The ActVA-ActVB system from Streptomyces coelicolor is a two-component flavin-dependent monooxygenase involved in the antibiotic actinorhodin biosynthesis. ActVB is a NADH:flavin oxidoreductase that provides a reduced FMN to ActVA, the monooxygenase that catalyzes the hydroxylation of dihydrokalafungin, the precursor of actinorhodin. In this work, using stopped-flow spectrophotometry, we investigated the mechanism of hydroxylation of dihydrokalafungin catalyzed by ActVA and that of the reduced FMN transfer from ActVB to ActVA. Our results show that the hydroxylation mechanism proceeds with the participation of two different reaction intermediates in ActVA active site. , 7), in the utilization of sulfur from aliphatic sulfonates in E. coli (8), and in the desulfurization of fossil fuels by Rhodococcus species (9), as well as the hydroxylation of 4-hydroxyphenylacetate in Acinetobacter baumannii (10). We have been investigating the mechanism of the FMN-dependent twocomponent enzyme system, ActVA-ActVB, which participates in the last steps of the biosynthesis of the antibiotic actinorhodin in Streptomyces coelicolor (11-13) (Scheme 1).The first enzyme of the two-component flavin-dependent oxygenases is a NAD(P)H:flavin oxidoreductase that catalyzes the reduction of free oxidized flavins (FAD and/or FMN) by the reduced pyridine nucleotides, NADPH or NADH (3,[14][15][16]. The second enzyme is an oxygenase that binds the resulting free reduced flavin and promotes its reaction with O 2 to hydroxylate the substrate (2,3,(5)(6)(7)(8)(9)(10)17). Therefore, in contrast to the single-component flavin hydroxylases, the two-component sys-* This work was supported by National Institutes of Health Grant GM64711 (to D. P. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom correspondence may be addressed.