Comparing ultrafast excited state quenching of flavin 1,N6-ethenoadenine dinucleotide and flavin adenine dinucleotide by optical spectroscopy and DFT calculations

Abstract

Flavins are photoenzymatic cofactors often exploiting the absorption of light to energize photoinduced redox chemistry in a variety of contexts. Both flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) are used for this function. The study of these photoenzymes has been facilitated using flavin analogs. Most of these analogs involve modification of the flavin ring, and there is recent evidence that adenine (Ade)-modified FAD can affect enzyme turnover, but so far this has only been shown for enzymes where the adenine and flavin rings are close to each other in a stacked conformation. FAD is also stacked in aqueous solution, and its photodynamics are quite different from unstacked FAD or FMN. Oxidized photoexcited FAD decays rapidly, presumably through PET with Ade as donor and Fl* as acceptor. Definitive identification of the spectral signatures of Ade •+ and Fl •− radicals is elusive. Here we use the FAD analog Flavin 1,N 6 -Ethenoadenine Dinucleotide (εFAD) to study how different photochemical outcomes depend on the identity of the Ade moiety in stacked FAD and its analog εFAD. We have used UV-Vis transient absorption spectroscopy complemented by TD-DFT calculations to investigate the excited state evolution of the flavins. In FAD*, no radicals were observed, suggesting that FAD* does not undergo PET. εFAD* kinetics showed a broad absorption band that suggests a charge transfer state exists upon photoexcitation with evidence for radical pair formation. Surprisingly, significant triplet flavin was produced from εFAD* We hypothesize that the dipolar (ε)Ade moieties differentially modulate the singlet-triplet energy gap, resulting in different intersystem crossing rates. The additional electron density on the etheno group of εFAD supplies better orbital overlap with the flavin S 1 state, accelerating charge transfer in that molecule.

Determining Excited-State Absorption Properties of a Quinoid Flavin by Polarization-Resolved Transient Spectroscopy

“…The absorption spectrum of 1 exhibits two broad absorption features in the UV/vis range, with peaks at 440 nm (22,727 cm –1 , S 0 → S 1 ) and 330 nm (30,303 cm –1 , S 0 → S 3 ). There is evidence for an optically dark n π* state (S 2 ) between these two bands. ,, The shape and central wavelength of the two absorption peaks are in agreement with previous works. ,− The determined molar extinction coefficients of 10,000 and 8000 M –1 cm –1 are in good agreement with literature values for flavin in aprotic solvents. , The 1300 cm –1 vibronic progression observed in both absorption and emission corresponds largely to C–C and C–N stretching and bending vibrations of the isoalloxazine ring . The fluorescence spectrum peaks around 530 nm (18,868 cm –1 ) and shows good mirror image symmetry with the absorption.…”

Electronic Structure Methods for Simulating Flavin’s Spectroscopy and Photophysics: Comparison of Multi-reference, TD-DFT, and Single-Reference Wave Function Methods

“…Multiple computational studies have investigated the excited-state electronic structure and photophysics of flavin. A nonexhaustive list includes refs , – , – . A more comprehensive list of computational studies can be found in a recent review article by Kar, Miller, and Mroginski .…”

“…Several methods, including semiempirical, TD-DFT, and multireference (often, MR-PT2) methods have been used already to calculate the excited-state properties of flavins. ,− ,− For multireference calculations, the issue of active space selection is a recurring theme and a decision is often made that balances computational cost and computational accuracy due to the steep increase in computing time with the size of the active space. Automated protocols to select the active space have been developed by several research groups. ,− Sayfutyarova and Hammes-Schiffer applied their automated protocol that is based on Hückel theory for the selection of a suitable active space for flavin’s π-conjugated orbitals .…”

Importance of Polarizable Embedding for Absorption Spectrum Calculations of Arabidopsis thaliana Cryptochrome 1

“…The light absorbing characteristics of FAD have been extensively documented in multiple proteins through computational simulations − and empirical methods. ,− Notably, transient absorption spectroscopy has elucidated the absorption attributes of FAD’s flavin moiety. , However, comprehensive studies regarding other redox states of FAD remain rare. , The semiquinone radical (FADH • ) state is particularly intriguing due to its association with the prolonged signaling phase in specific proteins, like cryptochromes, which play a pivotal role in circadian rhythm modulation and magnetoreception. , …”