The same type of CaverDock analysis with a smaller probing radius enabled tentative assignment of O 2 transport tunnels (Figure ). While several tunnels were identified which could act as O 2 transport tunnels (see above), a common tunnel structure formed by multiple hydrophobic residues close to the substrate tunnel was identified in all variants. The archi- tecture of the identified tunnels (Table ) as defined by the mean and bottleneck radii and throughput numbers by CaverDock agrees well with similar analyses of O 2 tunnel structures in lipoxygenases, another class of non-heme ferrous iron enzymes. We found that residues L238 and M248 are part of O 2 transport tunnels identified for wt 2NTDO. However, these residues no longer appear as part of the tunnels that were identified as most likely in the evolved enzyme variants. By contrast, we observed a slightly increased O 2 tunnel radius and a reduced tunnel curvature for the most likely tunnel in the variants M248I and L238V-M248I in comparison to the wild-type. O 2 binding energies calculated along these O 2 transport tunnels (Figure ) are smaller than those for the substrates in substrate transport tunnels (Figure ). Moreover, the O 2 binding energy differences are not as pronounced among the variants carrying different point mutations. In contrast to substrate transport tunnels (Figure ), the bottlenecks for O 2 transport are located at a greater distance from the active site and thus in closer proximity to L238 and M248 residues.