A "how-to" guide to the stark spectroscopy of flavins and flavoproteins

Flavins and flavoproteins have been studied by a plethora of spectroscopic techniques. Beginning with the characterization of DNA photolyases and the discovery of the diversity of roles played by excited-state flavins in photobiology, the characterization of the electronic excited state of flavins has become increasingly important. In this protocol, we provide a guide to using Stark spectroscopy in obtaining the degree of electronic charge redistribution in simple flavins and in flavoproteins. Stark spectroscopy is technically simpler than more common approaches used to explore the structure of the excited state, considerably cheaper to implement, and yet very powerful in its scope. At the end of this guide, we present data taken on non-photobiological flavoproteins, glutathione reductase and lipoamide dehydrogenase, that suggest that Stark spectroscopy is a unique way to elucidate the electrostatic environment that the flavin cofactor experiences bound inside the protein.