Pyoverdines are important siderophores that guarantee iron supply to important pathogenic and non-pathogenic pseudomonads in host habitats. A key characteristic of all pyoverdines is the fluorescent dihydroxyquinoline group that contributes two ligands to the iron complexes. Pyoverdines are derived from the non-ribosomally synthesized peptide ferribactin, and their fluorophore is generated by periplasmic oxidation and cyclization reactions of d-tyrosine and l-diaminobutyric acid. The formation of the fluorophore is known to be driven by the periplasmic tyrosinase PvdP. Here we report that the putative periplasmic oxidoreductase PvdO of Pseudomonas fluorescens A506 is required for the final oxidation of dihydropyoverdine to pyoverdine, which completes the fluorophore. The pvdO deletion mutant accumulates dihydropyoverdine, and this phenotype is fully complemented by recombinant PvdO. The autoxidation of dihydropyoverdine at alkaline pH and the presence of high copper concentrations can mask this phenotype. Mutagenesis of conserved residues with potential catalytic function identified Glu-260 as an essential residue whose mutation abolished function without affecting stability or transport. Glu-260 of PvdO is at the exact position of the active-site cysteine in the structurally related formylglycine-generating enzyme. Evolution thus used the same protein fold for two distinct functionalities. As purified PvdO was inactive, additional factors are required for catalysis.