How pH Modulates the Reactivity and Selectivity of a Siderophore-Associated Flavin Monooxygenase

[Image: see text] Flavin-containing monooxygenases (FMOs) catalyze the oxygenation of diverse organic molecules using O(2), NADPH, and the flavin adenine dinucleotide (FAD) cofactor. The fungal FMO SidA initiates peptidic siderophore biosynthesis via the highly selective hydroxylation of l-ornithine, while the related amino acid l-lysine is a potent effector of reaction uncoupling to generate H(2)O(2). We hypothesized that protonation states could critically influence both substrate-selective hydroxylation and H(2)O(2) release, and therefore undertook a study of SidA’s pH-dependent reaction kinetics. Consistent with other FMOs that stabilize a C4a-OO(H) intermediate, SidA’s reductive half reaction is pH independent. The rate constant for the formation of the reactive C4a-OO(H) intermediate from reduced SidA and O(2) is likewise independent of pH. However, the rate constants for C4a-OO(H) reactions, either to eliminate H(2)O(2) or to hydroxylate l-Orn, were strongly pH-dependent and influenced by the nature of the bound amino acid. Solvent kinetic isotope effects of 6.6 ± 0.3 and 1.9 ± 0.2 were measured for the C4a-OOH/H(2)O(2) conversion in the presence and absence of l-Lys, respectively. A model is proposed in which l-Lys accelerates H(2)O(2) release via an acid–base mechanism and where side-chain position determines whether H(2)O(2) or the hydroxylation product is observed.