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J. Phys. Chem. B 2008, 112, 13424–13432
Ultrafast Infrared Spectroscopy of Riboflavin: Dynamics, Electronic Structure, and Vibrational Mode Analysis Matthias M. N. Wolf, Christian Schumann, Ruth Gross, Tatiana Domratcheva,*,† and Rolf Diller* Fachbereich Physik, TU Kaiserslautern, D-67663 Kaiserslautern, Germany ReceiVed: May 13, 2008; ReVised Manuscript ReceiVed: August 8, 2008
Femtosecond time-resolved infrared spectroscopy was used to study the vibrational response of riboflavin in DMSO to photoexcitation at 387 nm. Vibrational cooling in the excited electronic state is observed and characterized by a time constant of 4.0 ( 0.1 ps. Its characteristic pattern of negative and positive IR difference signals allows the identification and determination of excited-state vibrational frequencies of riboflavin in the spectral region between 1100 and 1740 cm-1. Density functional theory (B3LYP), Hartree-Fock (HF) and configuration interaction singles (CIS) methods were employed to calculate the vibrational spectra of the electronic ground state and the first singlet excited ππ* state as well as respective electronic energies, structural parameters, electronic dipole moments and intrinsic force constants. The harmonic frequencies of the S1 excited state calculated by the CIS method are in satisfactory agreement with the observed band positions. There is a clear correspondence between computed ground- and excited-state vibrations. Major changes upon photoexcitation include the loss of the double bond between the C4a and N5 atoms, reflected in a downshift of related vibrations in the spectral region from 1450 to 1720 cm-1. Furthermore, the vibrational analysis reveals intra- and intermolecular hydrogen bonding of the riboflavin chromophore. 1. Introduction
SCHEME 1: Riboflavin
Flavin excited-state photochemistry regulates a number of important photobiological and photochemical processes. Among them are the initial steps of signal processing in the blue-light photoreceptors found in plants and other organisms, where flavins serve as chromophores. Prominent members of the class of flavins (7,8-dimethyl substituted isoalloxazines) are lumiflavin, riboflavin (vitamin B2, lactoflavin) (Scheme 1), flavin mononucleotide (FMN) and flavin-adenine-dinucleotide (FAD). In blue-light receptors, specific interactions between the protein and chromophore result in a broad manifold of excited-state processes. In light-oxygen-voltage (LOV) domains of the phototropin (Phot) receptors, the triplet FMN chromophore forms the covalent flavinsC(4a)-cysteinyl adduct.1,2 In bluelight-sensing-using-FAD (BLUF) proteins, formation of the singlet-exited flavin triggers the hydrogen-bond rearrangement in the core of the photoreceptor domain3-6 and possibly tautomerization of the Gln side chain.7,8 In cryptochromes, the photoexcited FAD cofactor undergoes one-electron reduction to semiquinone.9,10 Moreover, fluorescence of the flavin chromophores serves as a sensitive probe for the specific proteincofactor interaction, that is, as a marker for the chromophore environment. The understanding of these processes has been and still is subject of intense experimental and theoretical work requiring the study of simpler systems. To this end, photophysical and spectroscopic properties of riboflavin and analogues, such as the absorption and emission spectra, fluorescence lifetime and quantum yield of fluorescence and of triplet * To whom correspondence should be addressed. T.D.: E-mail,
[email protected]; phone, 49-6221-486-504; fax, 49-6221-486-585. R.D.: E-mail,
[email protected]; phone, 49631-205-2323; fax, 49-631-205-3902. † Max-Planck-Institut fu ¨ r medizinische Forschung, D-69120 Heidelberg, Germany.
formation etc. in various solvents, have been investigated in a number of studies (see refs 11 and 12 and references therein13-19). In this article we present a description of riboflavin in DMSO in terms of its electronic ground- and excited-state vibrational spectra and its vibrational dynamics on the picosecond time scale after photoexcitation in the UV. In addition, quantum chemical methods are applied to calculate the vibrational spectra of the electronic ground state and the singlet excited ππ* state as well as respective electronic energies, structural parameters, electronic dipole moments and intrinsic force constants. The characterized molecular response of riboflavin/DMSO to photoexcitation serves as a reference system for analogous transient IR experiments dealing with the primary processes of isoalloxazine-type chromophores either in a protein environment7 or in a solution environment.19 The results provide a basis for distinguishing chromophore vibrational modes from protein IR bands that are expected to be associated with the primary photochemistry. 2. Materials and Methods 2.1. Sample Preparation. Riboflavin (Sigma) was used as received and dissolved in dimethyl sulfoxide (DMSO, Fluka,
10.1021/jp804231c CCC: $40.75 2008 American Chemical Society Published on Web 09/27/2008
Ultrafast IR Spectroscopy of Riboflavin
J. Phys. Chem. B, Vol. 112, No. 42, 2008 13425
Figure 1. IR-difference spectra of riboflavin in DMSO (top) and DMSO-d6 (bottom) after photoexcitation at 387.5 nm for three different delay times. Note the different ordinate scaling left and right. Below 1350 cm-1, data are smoothed (3,5 SavitzkysGolay).
Figure 2. Absorbance transients of riboflavin/DMSO at selected wavenumbers. They show the fast kinetic component with a time constant τ1 ) 4 ( 0.1 ps as derived from a global fit.
99.8%,) at a concentration of 13 mM. A volume of ca. 200 µL of the solution was placed between two CaF2 windows (diameter 38.1 mm, thickness 2 mm) separated by a 200 µm Teflon spacer. To avoid strong infrared absorption of the solvent between 1300 and 1450 cm-1, DMSO-d6 (Euriso-top, 99.8%,
