Mechanistic Insights into the Dioxygen Activation and Hydroxylation of Aliphatic C–H Bonds Catalyzed by the Heterobimetallic Manganese–Iron Hydroxylase AibH1H2 from <i>Rhodococcus wratislaviensis</i>

The nonheme diiron enzymes usually employ the diiron cofactor to carry out the oxidative reactions; however, the monooxygenase AibH1H2, which represents the first documented Mn-dependent monooxygenase in biology, utilizes the Mn/Fe heterobimetallic cofactor to specifically catalyze the inert methyl hydroxylation of α-aminoisobutyric acid (Aib). To illuminate why AibH1H2 employs the heterobimetallic factor to activate the dioxygen and functionalize the strong aliphatic C-H bond, we constructed different reactant models and performed a series of quantum mechanical/molecular mechanical (QM/MM) calculations. Our calculations reveal that the heterobimetallic (Fe/Mn) active site of AibH1H2 forms a special coordinate structure in which the binding process of dioxygen can undergo an energetically favorable spin state crossing from triplet to singlet, resulting in the stable Fe(II)···O═O···Mn(II) complex, in which the O═O bond is significantly weakened with its bond length elongated from 1.21 to 1.39 Å, and the subsequent -O cleavage corresponds to an overall barrier of 13.6 kcal/mol. In the later hydroxylation process, the highly reactive Mn(II)-O complex is responsible for the hydrogen abstraction and OH rebound reactions. In addition, our calculations further indicated that the similar spin state crossing is energetically unfavorable for Fe(II)···O═O···Fe(II) and Mn(II)···O═O···Mn(II) complexes, and the following -O bond cleavage corresponds to higher overall energy barriers, which can well explain the weak catalytic activity of the homobimetallic cofactor of Fe/Fe and Mn/Mn. These findings reveal the importance of the coordinate architecture of the bimetallic enzymes and may provide useful information for the design of biocatalysts.