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Nitration of Tyrosine Channels Photoenergy Through A Conical Intersection in Water Longteng Tang, and Chong Fang J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.9b03464 • Publication Date (Web): 16 May 2019 Downloaded from http://pubs.acs.org on May 18, 2019
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Article for J. Phys. Chem. B (2019)
Nitration of Tyrosine Channels Photoenergy Through A Conical Intersection in Water
Longteng Tang and Chong Fang*
Department of Chemistry, Oregon State University, 153 Gilbert Hall, Corvallis, Oregon 973314003, United States
To whom correspondence should be addressed: E-mail:
[email protected] 1
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ABSTRACT. Nitration of tyrosine occurs under oxidative stress in vivo. The product, 3nitrotyrosine (3NY), has a dramatically decreased quantum yield and can be used as a molecular ruler. In this study, femtosecond transient absorption spectroscopy and quantum calculations were implemented to elucidate the photoinduced relaxation processes of anionic 3NY in water. Upon 400-nm excitation into an excited electronic state with notable charge-transfer (CT) character, a barrierless nitro-twisting motion rapidly (98% HPLC) and used without further purification. It was dissolved in pH=9 or 3 sodium phosphate buffer solution for spectral characterization, and the absorption spectra were measured by a Thermo Scientific Evolution 201 UV-visible (UV-Vis) spectrometer at room temperature. The fluorescence spectra with various excitation wavelengths were collected by a high-sensitivity Shimadzu RF-6000 fluorescence spectrophotometer with sample solution in a 1-cm-pathlength quartz fluorometer cell (3/Q/10, Starna Cells, Inc.). The fluorescence quantum yield of 3NY in basic solution was measured with a relative method that involves comparison of fluorescence intensity to a standard fluorescent dye in a similar absorption and emission range (the excitation/emission bandwidth was set at 3.0/3.0 nm with “low” sensitivity to avoid signal saturation from the highly fluorescent dye).28 For the excitation-dependent fluorescence detection of the extremely dim 3NY sample, we selected the excitation/emission bandwidth of 5.0/5.0 nm with “high” sensitivity to amplify the signal for comparison with the time-dependent data below. II.II. Femtosecond Transient Absorption (fs-TA) Spectroscopy. A ~40 fs, 400 nm pulse was used as the actinic pump for our fs-TA experiment. It was generated from the second harmonic of the 35 fs, 800 nm fundamental laser pulse (from a Legend Elite-USP-1K-HE regenerative amplifier system, Coherent, Inc.), followed by temporal compression through a prism pair (Suprasil-1, CVI Melles Griot).26,29,30 The pump power was then reduced to ~0.4 mW via a neutral density filter before an optical chopper operating at 500 Hz (synchronized with the laser repetition rate at 1 kHz). A broadband supercontinuum white light serves as the probe pulse, which was obtained by focusing a portion of the 800 nm fundamental pulse onto deionized water housed in a 2-mm-thick quartz cell (1-Q-2, Starna Cells), then compressed via a chirped mirror pair (DCM12, for the spectral range 400—700 nm, Laser Quantum, Inc.). The transmitted probe beam was dispersed by a reflective grating (300 grooves/mm with 300 nm blaze wavelength, i3-030-300-P)
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in an IsoPlane SCT-320 imaging spectrograph (Princeton Instruments, Inc.) equipped with a 1340´100, 20´20 µm size CCD array camera (PIXIS:100F front-illuminated CCD with Lumogen UV coating). During spectral data collection, the sample solution in a 1-mm-thick quartz cell was constantly stirred by a magnetic stir bar cut from a metal staple. During the subsequent data processing, coherent artifacts caused by the overlap between two strong and compressed fs pump and probe pulses near time zero27 were removed by polynomial fitting. We also performed a series of TA measurements for the anionic 3NY in basic aqueous solution (pH=9) with an OD of 0.4 – 1.2 per mm at 400 nm (our actinic pump wavelength) and the pump power of 0.3 – 0.5 mW. We found that a combination of OD at 1.0 and pump power of 0.4 mW yielded the highest TA signal intensity while avoiding potential issues related to higher sample concentration or higher power. For experimental error estimation of the lifetimes reported in this work (see Figures 2 and 3 below), we collected the 3NY in basic water solution for five times and with additional glycerol for three times using retweaked and reoptimized optical setups in the lab on different days. The corresponding error estimates (1 s.d., N=5 in water and N=3 in water-glycerol mixture) are provided below in the results and discussion section, substantiating the reproducibility of spectral data and robustness of least-squares fitting of the dynamic traces of fs-TA data. II.III. Quantum Calculations. The GS and ES calculations of the anionic 3NY chromophore (with a total charge of -2 at pH=9 aqueous solution, see the relevant pKa values in Figure S1 inset) were performed using DFT method with B3LYP functional as well as time-dependent (TD)-DFT method with the PBE0 functional (PBE1PBE) using Gaussian 09 software,31 respectively. The PBE0 hybrid functional mixes 25% Hartree-Fock exchange energy, 75% Perdew–BurkeErnzerhof (PBE) exchange energy, and full PBE correlation energy, which was shown to provide rather accurate excited state energies, singlet and triplet state ordering, and vertical absorption
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spectra of solute molecules in solution.15,16,20,32,33 The 6-311++G(d,p) basis sets and water solvent (bulk effect with the polarizable continuum model using the integral equation formalism variant, termed as the IEFPCM method) were chosen for all calculations with default solvent parameters provided in Gaussian 09. For the optimization calculations on GS and ES, the solvent effect was modeled by the equilibrium IEFPCM with the self-consistent reaction field (SCRF) method. For the energy calculations only on the ES, the electrostatic non-equilibrium (NE)–IEFPCM was implemented in Gaussian as default. The nitroaromatic twisting angle was set as the main nuclear coordinate to scan the GS and ES potential energies of anionic 3NY due to the flexibility and importance of the –NO2 internal rotation angle (being a nontotally symmetric mode, which could receive energy from the impulsively excited totally symmetric modes that drive the system out of the initial Franck-Condon region) as reported and validated in literature.14-17,19-22,25,34-37 For additional control, we also performed the same-level calculations on the anionic 3NY with a protonated aamine group so the total charge of the compound is -1 (see additional details in Figure S6).
III. Results and Discussion III.I. Nitration Modifies the Electronic Structure of Tyrosine. Natural tyrosine absorbs in the UV region at ~275 nm and emits at 304 nm with a fluorescence quantum yield of 0.14±0.01 as measured in pH=6 water at room temperature (23 ºC).38 Addition of a strong electron-withdrawing nitro group on the aromatic ring leads to a notably red-shifted absorption band in the near-visible region while decreasing the pKa of the phenolic OH group to ~7, which can be considered as the electron donor site especially after chromophore deprotonation.13,39,40 In acidic (pH=3) aqueous solution, neutral 3NY absorbs strongly at 356 nm (Figure 1). In basic (pH=9) aqueous solution, the visible absorption band of 3NY is centered around 420 nm with a much enhanced intensity 8
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ratio between the visible absorption band and the UV absorption band when compared to the neutral 3NY case.1 Considering the sample concentration of ~1 mM and using the Beer-Lambert law, we estimated the extinction coefficient (molar absorption coefficient) of neutral 3NY in pH=3 aqueous solution at 356 nm peak (see Figure 1) to be ~2900 M-1·cm-1, while the value for the anionic 3NY in pH=9 aqueous solution at 423 nm peak is ~4500 M-1·cm-1 that largely matches the reported value of ~4200 M-1·cm-1 by measuring the nitrotyrosine absorbance at 428 nm in pure proteins under alkaline conditions.1 The fluorescence quantum yield of 3NY also decreases drastically, and in particular, we measured it to be
