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

Through QM/MM modeling, we discovered that C(sp )-H β-hydroxylation in the diiron hydroxylase CmlA transpires by traceless amine-assisted O activation. Different from the canonical diiron hydroxylase sMMO, this aliphatic-amine-assisted O activation avoids generating the high-valent diferryl Fe O species, but alternatively renders a diferric Fe O species as the reactive oxidant. From this unprecedented O activation mode, the derived C(sp )-H hydroxylation mechanism in CmlA also differs drastically from the toluene aromatic C(sp )-H hydroxylation in the diiron hydroxylase T4MO. This substrate-modulated O 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 binding in CmlA opens up more structural and mechanistic possibilities for O activation in non-heme diiron enzymes.