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

Through QM/MM modeling, we discovered that C(sp 3 )-H β-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 Fe IV 2 O 2 species, but alternatively renders a diferric Fe III 2 O 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.