AbstractSiderophore A (SidA) from Aspergillus fumigatus is a flavin‐containing monooxygenase that hydroxylates ornithine (Orn) at the amino group of the side chain. Lysine (Lys) also binds to the active site of SidA; however, hydroxylation is not efficient and H2O2 is the main product. The effect of pH on steady‐state kinetic parameters was measured and the results were consistent with Orn binding with the side chain amino group in the neutral form. From the pH dependence on flavin oxidation in the absence of Orn, a pKa value >9 was determined and assigned to the FAD‐N5 atom. In the presence of Orn, the pH dependence displayed a pKa value of 6.7 ±0.1 and of 7.70 ±0.10 in the presence of Lys. Q102 interacts with NADPH and, upon mutation to alanine, leads to destabilization of the C4a‐hydroperoxyflavin (FADOOH). Flavin oxidation with Q102A showed a pKa value of ~8.0. The data are consistent with the pKa of the FAD N5‐atom being modulated to a value >9 in the absence of Orn, which aids in the stabilization of FADOOH. Changes in the FAD‐N5 environment lead to a decrease in the pKa value, which facilitates elimination of H2O2 or H2O. These findings are supported by solvent kinetic isotope effect experiments, which show that proton transfer from the FAD N5‐atom is rate limiting in the absence of a substrate, however, is significantly less rate limiting in the presence of Orn and or Lys.