C(sp(3))-H Hydroxylation in Diiron beta-Hydroxylase CmlA Transpires by Amine-Assisted O-2 Activation Avoiding (Fe2O2)-O-IV Species

Through QM/MM modeling, we discovered that C(sp(3))-H beta-hydroxylation in the diiron hydroxylase CmlA transpires by traceless amine-assisted O-2 activation. Different from the canonical diiron hydroxylase sMMO, this aliphatic-amine-assisted O-2 activation avoids generating the high-valent diferryl (Fe22O2)-O-IV species, but alternatively renders a diferric (Fe2O2)-O-III species as the reactive oxidant. From this unprecedented O-2 activation mode, the derived C(sp(3))-H hydroxylation mechanism in CmlA also differs drastically from the toluene aromatic C(sp(2))-H hydroxylation in the diiron hydroxylase T4MO. This substrate-modulated O-2 activation in CmlA has rich mechanistic implications for other diiron hydroxylases with an amine group adjacent to the C-H bond under hydroxylation in substrates, such as hDOHH. Furthermore, the adapted coordination environment of the diiron cofactor upon O-2 binding in CmlA opens up more structural and mechanistic possibilities for O-2 activation in non-heme diiron enzymes.