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

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.