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.

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.