LAUSR

Significance Flavoproteins are colored proteins involved in a large variety of biochemical reactions. They can perform photochemical reactions, which are increasingly exploited for bioengineering new protein-derived photocatalysts. In particular, light-induced reduction of the resting oxidized state of the flavin by close-lying amino acids or substrates is extensively studied. Here, we demonstrate that the reverse and previously unknown reaction photooxidation of the anionic semireduced flavin radical, a short-lived reaction intermediate in many biochemical reactions, efficiently occurs in flavoprotein oxidases. We anticipate that this finding will allow photoreduction of external reactants and lead to exploration of novel photocatalytic pathways. The photophysical properties of anionic semireduced flavin radicals are largely unknown despite their importance in numerous biochemical reactions. Here, we studied the photoproducts of these intrinsically unstable species in five different flavoprotein oxidases where they can be stabilized, including the well-characterized glucose oxidase. Using ultrafast absorption and fluorescence spectroscopy, we unexpectedly found that photoexcitation systematically results in the oxidation of protein-bound anionic flavin radicals on a time scale of less than ∼100 fs. The thus generated photoproducts decay back in the remarkably narrow 10- to 20-ps time range. Based on molecular dynamics and quantum mechanics computations, positively charged active-site histidine and arginine residues are proposed to be the electron acceptor candidates. Altogether, we established that, in addition to the commonly known and extensively studied photoreduction of oxidized flavins in flavoproteins, the reverse process (i.e., the photooxidation of anionic flavin radicals) can also occur. We propose that this process may constitute an excited-state deactivation pathway for protein-bound anionic flavin radicals in general. This hitherto undocumented photochemical reaction in flavoproteins further extends the family of flavin photocycles.