have proved that the PCET reactions of hydrogen-bonded phenols are well described by the semiclassical Marcus ET theory.71 The first one-electron-reduction peak potential of Atto655 is not available in the literature. However, Sauer and co-workers reported that Atto655 exhibited very similar spectroscopic characteristics as the oxazine fluorophore MR121, and that the peak potential for one-electron reduction of MR121 is −0.18 V (vs NHE).16,29 From the literature,42,49,55 it is found that the first one-electron-oxidation peak potential of AcTyrOH in pH = 8 solution is 0.89 V (vs NHE), the first one-electronoxidation peak potential of AcTyrO− in pH = 12 solution is 0.72 V (vs NHE), and the first one-electron-oxidation peak potential of AcTrpNH in pH = 8 solution is 0.95 V (vs NHE), respectively. Besides, from the ensemble steady-state absorption and fluorescence spectra shown in Figure 1, it is found that the zero−zero transition energy of Atto665 in aqueous solution to be 1.85 eV (∼670 nm). Substituting these values into eq 2, we derived that ΔG = −0.78 eV for the PCET reaction between Atto655 and AcTyrOH, ΔG = −0.95 eV for the ET reaction between Atto655 and AcTyrO−, and ΔG = −0.72 eV for the ET reaction between Atto655 and AcTrpNH. Assuming a typical reorganization energy of λ = 1.2 eV for aqueous solution71−73 and substituting the PCET or ET rates in the complexes between Atto655 and respective quenchers into eq 1, we derived that the electronic coupling constant for the PCET reaction between Atto655 and AcTyrOH is 8.3 cm−1, the electronic coupling constant for the ET reaction between Atto655 and AcTyrO− is 48 cm−1, and the electronic coupling constant for the ET reaction between Atto655 and AcTrpNH is 162 cm−1. Clearly, the electronic coupling constant for the reaction between Atto655 and AcTyrOH is smaller than that between Atto655 and AcTrpNH or AcTyrO−. Gotz et al.30 reported that the electronic coupling constant of an ET reaction between labeled fluorescein and Trp residue in the

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CONCLUSIONS In summary, we measured the first electronic excited-state decays of Atto655 in the presence of different concentrations of AcTyr and AcTrp and investigated the ultrafast fluorescence quenching kinetics of Atto655 by the respective quencher in aqueous solution with femtosecond transient absorption spectroscopy. The pH value and D2O dependences of the excited-state decay kinetics of Atto655 in 200 mM AcTyr and AcTrp solutions reveal that the fluorescence quenching of Atto655 by AcTyr in pH = 8 solution is via a PCET process and that the fluorescence quenching of Atto655 by AcTrp in pH = 8 solution is via an ET process. The PCET rate between Atto655 and AcTyr in pH = 8 solution is about 240 times smaller than the ET rate between Atto655 and AcTrp in pH = 8 solution. With the version of the semiclassical Marcus ET theory, we derived that the electronic coupling constant for the PCET reaction between Atto655 and AcTyr in pH = 8 solution is 8.3 cm−1. The smaller electronic coupling constant indicates that the PCET reaction between Atto655 and AcTyr in aqueous solution is nonadiabatic.

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ASSOCIATED CONTENT

S Supporting Information *

Time-correlated single-photon-counting measurement, cyclic voltammetry measurement, additional figures, and additional tables. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86-10-6251-4601. Fax: +86-10-6251-6444. E-mail: a. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Fundamental Research Funds for the Central Universities and the Research Funds of Renmin University of China (10XNJ047).

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