In this study, we aimed to establish the relationship betweeen microbioal adapaptation to alternative substrates, modification of enzyme sequence and structure, and the oxygenation efficiency of contaminant-degrading oxygenases. To that end, we studied Rieske non-heme ferrous iron oxygenases (RO), an important class of contaminant-degrading enzymes. Rieske oxygenases catalyze oxygenations and oxidative heteroatom dealkylations of a broad number of contaminants including carboxylated, nitrated, halogenated, as well as N -and O-alkylated (poly)aromatic structures. These substrate structures reflect the wide range of man-made chemicals such as pesticides, pharmaceuticals, industrial chemicals, and explosives. Despite the well-known role of Rieske oxygenases for biocatalysis, the factors that lead to successful substrate oxygenation are still largely elusive. Both, structural elements of the oxygenase such as substrate tunnels and flexible loops as well as electronic interactions in the active site between substrate and the non-heme Fe center appear critical for successful O 2 activation and substrate hydroxylation. Yet it remains unclear whether any of these structural and electronic factors are optimized in Rieske oxygenases as microorganisms adapt to alternative contaminants as primary substrates.