INTRODUCTION

Article pubs.acs.org/JPCB

Ultrafast Spectroscopic Dynamics of Quinacrine-Riboflavin Binding Protein Interactions Shiori Yamazaki, Matthew A. Diaz, Thomas M. Carlino, Chitra Gotluru, Mercedes M. A. Mazza, and Amy M. Scott* University of Miami, Department of Chemistry, 1301 Memorial Drive, Coral Gables, Florida 33146, United States S Supporting Information *

ABSTRACT: Redox active cofactors play a dynamic role inside protein binding active sites because the amino acids responsible for binding participate in electron transfer (ET) reactions. Here, we use femtosecond transient absorption (FsTA) spectroscopy to examine the ultrafast ET between quinacrine (Qc), an antimalarial drug with potential anticancer activity, and riboflavin binding protein (RfBP) with a known Kd = 264 nM. Steady-state absorption reveals a ∼ 10 nm red-shift in the ground state when QcH32+ is titrated with RfBP, and a Stern−Volmer analysis shows ∼84% quenching and a blue-shift of the QcH32+ photoluminescence to form a 1:1 binding ratio of the QcH32+−RfBP complex. Upon selective photoexcitation of QcH32+ in the QcH32+−RfBP complex, we observe charge separation in 7 ps to form 1 [QcH3_red•+−RfBP•+], which persists for 138 ps. The FsTA spectra show the spectroscopic identification of QcH3_red•+, determined from spectroelectrochemical measurements in DMSO. We correlate our results to literature and report lifetimes that are 10−20× slower than the natural riboflavin, Rf−RfBP, complex and are oxygen independent. Driving force (ΔG) calculations, corrected for estimated dielectric constants for protein hydrophobic pockets, and Marcus theory depict a favorable one-electron ET process between QcH32+ and nearby redox active tyrosine (Tyr) or tryptophan (Trp) residues.

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INTRODUCTION Electron transfer (ET) reactions of flavin chromophore cofactors are critical processes that control biological activity in enzymes, proteins, and cellular function.1−6 However, studying the dynamical complex with respect to reactivity, binding and redox activity is challenging because the mechanistic details cannot be fully understood through crystallography, which provides only static information about the cofactor-protein interaction. In this work, quinacrine (Qc), a potential anticancer drug with similar chemical structure to riboflavin (Rf, vitamin B2) and a large binding affinity to riboflavin binding protein (RfBP), was studied using electrochemical and time-resolved spectroscopic techniques to gain a fundamental understanding of the light-induced processes and dynamics of ET between QcH32+ and RfBP with femtosecond resolution. The studies here are critical for understanding drug−protein toxicity in light-sensitive biological systems, such as the retina,7 and to facilitate theoretical calculations aimed at developing predictive drug−protein binding models. The results we obtained are explained and correlated to previously published femtosecond studies on cofactor−RfBP to give perspective of the factors that control ultrafast ET and binding of redox active cofactors or drugs inside protein pockets. Rf is a water-soluble metabolite involved in the biosynthesis of flavin cofactors, which participate in electron transfer reactions in binding proteins to regulate cellular metabolism.8−20 Rf is also a light-sensitive redox active molecule with π−π*, ground state absorption of the aromatic core centered at © 2017 American Chemical Society

400 nm, and thus, it serves as a reliable spectroscopic probe of the binding pocket and side-reactions driven by light. According to the reported X-ray crystal structure of the Rf− RfBP complex,12 the RfBP hydrophobic pocket interacts primarily with the xylene domain of the isoalloxazine ring of Rf, which is juxtaposed between Tyr-75 and Trp-156 residues found deep inside the RfBP globular protein (Scheme S1). The Rf ribityl side chain contains several hydroxyl groups that can form hydrogen bonds with the protein binding active site. Several studies have shown that Rf antagonists can bind within membrane binding proteins like RfBP and can be taken-up in human cancer KB cells,21−26 suggesting that small molecules similar in chemical structure to Rf can be delivered by interfering with the cellular functions of flavin cofactors.20,27,28 To understand the structure−function relationship of cofactor−RfBP interactions, we have identified and studied here the ultrafast dynamics between QcH32+ and RfBP. Recently, it was reported that QcH32+ is an artificial redox cofactor upon binding to RfBP with a reported Kd = 264 nM, determined through isothermal titration calorimetry and surface plasmon resonance.29 The chemical structure of QcH32+ is composed of a tricyclic heterocycle acridine derivative with a reported30 absorption band centered at 425 nm, which was attributed to π−π* ground state absorption of the aromatic core, and is Received: May 31, 2017 Revised: July 19, 2017 Published: August 1, 2017 8291

DOI: 10.1021/acs.jpcb.7b05304 J. Phys. Chem. B 2017, 121, 8291−8299

Article

The Journal of Physical Chemistry B

nm, and an aqueous background solution was collected prior to the samples. Steady-state emission and excitation spectra were collected on a RF-6000 Shimadzu spectrofluorophotometer using a 1 cm quart cuvette in the presence of air. The excitation wavelengths were selected as the absorption maxima of QcH32+ at 275, 345, and 425 nm. The temperature of the solutions was adjusted to 297, 309, and 317 K by a temperature controller (Quantum) and the temperature was monitored during the experiment with a digital thermometer. Electrochemistry and Spectroelectrochemistry. Cyclic voltammetry (CV) and square wave voltammetry (SWV) experiments were conducted on an electrochemical analyzer potentiostat, 650E, from CH Instruments. Solutions were deareated with high purity Ar gas for 10 min prior to each experiment. The working, reference, and counter electrodes were Pt or glassy carbon, Ag/AgCl or Ag wire, and a Pt wire, respectively. A background scan of the electrolyte ((TBA)PF6) and solvent (DMSO) was conducted at both positive and negative potentials prior to the sample voltammograms. To determine the electronic absorption spectra of reduced QcH32+, spectroelectrochemistry was performed using an external bulk electrolysis flow-cell setup with a high surface area, reticulated vitreous carbon (RVC) working electrode, Pt counter, and a Ag/AgCl reference electrode. The applied potential was determined from previous CV experiments, and the absorption spectra were collected on a Shimadzu 1800 spectrophotometer using a 2 mm flow cell cuvette. Transient Absorption Spectroscopy. Femtosecond transient absorption (FsTA) measurements were performed with an instrument based on a commercial Ti:sapphire oscillator (MaiTai, Spectra Physics) and a mode-locked Ti:sapphire regenerative amplifier (Spitfire Ace, Spectra Physics) pumped by a 527 nm Nd:YLE laser (Empower, Spectra Physics). The amplified pulse output is 5 W of 35 fs (fwhm) light centered at 800 nm at a repetition rate of 1 kHz. The output of the ultrafast laser system was divided into two beams, for the pump and the probe. A 400 nm pump beam was generated from the 800 nm fundamental using a SHG crystal, with an instrument response time of ∼70 fs (fwhm). The white light probe was generated from the split 800 nm beam by focusing on a sapphire window. The pump and probe beams were sent to a commercial FsTA setup (Helios, Ultrafast Systems) in which the probe was delayed relative to the pump using a mechanical delay track. The nanosecond transient absorption (NsTA) measurements were performed using a commercial transient absorption spectrometer (EOS, Ultrafast Systems). A photonic crystal fiber based supercontinuum pulsed light source was used for probe generation and the delay time was controlled electronically. The FsTA and NsTA measurements were conducted using a 2 mm quart cuvette and the pump beam diameter at the sample was ∼400 mm. No pump power dependency (1.0−3.0 μJ/pulse) was observed on the FsTA kinetics or spectra and those results are shown in the Supporting Information. A 50 μM QcH32+ solution was prepared in pH 7.0 PBS, and RfBP was dissolve to obtain a 50 μM QcH32+ - 400 μM RfBP solution. Samples were freshly prepared for each measurement and were prepared by deareating with Ar gas for 15 min. The experiments were conducted at room temperature, and the data were background subtracted and chirp-corrected using the Ultrafast Systems Surface Xplorer Software.

similar but not identical to the tricyclic heterocycle isoalloxazine chromophore found in Rf (Figure 1).

Figure 1. Chemical structures of redox active cofactors (A) riboflavin, Rf, and (B) quinacrine, Qc, that were studied upon binding inside the riboflavin binding protein (RfBP).

The relationship between Rf and RfBP has been examined extensively, including femtosecond studies by Mataga31,32 and Zewail33,34 over the past few decades.10,33−37 Upon photoexcitation of the Rf−RfBP complex, ultrafast electron transfer from Rf to Trp in the RfBP occurs in ∼500 fs and results in a short-lived radical pair, Rf•− RfBP•+, that recombines in 8 ps.34 Therefore, Qc is expected to have similar ET dynamics and πstacking interactions with Tyr-75 and Trp-156 to form CT states in the RfBP pocket, based on a similar argument made by Zewail and co-workers33,34 in their femtosecond studies on DM−RfBP, a drug similar in chemical structure to Rf (Scheme S1). However, they observed lifetimes for DM•−−RfBP•+ in the absence of molecular oxygen that are similar to the lifetimes to the QcH3_red•+−RfBP•+ complex reported here, and together both have lifetimes that are 10−20× slower than the Rf−RfBP complex. Here, we combine FsTA, UV−vis, photoluminescence, and electrochemistry to study QcH32+−RfBP dynamics in aqueous solutions through detection of the photogenerated radical pair that persists on the hundreds of picosecond time scales that we model using Marcus theory. We observed new spectroscopic shifts in the ground state absorption and photoluminescence spectra, and together, discuss the driving force, dielectric constant and electron spin dynamics as factors that influence binding and ET of redox active cofactors inside the RfBP hydrophobic pocket. Finally, the ultrafast results explain that QcH32+ does not undergo oxygen- and lightsensitive side reactions because there are no triplet excited states that were detected, and thus, it does not undergo cytotoxic side reactions like other drug−protein systems.

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METHODS Materials. (−)-Riboflavin (≥98%, Rf), riboflavin binding protein (riboflavin-depleted apo-form, RfBP), quinacine dihydrochloride (≥90%, Qc) were purchased from SigmaAldrich. Qc was purified through recrystallization from an ethanol solution. The 11 μM phosphate buffer solution (PBS) was prepared by dissolving 0.784 g of KH2PO4 into 500 mL of deionized water. The pH values of the buffer solutions were adjusted using NaOH and H3PO4. Steady-State Spectroscopy. A 15.00 μM QcH32+ solution was prepared in pH 7.0 PBS, and RfBP powder was dissolved to make 1.875, 3.750, 7.500, 15.00, and 60.00 μM RfBP−15.00 μM QcH32+ samples. UV−vis absorption spectra were measured using a UV-1800 Shimadzu spectrophotometer in a 1 cm quart cuvette (Starna) at room temperature and in the presence of air. The excitation and detection slits were set to 5 8292


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Figure 2. Structures of quinacrine following prototropic reactions in water (QcH43+, QcH32+, QcH2+, and QcH). The values of each pKa were previously reported.30

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RESULTS AND DISCUSSION Quinacrine (Qc) can form four protonated states under varying pH conditions and the pKa values have been determined previously (QcH, QcH2+, QcH32+, and QcH43+) as shown in Figure 2. Under physiological conditions and for the studies presented here, QcH32+ is the favorable form at pH 7.0 and in DMSO and was used to study the binding with RfBP using steady-state and time-resolved spectroscopy. Steady-State Spectroscopy. Figure 3 shows the steadystate absorption and photoluminescence spectra of QcH32+ Figure 4. Photoluminescence spectra of 15.00 μM QcH32+ with various concentrations of RfBP (0.000, 1.875, 3.50, 7.500, and 15.00 μM) in pH 7.0 PBS at room temperature in the presence of air at λexc = 425 nm.

discussed in more detail later. In order to determine the quenching mechanism between QcH32+ and RfBP, the Stern− Volmer relationship38,39 was used to analyze a series of concentration and temperature dependent measurements on photoluminescence intensity (see details in Supporting Information). We observed that the Stern−Volmer quenching constant, KSV, decreases with increasing temperature; the inverse of the effective quenching constant, 1/Ka, increases with high temperature as shown in Figure S3. These trends suggest a static quenching mechanism, and the opposite trends can be observed for collisional quenching. The binding constant, K, and the number of binding sites, n, was calculated by using the equation below:40

Figure 3. Steady-state absorption spectra (pink lines) and photoluminescence spectra (blue lines) of 15 μM QcH32+ without RfBP (solid lines) and 15 μM QcH32+ with 60 μM RfBP (dash−dot lines) in pH 7.0 PBS at room temperature in the presence of air. The photoluminescence experiments were acquired upon λexc = 400 nm.

(solid lines) and the QcH32+−RfBP complex at a 1:4 ratio (dash−dot line) in pH 7.0 PBS at room temperature in the presence of air. The absorption spectrum of QcH32+ exhibits broad absorption bands with a vibronic progression centered at 450 nm (0−0 transition of 1La) and 425 nm (0−1 transition of 1 La). The photoluminescence maximum was observed at 505 nm upon photoexcitation at 400 nm. Upon the addition of RfBP to the QcH32+ solution, the absorbance spectrum of the QcH32+ shows a ∼ 10 nm red-shift in the visible region, and the intensity of the photoluminescence peak at 505 nm is dramatically reduced and the quenching indicates a strong interaction between QcH32+ and RfBP. Stern−Volmer analysis of the photoluminescence quenching as a function of temperature was used to determine the quenching mechanism. The photoluminescence spectrum of a 15.00 μM QcH32+ solution with various concentrations of RfBP at pH 7.0, photoexcited at 425 nm and recorded at 298 K in the presence of air, is graphed in Figure 4. Figure S2 shows the photoluminescence spectra photoexcited at 275 and 345 nm and under the same conditions as described previously for Figure 4. Following the QcH32+−RfBP complexation, the photoluminescence decreased by ∼84% and blue-shifted from 505 to 490 nm. This suggests there is a small perturbation of the QcH32+ ground state structure with RfBP, and this will be

⎛F − F⎞ ⎟ = log K + n log[RfBP ] log⎜ 0 ⎝ F ⎠

(1)

QcH32+

and RfBP bind with a 1:1 ratio at three difference temperatures as shown in Table S3. Therefore, QcH32+ appears to have a similar binding ratio as Rf inside the hydrophobic pocket of RfBP. Circular dichroism (CD) experiments, not presented here, show no difference in the spectrum with and without QcH32+. This is in agreement with previous CD literature for Rf−RfBP binding.37 Finally, the amino acids inside the hydrophobic pocket of RfBP absorb and emit light in the UV region,16,41,42 and there is no spectral overlap with QcH32+. Thus, we ruled out possible energy transfer pathways as the primary mechanism for photoluminescence quenching and focus our discussion on static quenching through electron transfer. Electrochemistry and Spectroelectrochemistry. Cyclic voltammetry (CV) and square wave voltammetry (SWV) measurements were conducted on QcH32+ in DMSO to determine the electrochemical reduction potentials, in a polar aprotic solvent, and used to estimate the driving for electron transfer between Trp and Tyr amino acids inside the protein. The CV of a 300 μM QcH32+ solution in DMSO with 0.1 M 8293


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The Journal of Physical Chemistry B TBPF6 with the corresponding SWV, scanning from 0 to −2.0 V vs Ag/AgCl is presented in Figure S5 using a Pt working electrode and a Ag wire pseudoreference electrode. We examined various experimental conditions and working and reference electrodes to facilitate the interpretation of the electrochemistry. On the basis of our control experiments, and with the addition of ferrocene as an internal standard, the two reduction potentials that were extracted for QcH32+ are −0.931 and −1.65 V vs Ag/AgCl, and are similar to the half-wave potentials, E1/2, obtained from SWV. Spectroelectrochemistry measurements facilitate in the identification of new absorption bands upon electrochemical reduction and are used to approximate the transient absorption spectra formed as intermediates during the ultrafast laser experiment, similar to the chemical intermediate assignment for the photogeneration of anionic flavin semiquinone that undergo ET inside BLUF proteins.43 Figure 5A shows the spectroelectrochemical results

Figure 6. Femtosecond transient absorption (FsTA) spectra of QcH32+ (50 μM, blue lines) and QcH32+ with RfBP (50 μM:400 μM, orange lines) in pH 7.0 PBS at 1 and 5 ps after 400 nm photoexcitation with 3.0 μJ/pulse (IRF ∼ 70 fs) at room temperature. The samples were deaerated with Ar gas. Change in absorbance of QcH32+ in DMSO with 0.1 M (TBA)PF6 following electrochemical reduction at a potential of −1.4 V vs Ag/AgCl was obtained from the spectroelectrochemistry measurement (green line).

(band I) and one centered at 520 nm (band II), and a positive broadband band from 550 to 725 nm (band III). The FsTA spectra of the QcH32+−RfBP complex (orange lines in Figure 6) also show a negative band from 450 to 475 nm (band i) and a positive broadband from 525 to 725 nm (band iii). We attribute band iii to the absorption profile of the CT state. The negative band at 520 nm as seen in QcH32+ is absent. Rather, the FsTA spectra of the QcH32+−RfBP complex have a weak, positive band centered at 480 nm (band ii) and are similar in absorbance as the spectroelectrochemical results for the reduction of QcH32+. Using the steady-state absorption and photoluminescence spectra shown in Figure 3, we determined that the negative bands I and i correspond to the ground state bleach (GSB) of QcH32+, and band II is attributed to the stimulated emission (SE) of QcH32+. The GSB band i shifts 10 nm over the 5 ps time scale, and the broad absorption bands III and iii have different spectra shape and intensity, which indicates that the excited states for QcH32+ are different from the CT state of QcH32+−RfBP. The FsTA kinetics were analyzed for QcH32+ and QcH32+−RfBP at 520 and 700 nm, which represent bands II and ii, and bands III and iii, respectively. We were not able to fit the FsTA kinetics of bands I and i because of low signal-to-noise. The FsTA kinetics of QcH32+ for band II and III show biexponential decay components of τ1 = 293 ps (38%) and τ2 = 2 ns (62%) at both 520 and 700 nm, as shown in Figure 7 (blue). As seen in Figure 7, the kinetics of QcH32+−RfBP decay faster than the kinetics of QcH32+alone at 520 and 700 nm. The kinetics of QcH32+−RfBP at 520 nm (band ii) rise in τ = 7 ps and decay in τ = 120 ps and is presented in Figure 7A (orange). We also observed similar time components of the transient of QcH32+− RfBP at 520 and 700 nm. The excited state absorption (ESA) signal at 700 nm (band iii) reveals two exponential decay components of τ = 8 ps (31%) and τ = 138 ps (69%) as shown in Figure 7B (orange). The FsTA results indicate that the QcH32+−RfBP complex is not expected to undergo a singlet− singlet energy transfer process because the photoluminescence of QcH32+ does not have any spectral overlap with RfBP absorption. This is consistent with previous33 femtosecond experiments on DM−RfBP. Finally, we performed a laser pump power dependency and determined the processes studied here occur via a one photon absorption process (Figure S8), and the

Figure 5. (A) Spectroelectrochemistry measurements showing the absorbance spectrum of QcH32+ in DMSO at a potential of −1.4 V vs Ag/AgCl with 0.1 M (TBA)PF6. Upon applying the potential, the absorption spectra changed from blue line (initial), to the red line (final). The black dash line is the steady-state absorption spectra of QcH32+, which was achieved by preparing QcH32+ solution in pH 7.0 PBS. (B) Protonated species of Qc with proton sources. [QcH3_red]+ representing the first reduction of QcH32+.

when QcH32+ is reduced at −1.4 V vs Ag/AgCl in DMSO. Following the reduction, the shape of the absorption spectra changed dramatically (blue line to red line). First, the featured vibronic band centered at 452 nm dissipated to form two new bands at 350 and 500 nm. Figure 5B shows the protonated quinacrine species with proton sources. [QcH3_red]+ represents the first reduction of QcH32+. The spectroelectrochemistry measurements are used to identify transient species formed as intermediates in the QcH32+−RfBP upon laser excitation in transient absorption experiments. Ultrafast Spectra and Kinetic Analysis of Quinacrine− RfBP Electron Transfer. We have previously used transient absorption (TA) spectroscopy to investigate the electronic and photophysical properties of excited states, radical pairs and redox states, and electron spin states as a function of absorption, time, and wavelength. Here, we describe the spectral and temporal response from the interaction between QcH32+ and RfBP upon selected photoexcitation of QcH32+ at 400 nm. Figure 6 shows the FsTA spectra of QcH32+ and QcH32+−RfBP in pH 7.0 PBS recorded at 1 and 5 ps after photoexcitation, and are overlaid with the change in absorbance following electrochemical reduction of QcH32+ in DMSO (−1.4 V vs Ag/AgCl). The FsTA spectra of QcH32+ (blue lines in Figure 6) display two negative bands, from 450 to 470 nm 8294


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proteins is expected to be significantly lower than water and has recently been determined experimentally46 as ∼8.9 and theoretically47 as ∼10. Here, we used a static dielectric of ∼8.9 for εp and the εDMSO from the electrochemical measurements were used to correct ΔG for ET and those equations and results are located in Figure S11.46 The 0−0 transition (S1 ← S0) energy is calculated as 2.65 eV using the steady-state absorption and photoluminescence spectra shown in Figure 3. The Trp/Trp•+ and Tyr/Tyr•+ oxidation potentials48 were reported as +0.953 V vs Ag/AgCl and +1.20 vs Ag/AgCl at pH 7.0, respectively. Therefore, the driving force for electron transfer, ΔG (εp = 8.9), is calculated for the one- electron process to form QcH3_red•+−RfBP•+ for CSTrp = −1.05 eV and CRTrp = −1.60 eV. ΔG values for CSTyr = −0.81 eV and CRTyr = −1.84 eV. Using the functional form of the semiclassical expression for electron transfer rate and neglecting the proton coupled electron transfer, which is not been observed here or in previous studies, eq 2,49−51

Figure 7. Femtosecond transient absorption kinetics of QcH32+ (blue) and QcH32+ with RfBP (orange) in pH 7.0 PBS at (A) 520 nm and (B) 700 nm, excited at 400 nm with 3.0 μJ/pulse (IRF ∼ 70 fs) at room temperature. The FsTA kinetics at 520 nm represent bands II and ii, and the FsTA kinetics at 700 nm represent bands III and iii. The samples were deareated with Ar gas before each measurement.

k=

2π |VDA|2 ℏ

2 1 e[−(ΔG + λ) /4λkBT ] 4πλskBT

(2)

we determined the rate of charge recombination, kCS, and charge recombination, kCR, for the QcH32+−RfBP complex using interatomic distance parameters of the π-stacked donor (D)−acceptor (A) pair, where RDA = 4 Å, from the X-ray crystal structure of Rf−RfBP.12,32 Here, the electronic coupling, VDA2, describes the orbital coupling between the QcH32+ and each of the Tyr and Trp redox active amino acids in the π-stack that are responsible for the ultrafast ET and we explore and vary the magnitude of VDA2 within realistic values previously determined experimentally for model, D-bridge (B)-A systems.52 Using the experimental CS and CR values, in a similar fashion as others,53 we determined VDA2 as ∼22 cm−1, and is approximately 1 order of magnitude larger than VDA2 determined for weakly bound hydrogen bonded D−A systems.53 λ is the total reorganization energy that accounts for both internal and solvent and contribution of the vibrational reorganization energy, where λ = λi + λs. The value of λs was approximated by taking into account the dielectric constant calculated and determined for protein hydrophobic pockets and calculated in Figure S11. The value of λi was adjusted to give a total λ ∼ 1.2 eV, determined from previous ultrafast photoinduced ET between tryptophan residues in Arabidopsis thaliana cryptochrome and flavin π stacks.54 Utilizing eq 2 and VDA2 as ∼22 cm−1, we determined lifetimes for the Qc−Trp pair as 1/kCS = 9 ps and 1/kCS = 53 ps, and for the Qc−Tyr pair, 1/kCS = 21 ps and 1/kCS = 628 ps. Thus, varying VDA2 suggests that either Trp or Tyr is responsible for the experimental ET lifetimes observed in the QcH32+−RfBP complex. Here, the results are discussed and correlated to previous ultrafast spectroscopic studies suggesting the RfBP pocket has sufficient structural flexibility to bind artificial redox active cofactors and our analysis is based on the following discussion. Previous femtosecond measurements on Rf−RfBP reveal CS = ∼ 500 fs and CR = 8 ps for a known distance between Trp-156, Tyr-75 and Rf of 3.7−4 Å from the X-ray crystal structure. The reduction potential for Rf/Rf•− is ∼ −0.95 V vs Ag/AgCl and is similar to QcH32+ studied here. If the redox potentials for Rf and QcH32+ are not significantly altered by inclusion inside the protein and an identical π-stacking motif is expected for each cofactor chromophore, the lifetimes of CS and CR should not

Jablonski diagram illustrating the photophysics observed for QcH32+ and the QcH32+−RfBP complex is shown in Figure 8.

Figure 8. Jablonski diagrams illustrating the photoinduced dynamics of (A) QcH32+ and (B) the QcH32+−RfBP singlet complex.

Driving Force and Marcus Equation Calculations: Structural Flexibility of the RfBP Pocket to Bind Small Molecules. On the basis of the previously discussed steadystate and time-resolved data presented here, a photoinduced ET between QcH32+ and RfBP through a cofacial π-stack with Trp or Tyr is considered the primary mechanism of binding to form a CT state. However, the RfBP binding pocket has known dimensions of 20 Å × 15 Å and we suggest here that the binding site has flexibility for QcH32+ binding and interaction with amino acids based on a correlation of previously published work by others33,34,44 and by calculations presented here on the driving force, ΔG, and predicted ET rates for calculated charge separation (CS) and charge recombination (CR). Thus, our explanations and calculations presented below include QcH32+ interaction with both Trp and Tyr, since both are redox active and involved in the stacking interaction with RfBP.9,12 Although others have studied related FMN−RfBP and observed photoluminescence quenching in protein mutants with Trp primarily responsible for femtosecond quenching, replacement with Tyr does show ET but on slower time scales.45 First, we evaluated ΔG for ET by taking into account correction factors for the Coulombic, ΔGcoul, and solvent interaction, ΔGsolv, which has not been corrected or reported for CS or CR in previous ultrafast studies of cofactor−RfBP. The effective dielectric constant, εp, inside of hydrophobic pocket of 8295


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excited states from a vibronically coupled 1π−π* S1 state and dark 1n-π* state.58 Thus, flavin redox cofactors have the potential to participate in complex photophysics and photochemistry over many orders of magnitude in time, and these processes are not fully understood given the large number of flavin derivatives, binding proteins and environmental conditions that influence the dynamics inside proteins. Although the majority of ET experiments performed on flavin-binding proteins have reported rate constants, only recently have the electron spin states been evaluated with respect to the formation of triplet radical pair states,59 which are affected by external magnetic fields and have been previously studied mechanistically by us in D−B−A systems.52,60 Therefore, we performed nanosecond transient absorption (NsTA) experiments for QcH32+ and the QcH32+−RfBP complex in the absence of air to study the possibility of oxygen-sensitive reactions from ISC. The ultrafast kinetic traces for QcH32+−RfBP are noisier but the decay lifetimes are identical to those results we obtained in the presence of air. The NsTA experiments, shown in the Supporting Information, of QcH32+ and Rf for comparison, were conducted with and without air in pH 7.0 PBS to study the long-lived decay components because 1Rf* undergoes intersystem crossing to form 3Rf*. The GSB of Rf was observed at 445 nm and include three decay components in the presence of oxygen and two in the absence of oxygen. The shortest decay component was ∼6 ns and corresponds to the lifetime of 1Rf*. The second decay component was ∼3.6 μs, in the presence of oxygen and is the concentration dependent lifetime from annihilation, rather than the intrinsic 3Rf* decay. The NsTA results indicate that the excited state dynamics of QcH32+ decays through a singlet state and does not show oxygen effects. The GSB, SE, and ESA of QcH32+ were observed at 430, 520, and both 465 and 675 nm, respectively; all of them have a 3 ns decay corresponding to the lifetime of 1QcH32+*. In the case for drug−protein complexes, photosensitivity can lead to drug cytotoxicity by O2− formation as it does in the DM−RfBP complex. In that particular study, ET from Trp to DM was observed on the 1−6 ps time scale while the CR process was found to be sensitive to side reaction with inclusion oxygen, thus reducing the CR from >100 to 10 ps. Interestingly, both DM and Rf chemical structures contain carboxyl groups, known to form 1n-π* states, and we believe there is the possibility to form 3[DM•−−RfBP•+] or 3[Rf•−− RfBP•+] as intermediate states, which could explain the selective reactivity affecting only CR for DM, since previous studies suggest that water is not present in the protein pocket and not able to form chromophore-water adducts. In another study of Arabidopsis thaliana cryptochrome and flavin (FAD), the initial 1FAD* was quenched prior to the intersystem crossing (ISC), resulting in the formation of singlet radical pair states.59 Thus, the electron spin configuration is important, yet not widely studied or understood parameter for explaining chemical reactivity and photosensitivity of the photogenerated flavin-amino acid radical pairs. Finally, a comparison of the chemical structure for Rf and QcH32+ shows that the N-acridine redox active moiety in QcH32+ does not contain carbonyl groups and thus is not likely to produce a large spin−orbit effect for ISC, as we observed in our NsTA results, and no ROS or byproduct was observed in the steady-state or time-resolved studies after irradiating with ultrafast laser pulses for >12 h. However, in other studies of p-benzoquinone (BQ) and Trp in a model globular protein, photoinduced electron transfer resulted in the formation of a Trp byproduct, kynurenine

be significantly different. Yet, we observe a factor of 10−20× slower ET dynamics compared to Rf−RfBP. Thus, we can postulate a slight change in the binding pocket where Qc preferentially binds with Trp, which results in slower ET dynamics but maintains the same D−A interaction distance, as previously observed with FMN−RfBP. However, we have considered other contributions to the photoinduced ET lifetimes and these are discussed below and in the next section. The ΔG values and predicted lifetimes reported here for CS and CR provide an upper limit for a purely adiabatic system, where V ≫ kbT (200 cm−1), values for CS ∼ 100−250 fs and for CR ∼ 600−7300 fs for CR, for Trp and Tyr, respectively. Therefore, by adjusting VDA2 in this way, we can account for a weaker electronic coupling and binding for QcH32+. The lifetimes obtained in the strong coupling regime are similar to the femtosecond ET dynamics that have been observed34 experimentally for Rf−RfBP and for D. vulgaris flavodoxin44 for similar interaction distances of ∼4 Å. In the latter example, the short-range ET lifetimes were studied with 11 amino acid mutations at the active site, and rate constants appear to depend primarily on the redox potentials more than the minor changes in the mutated active site. Thus, the redox chemistry for Rf and QcH32+ inside the RfBP may not be identical. To understand the nature of the π stacking interaction, previously studied31−34,45 Rf−RfBP and DM−RfBP do not show red and blue shifts in the ground state absorption and photoluminescence, respectively, and our results for QcH32+ strongly suggest a new electronic interaction between Trp and Tyr, or other amino acids. Alternatively, a water molecule could be present inside the binding pocket. If a water inclusion molecule were present at the binding site, red-shifting of the absorption and emission bands is explained by a more polar binding pocket and could be estimated by adjusting the dielectric constant in the previous calculations. However, another explanation from recent studies on carbazole− carborane D−A chromophores55 may be a more plausible argument for the unusual shifts in the ground state electronic spectra. A similar red-shift in the UV−vis and blue-shift in the photoluminescence was observed because of a combination of the local excited (LE) state and charge-transfer (CT) emission from a π-bridge extension; the CT emission was also diminished when studied in polar solvents.55 Therefore, we can likely exclude the role of an inclusion water molecule in the binding pocket of the RfBP, as others have suggested,45 and we explain the blue-shift in the photoluminescence when QcH32+, initially in water at εs ∼ 80, binds inside the hydrophobic RfBP pocket with a much lower dielectric constant, εp ∼ 8.9, and raises the energy of the CT state. Role of Electron Spin States for Charge Separation and Charge Recombination. Recent literature suggests that Rf and other flavin analogues participate in complex ET that can influence the dynamics inside the protein from two primary mechanisms: (1) photoreaction in a triplet excited or charge transfer state via ISC of carbonyl containing chromophores that react with 3O2 to form reactive oxygen species (ROS) such as superoxide, O2−, and singlet oxygen, 1O2 that are responsible in the formation of a number of byproducts33 or (2) photoreaction of water-chromophore adducts that produce OH• or semiquinone ROS. For example, Rf has recently shown homogeneous proton coupled electron transfer reactions in solution56 and the isoalloxazine structure can undergo tautomerization to form the lactam and lactim forms upon binding to other substrates.57 In addition, Rf can form triplet 8296


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variety of ailments and is used in biomedical applications,18,20 the light-induced studies performed here are the first to explore their reactivity and safety with no expected cytotoxicity observed inside binding proteins. Thus, these studies could be useful for understanding the structure, function and regulation of flavin, and drugs similar in chemical structure, inside retbindin, Retb, a protein located near the rod photoreceptor in the retina that has recently shown to bind flavins.7

(Kyn), in 290 ps with new absorption and photoluminescence maxima.61 These oxidative modifications are possible through the generation of ROS that participate in the cleavage of the Trp indole ring,62 and new modeling of the photochemical reaction of BQ63 and N-acridine64 have suggested that water splitting plays a vital role. For our studies of QcH32+, an Nacridine derivative, deprotonation of the nitrogen would be required to form the water adduct and subsequent ROS, which is not the case for our results shown here at pH = 7.0. Thus, we exclude the possibility of ROS and byproduct formation and are able to assign the spin state of the one electron charge transfer as 1[QcH3_red•+−RfBP•+] that recombines spin-selectively to the singlet ground state.

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ASSOCIATED CONTENT

S Supporting Information *

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.7b05304. Steady-state absorption and photoluminescence quenching analysis, electrochemistry results, femtosecond and nanosecond transient absorption results, and driving force and Marcus equation calculations (PDF)

CONCLUSIONS Here, we have described the ultrafast ET dynamics between QcH32+ and the Tyr and Trp amino acids responsible for binding inside RfBP. Steady-state spectroscopy with Stern− Volmer analysis of the photoluminescence quenching determined a 1:1 binding ratio of the QcH32+−RfBP complex. The shifts in the ground state absorption and photoluminescence spectra of the QcH32+−RfBP complex is attributed to a combination of LE and CT states. More specifically, the blueshift in the emission is explained by an increase in energy of the CT state because of the decrease in dielectric constant inside the protein binding pocket. Electrochemistry and spectroelectrochemistry experiments performed on QcH32+ in DMSO were used to facilitate the spectroscopic identification of the ultrafast spectroscopy. Upon QcH32+ binding with RfBP, we observed lifetimes for 1[QcH3_red•+−RfBP•+] that are similar to other drug−RfBP dynamics and together are 10−20× slower than Rf−RfBP. We determined the driving force for ET and modeled the lifetimes using Marcus theory for CS and CR of singlet states, in absence of competing side photochemical reactions that influence CS and CR, and these results are in sufficient agreement with the experimental values we obtained through FsTA spectroscopy for a primary interaction between QcH32+ and Trp. The projected upper limit for ET is similar to those experimental results obtained for Rf−RfBP but modeled with larger electronic coupling to reflect tighter binding inside the pocket. Current work to isolate the photoinduced CT interactions between Qc−Tyr and Qc−Trp in host systems is in progress to further explain the dynamics of drugs binding in protein pockets in a biomimetic approach. Finally, it has been shown previously for DM−RfBP that oxygen sensitivity from either 1[DM•−−RfBP•+] or 3[DM•−− RfBP•+], are responsible for side reactions and directly influence CR lifetimes through photoinduced reactions to form ROS. Interestingly, Therian and co-workers have recently discovered that a decrease in dielectric constant after CS is responsible for slower CR in a de novo designed protein and future work in this field should simultaneously explore three primary factors affecting CS and CR dynamics: (1) dielectric changes in the protein pocket, (2) photochemical byproduct formation through ROS, and (3) electron spin dynamics.46 Thus, research over the past decade has shed new light on the factors that influence and ultimately control ultrafast CS and CR in cofactor-protein systems. To conclude, our results show that there is no oxygen dependence and we are able to assign the radical pair as a singlet state, 1[QcH3_red•+−RfBP•+], which is important for understanding the light-sensitive side reaction that may occur in the triplet manifold. Since Qc is one of many aminoacridines that have therapeutic and drug activity toward a

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AUTHOR INFORMATION

Corresponding Author

*(A.M.S.) E-mail: [email protected]. ORCID

Amy M. Scott: 0000-0002-3316-8315 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Science Foundation (CHE1560103).

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REFERENCES

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