Ultrafast Vibrational Spectroscopy of the Flavin Chromophore

School of Chemical Sciences and Pharmacy, UniVersity of East Anglia, Norwich NR4 7TJ, U.K.,. Rutherford Appleton Laboratory, Central Laser Facility, C...
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20107

2006, 110, 20107-20110 Published on Web 09/23/2006

Ultrafast Vibrational Spectroscopy of the Flavin Chromophore Minako Kondo,† Je´ roˆ me Nappa,† Kate L. Ronayne,‡ Allison L. Stelling,§ Peter J. Tonge,§ and Stephen R. Meech*,† School of Chemical Sciences and Pharmacy, UniVersity of East Anglia, Norwich NR4 7TJ, U.K., Rutherford Appleton Laboratory, Central Laser Facility, CCLRC, Didcot, Oxon, OX11 0QX, U.K., and Department of Chemistry, Stony Brook UniVersity, Stony Brook, New York 11794-3400 ReceiVed: August 7, 2006; In Final Form: September 4, 2006

Ultrafast time-resolved infrared (TRIR) spectra of flavin adenine dinucleotide (FAD) and the anion of lumiflavin (Lf-) are described. Ground-state recovery and excited-state decay of FAD reveal a common dominant ultrafast relaxation and a minor slower component. The Lf- transient lacks a fast component. No intermediate species are observed, suggesting that the quenching mechanism is internal conversion promoted by interaction of the adenine and isoalloxazine rings in FAD. Modes are assigned, and the potential for extension of the TRIR method to photoactive proteins is discussed.

Introduction The flavin moiety plays an important role in a variety of biochemical processes, perhaps most significantly in redox chemistry.1 The photophysics and photochemistry of flavins are of particular significance in understanding the behavior of a range of blue light receptors, which play an important role in regulating photosynthesis and phototropism.2,3 For example, the light-oxygen-voltage (LOV) domains of the plant phototropins bind the chromophore flavin mononucleotide (FMN),4 while the flavin-adenine-dinucleotide (FAD) chromophore is bound by the BLUF (blue light using FAD) proteins found in a variety of organisms.5 In both cases, the photosensing capability relies upon the strong absorption of the isoalloxazine ring in the 400500 nm spectral region. In this letter, we describe an ultrafast time-resolved infrared (TRIR) study of the excited-state decay and ground-state recovery of the FAD chromophore and contrast it with that of the simpler Lf derivative (structures are shown in Figure 1). The excited-state chemistry of FAD has been studied intensely for a number of years. Weber and co-workers reported long ago that FAD in neutral aqueous solution had a quantum yield only 2% of that of FMN,6 indicating that the FAD adenine ring quenches the fluorescence of the isoalloxazine ring through an intramolecular mechanism (Figure 1).7 In a recent detailed study of the ultrafast fluorescence decay of FAD, an excited-state lifetime of 9 ps was reported and it was proposed on energetic grounds that the mechanism of quenching was electron transfer from adenine to the electronically excited isoalloxazine ring.8 Simulations show that these two rings have a high probability of close approach in aqueous solvents.9 In the following, we observe the FAD excited-state decay and ground-state recovery with vibrational resolution. These data provide new insights into the rate and mechanism of excited* Corresponding author. E-mail: [email protected]. † University of East Anglia. ‡ Rutherford Appleton Laboratory. § Stony Brook University.

10.1021/jp0650735 CCC: $33.50

Figure 1. Structures of FAD and Lf. The quaternary carbon atoms are labeled na (where n is the preceding atom number) in spectroscopic assignments.

state quenching in FAD and point the way to TRIR studies of the photoproteins themselves. Experimental Section The TRIR apparatus has been described in detail elsewhere.10 Excitation pulses at 400 nm with a repetition rate of 500 kHz had pulse widths of