O₂ Activation and Enzymatic C-H Bond Activation Mediated by a Dimanganese Cofactor

Dioxygen (O₂) is a potent oxidant used by aerobic organisms for energy transduction and critical biosynthetic processes. Numerous metalloenzymes harness O₂ to mediate C-H bond hydroxylation reactions, but most commonly feature iron or copper ions in their active site cofactors. In contrast, many manganese-activated enzymes─such as glutamine synthetase and isocitrate lyase─perform redox neutral chemical transformations and very few are known to activate O₂ or C-H bonds. Here, we report that the dimanganese-metalated form of the cambialistic monooxygenase SfbO (Mn₂-SfbO) can efficiently mediate enzymatic C-H bond hydroxylation. The activity of the dimanganese form of SfbO toward substrate hydroxylation is comparable to that of its heterobimetallic Mn/Fe form but exhibits distinct kinetic profiles. Kinetic, spectroscopic, and structural studies invoke a mixed-valent dimanganese cofactor (Mn^IIMn^III) in O₂ activation and evidence a stoichiometric role for superoxide in maturing an O₂-inert Mn^II₂ cofactor. Computational studies support a hypothesis wherein superoxide addition to the Mn^II₂ cofactor installs a critical bridging hydroxide ligand that stabilizes higher-valent manganese oxidation states. These findings establish the viability of proteinaceous dimanganese cofactors in mediating complex, multistep redox transformations.