SUMMARYMononuclear 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 FeIV=O species, which has been trapped for a number of NHFe enzymes4–8 including the halogenase SyrB2, the subject of this study. Computational studies to understand the reactivity of the enzymatic NHFe FeIV=O intermediate9–13 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 FeIV=O active site.14–16 Here we present the first NRVS structural characterisation of the reactive FeIV=O intermediate of a NHFe enzyme. This FeIV=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 FeIV=O intermediate, presenting specific frontier molecular orbitals (FMOs) which can activate the selective halogenation versus hydroxylation reactivity.

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[FeFe]-hydrogenases catalyze the reversible reduction of protons to molecular hydrogen with extremely high efficiency. The active site (“H-cluster”) consists of a [4Fe–4S]H cluster linked through a bridging cysteine to a [2Fe]H subsite coordinated by CN− and CO ligands featuring a dithiol-amine moiety that serves as proton shuttle between the protein proton channel and the catalytic distal iron site (Fed). Although there is broad consensus that an iron-bound terminal hydride species must occur in the catalytic mechanism, such a species has never been directly observed experimentally. Here, we present FTIR and nuclear resonance vibrational spectroscopy (NRVS) experiments in conjunction with density functional theory (DFT) calculations on an [FeFe]-hydrogenase variant lacking the amine proton shuttle which is stabilizing a putative hydride state. The NRVS spectra unequivocally show the bending modes of the terminal Fe–H species fully consistent with widely accepted models of the catalytic cycle.