Ab initio QM/MM calculations show an intersystem crossing in the hydrogen abstraction step in dealkylation catalyzed by AlkB.

AlkB is a bacterial enzyme that catalyzes the dealkylation of alkylated DNA bases. The rate-limiting step is known to be the abstraction of an H atom from the alkyl group on the damaged base by a Fe(IV)-oxo species in the active site. We have used hybrid ab initio quantum mechanical/molecular mechanical methods to study this step in AlkB. Instead of forming an Fe(III)-oxyl radical from Fe(IV)-oxo near the C-H activation transition state, the reactant is found to be an Fe(III)-oxyl with an intermediate-spin Fe (S = 3/2) ferromagnetically coupled to the oxyl radical, which we explore in detail using molecular orbital and quantum topological analyses. The minimum energy pathway remains on the quintet surface, but there is a transition between (IS)Fe(III)-oxyl and the state with a high-spin Fe (S = 5/2) antiferromagnetically coupled to the oxyl radical. These findings provide clarity for the evolution of the well-known π and σ channels on the quintet surface in the enzyme environment. Additionally, an energy decomposition analysis reveals nine catalytically important residues for the C-H activation step, some of which are conserved in two human homologues. These conserved residues are proposed as targets for experimental mutagenesis studies.