Flavin Charge Redistribution upon Optical Excitation Is Independent of Solvent Polarity.

Flavin absorption spectra encode molecular details of the flavin's local environment through coupling of local electric fields with the chromophore's charge redistribution upon optical excitation. Translating experimentally measured field-tuned transition energies to local electric field magnitudes and directions across a wide range of field magnitudes requires that the charge redistribution be independent of the local field. We have measured the charge redistribution upon optical excitation of the derivatized flavin TPARF in the non-hydrogen-bonding, nonpolar solvent toluene, with and without a tridentate hydrogen-bonding ligand, DBAP, using electronic Stark spectroscopy. These measurements were interpreted using TD-DFT finite field and difference density calculations. In comparing our present results to previous Stark spectroscopic analyses of flavin in more polar solvents, we conclude that flavin charge redistribution upon optical excitation is independent of solvent polarity, indicating that dependence of flavin transition energies on local field magnitude is linear with local field magnitude.

Unusual reactivity of a flavin in a bifurcating electron-transferring flavoprotein leads to flavin modification and a charge-transfer complex.

From the outset, canonical electron transferring flavoproteins (ETFs) earned a reputation for containing modified flavin. We now show that modification occurs in the recently recognized bifurcating (Bf) ETFs as well. In Bf ETFs, the 'electron transfer' (ET) flavin mediates single electron transfer via a stable anionic semiquinone state, akin to the FAD of canonical ETFs, whereas a second flavin mediates bifurcation (the Bf FAD). We demonstrate that the ET FAD undergoes transformation to two different modified flavins by a sequence of protein-catalyzed reactions that occurs specifically in the ET site, when the enzyme is maintained at pH 9 in an amine-based buffer. Our optical and mass spectrometric characterizations identify 8-formyl flavin early in the process and 8-amino flavins (8AFs) at later times. The latter have not previously been documented in an ETF to our knowledge. Mass spectrometry of flavin products formed in Tris or bis-tris-aminopropane solutions demonstrates that the source of the amine adduct is the buffer. Stepwise reduction of the 8AF demonstrates that it can explain a charge transfer band observed near 726 nm in Bf ETF, as a complex involving the hydroquinone state of the 8AF in the ET site with the oxidized state of unmodified flavin in the Bf site. This supports the possibility that Bf ETF can populate a conformation enabling direct electron transfer between its two flavins, as has been proposed for cofactors brought together in complexes between ETF and its partner proteins.

Stark Spectroscopy of Lumichrome: A Possible Candidate for Stand-Off Detection of Bacterial Quorum Sensing.

Lumichrome (7,8-dimethylalloxazine, LC) is a natural photodegradation product and catabolite of flavin coenzymes. Although not a coenzyme itself, LC is used for biosignaling in plants and single-celled organisms, including quorum sensing in the formation of biofilms. The noninvasive detection of in vivo lumichrome would be useful for monitoring this signaling event. For molecules that undergo significant charge redistribution upon light excitation (e.g., intramolecular charge transfer), there are optical detection methods (e.g., second-harmonic generation) that would be well suited to this task. Here, we have used Stark spectroscopy to measure the extent and direction of charge redistribution in photoexcited LC. Stark and low-temperature absorption spectra were obtained at 77 K on LC in ethanol glasses and analyzed using the Liptay analysis to obtain the difference dipole moments and polarizabilities. These data were complemented by a computational analysis of the excited states using density functional theory (DFT) at the TD-B3LYP/6-311+G(2d,p) level of theory.

Measuring electronic structure properties of flavins and flavoproteins by electronic Stark spectroscopy.

The optical spectrum of a flavoprotein is one of its signature properties. No two flavoprotein absorption spectra are exactly alike as each encodes the details of the interaction of the flavin cofactor electronic structure with the specific protein binding pocket. Electronic Stark spectroscopy has the potential to elucidate these interactions with high sensitivity, at low cost, and requiring minimal technical sophistication. In this chapter we will outline the theoretical basis for Stark spectroscopy and describe the construction of the Stark spectrometer. Step-by-step instructions are given for acquiring and interpreting Stark spectra to retrieve difference moments of the flavin ground versus excited state charge distributions.