Biochemical Establishment and Characterization of EncM’s Flavin-N5-oxide Cofactor

The ubiquitous flavin-dependent monooxygenases commonly catalyze oxygenation reactions by means of a transient C4a–peroxyflavin. A recent study, however, suggested an unprecedented flavin-oxygenating species, proposed as the flavin-N5-oxide (Fl<sub>N5[O]</sub>), as key to an oxidative Favorskii-type rearrangement in the biosynthesis of the bacterial polyketide antibiotic enterocin. This stable superoxidized flavin is covalently tethered to the enzyme EncM and converted into FADH<sub>2</sub> (Fl<sub>red</sub>) during substrate turnover. Subsequent reaction of Fl<sub>red</sub> with molecular oxygen restores the postulated Fl<sub>N5[O]</sub> via an unknown pathway. Here, we provide direct evidence for the Fl<sub>N5[O]</sub> species via isotope labeling, proteolytic digestion, and high-resolution tandem mass spectrometry of EncM. We propose that formation of this species occurs by hydrogen-transfer from Fl<sub>red</sub> to molecular oxygen, allowing radical coupling of the formed protonated superoxide and anionic flavin semiquinone at N5, before elimination of water affords the Fl<sub>N5[O]</sub> cofactor. Further biochemical and spectroscopic investigations reveal important features of the Fl<sub>N5[O]</sub> species and the EncM catalytic mechanism. We speculate that flavin-N5-oxides may be intermediates or catalytically active species in other flavoproteins that form the anionic semiquinone and promote access of oxygen to N5.