To gain insights into the highly conserved catalytic residue E158, we tested the variants E158A and E158D. E158A led to the expected loss of NBD activity. To retain the deprotonation of the peroxo-iron complex, we introduced an aspartate as a shortened amino acid. Astonishingly, variant E158D proved to be active with a decelerated but prolonged product formation. Whereas the MthUPO wildtype reached the maximum product formation after a few minutes, the variant E158D converted the substrate overnight yielding 1.5-fold improved TONs relative to the wildtype. This variant represents the first exchange of the catalytic glutamate residue while preserving the UPO activity. Computational analysis based on MD simulations revealed that the E158D mutation induces higher rigidity to the α-7-helix than the wildtype (Figs. S10 and S11), which might be responsible for its prolonged conversion. The 23.6-fold decreased kcat value of variant E158D (Tab. 2) indicates that the mutation has a severe impact on the ratelimiting step of the reaction. This impact is most likely related to the H2O2 activation via proton transfer steps that are mediated by this residue. To assess the influence of the peroxide species with these variants, we employed cumene hydroperoxide and tert-butyl hydroperoxide as oxygen source. These experiments revealed diminished activities using the alternative peroxides with the wildtype and E158D and no activity with E158A (Fig. ).