C(sp3)-H Hydroxylation in Diiron β-Hydroxylase CmlA Transpires by Amine-Assisted O2 Activation Avoiding Fe2IVO2 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.

Automated summary

Through QM/MM modeling, it was discovered that C(sp(3))-H beta-hydroxylation in the diiron hydroxylase CmlA occurs through traceless amine-assisted O-2 activation. This unique mode of O-2 activation generates a diferric (Fe2O2)-O-III species instead of the conventional diferryl (Fe22O2)-O-IV species, resulting in a different C(sp(3))-H hydroxylation mechanism compared to other diiron hydroxylases. This substrate-modulated O-2 activation in CmlA has significant implications for understanding the mechanism of other diiron hydroxylases with similar amine groups adjacent to C-H bonds. Furthermore, the adapted coordination environment of the diiron cofactor upon O-2 binding in CmlA opens up possibilities for O-2 activation in non-heme diiron enzymes.