Delineating Ground to Excited State Vibrational Signatures of Anionic Flavin Adenine Dinucleotide in Aqueous Solution

Flavin adenine dinucleotide (FAD) is prevalent within numerous light-sensitive protein systems. One of the underexplored areas concerns the different protonation states of the fully oxidized FAD powering many photoreceptors. We implemented an integral electronic and vibrational spectroscopic toolset to investigate FAD at different pH conditions, and uncovered several distinct properties of the anionic FAD. Guided by femtosecond transient absorption (fs-TA) results, we performed wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS) from the ground to excited state to delineate vibrational signatures of a series of cationic, acidic, neutral, and anionic FAD in aqueous solution. The deprotonation of the flavin isoalloxazine polar ring moiety leads to notable electron density redistribution, enhancing the C4═O4' stretching motion while shifting the absorption band. After 400 and 480 nm excitations, the excited-state (ES)-FSRS of anionic FAD reveals a key vibrational peak around 1368 cm^-1 in S₁, which represents a 21 cm^-1 blueshift from the 1347 cm^-1 mode in S₀. Such a site-specific spectroscopic study of FAD in the physiologically-relevant water environment by tuning both pH and excitation wavelengths complements the current understanding of correlated FAD electronic and structural dynamics, better bridging the more characterized FAD redox behavior to fundamental insights into photophysical properties of this versatile biological cofactor.