Biofuel Production by Aldehyde-Deformylating Oxygenase: Insights into Spin States, Spin Coupling, and Diiron Cofactor Reactivity Using Multiscale Modeling

Understanding the mechanism of metalloenzymes, particularly multimetallic active sites, remains a significant challenge due to complex spin states, spin coupling, and spin-dependent reactivity. Nevertheless, such systems, exemplified by soluble Methane Monooxygenase (sMMO) and Photosystem II, demonstrate nature’s preference for multimetallic centers to perform chemically demanding transformations. Aldehyde-Deformylating Oxygenase (ADO) catalyzes an unusual redox-neutral, O 2 -dependent conversion of fatty aldehydes into alkanes and formate. Despite its superficial simplicity, the reaction mechanism involves cryptic redox steps, spin-state complexity, and a nonheme {Fe–Fe} diiron center similar to sMMO. Here, we elucidate the mechanism of ADO using multiscale QM/MM modeling combined with spectroscopic calculations. We reveal an unusual μ-peroxo Fe 2 (III/III) intermediate with asymmetric μ-η 1:η 2 binding, with antiferromagnetic Fe–Fe coupling that facilitates nucleophilic attack on the aldehyde carbonyl, lowering the activation barrier. An energetics–spectroscopy approach, adapted here, shows excellent agreement, validating the proposed intermediates. The formation of a thermodynamically stable peroxyhemiacetal necessitates the use of external reducing agents for turnover catalysis. Energetic analysis of all plausible metal combinations with Fe and Mn confirms that only the diiron configuration supports efficient catalysis, providing strong theoretical support for the native cofactor. These insights resolve the ADO mechanism and provide design principles for diiron enzymes, advancing biofuel production.