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

Abstract

Flavin-containing monooxygenases (FMOs) catalyze the oxygenation of diverse organic molecules using O₂, 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 H₂O₂. We hypothesized that protonation states could critically influence both substrate-selective hydroxylation and H₂O₂ 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 O₂ is likewise independent of pH. However, the rate constants for C4a-OO­(H) reactions, either to eliminate H₂O₂ 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/H₂O₂ conversion in the presence and absence of l-Lys, respectively. A model is proposed in which l-Lys accelerates H₂O₂ release via an acid–base mechanism and where side-chain position determines whether H₂O₂ or the hydroxylation product is observed