Elucidation of the iron(IV)–oxo intermediate in the non-haem iron halogenase SyrB2

Mononuclear non-haem iron (NHFe) enzymes catalyse a wide variety of oxidative reactions including halogenation, hydroxylation, ring closure, desaturation, and aromatic ring cleavage. These are highly important for mammalian somatic processes such as phenylalanine metabolism, production of neurotransmitters, hypoxic response, and the biosynthesis of natural products.(1–3) The key reactive intermediate in the catalytic cycles of these enzymes is an S = 2 Fe(IV)=O species, which has been trapped for a number of NHFe enzymes(4–8) including the halogenase SyrB2, the subject of this study. Computational studies to understand the reactivity of the enzymatic NHFe Fe(IV)=O intermediate(9–13) are limited in applicability due to the paucity of experimental knowledge regarding its geometric and electronic structures, which determine its reactivity. Synchrotron-based nuclear resonance vibrational spectroscopy (NRVS) is a sensitive and effective method that defines the dependence of the vibrational modes of Fe on the nature of the Fe(IV)=O active site.(14–16) Here we present the first NRVS structural characterisation of the reactive Fe(IV)=O intermediate of a NHFe enzyme. This Fe(IV)=O intermediate reacts via an initial H-atom abstraction step, with its subsquent halogenation (native) or hydroxylation (non-native) rebound reactivity being dependent on the substrate.(17) A correlation of the experimental NRVS data to electronic structure calculations indicates that the substrate is able to direct the orientation of the Fe(IV)=O intermediate, presenting specific frontier molecular orbitals (FMOs) which can activate the selective halogenation versus hydroxylation reactivity.