We explored whether structural changes in the enzymes upon amino acid substitutions related to the observable changes in oxygenation efficiency from two perspectives. First, we studied if the observed point mutations affected substrate and O 2 transport from the protein surface to the active site. In addition, we evaluated changes in the binding of nitroaromatic substrates in the active site through molecular docking by constraining the docking modes, based on the interaction between Asn258 and the nitro group via hydrogen bonding. The H-bonding interaction with Asn258 is not only a known prerequisite for catalytic activity of nitroarene dioxygenases but could also modulates the ability of the enzyme to hold the substrate in place for efficient hydroxylation by reactive Fe-oxygen species. Following procedures used in previous studies to identify small-molecule tunnels in nonheme Fe enzymes, we characterized possible substrate and O 2 transport tunnels in wt 2NTDO and the five variants using CaverDock 112 with minimum probing radii of 0.9 and 0.6 Å for substrates and O 2 , respectively. In all homology models, tunnels from the catalytic nonheme Fe site to the surface were localized and could potentially be assigned as nitroaromatic substrate tunnels. Tunnel dimensions and throughput derived with CaverDock are compiled in Table and show lengths between 20 and 23 Å, as well as mean and bottleneck radii of 1.6 -1.9 Å and 1.1 -1.5 Å, respectively. Figures and show examples of substrate tunnels for 2NTDO, as well as variants L238V-M248I and I204A from the 4NT + and 3NT + experiments, respectively. As evident from this structural analysis (Figure ), I204 is located at the substrate tunnel whereas residues L238 and M248 are not. The complete list of amino acid residues in the various tunnels is compiled in Table . Evidence for I204 acting as a bottleneck residue was obtained from ligand transport analyses. Substrate binding energies calculated with CaverDock along the substrate tunnels are shown in Figure . These data indeed revealed lower substrate binding energies at approx. 12 Å distance from the non-heme Fe site through substitutions at the I204 residue.