Flavin Redox Chemistry Precedes Substrate Chlorination during the Reaction of the Flavin-Dependent Halogenase RebH †

The flavin-dependent halogenase RebH catalyzes chlorination at the C7 position of tryptophan as the initial step in the biosynthesis of the chemotherapeutic agent rebeccamycin. The reaction requires reduced FADH2 (provided by a partner flavin reductase), chloride ion, and oxygen as cosubstrates. Given the similarity of its sequence to those of flavoprotein monooxygenases and their common cosubstrate requirements, the reaction of FADH2 and O2 in the halogenase active site was presumed to form the typical FAD(C4a)−OOH intermediate observed in monooxygenase reactions. By using stopped-flow spectroscopy, formation of a FAD(C4a)−OOH intermediate was detected during the RebH reaction. This intermediate decayed to yield a FAD(C4a)−OH intermediate. The order of addition of FADH2 and O2 was critical for accumulation of the FAD(C4a)-OOH intermediate and for subsequent product formation, indicating that conformational dynamics may be important for protection of labile intermediates formed during the reaction. Format...