Elucidation of the Fe(IV)=O intermediate in the catalytic cycle of the halogenase SyrB2

Abstract

Mononuclear non-haem iron (NHFe) enzymes catalyse a broad range of oxidative reactions, including halogenation, hydroxylation, ring closure, desaturation and aromatic ring cleavage reactions. They are involved in a number of biological processes, including phenylalanine metabolism, the production of neuro-transmitters, the hypoxic response and the biosynthesis of secondary metabolites(1-3). The reactive intermediate in the catalytic cycles of these enzymes is a high-spin S = 2 Fe(IV)=O species, which has been trapped for a number of NHFe enzymes(4-8), including the halogenase SyrB2 (syringomycin biosynthesis enzyme 2). Computational studies aimed at understanding the reactivity of this Fe(IV)=O intermediate(9-13) are limited in applicability owing to the paucity of experimental knowledge about its geometric and electronic structure. Synchrotron-based nuclear resonance vibrational spectroscopy (NRVS) is a sensitive and effective method that defines the dependence of the vibrational modes involving Fe on the nature of the Fe(IV)=O active site(14-16). Here we present NRVS structural characterization of the reactive Fe(IV)=O intermediate of a NHFe enzyme, namely the halogenase SyrB2 from the bacterium Pseudomonas syringae pv. syringae. This intermediate reacts via an initial hydrogen-atom abstraction step, performing subsequent halogenation of the native substrate or hydroxylation of non-native substrates(17). Acorrelation of the experimental NRVS data to electronic structure calculations indicates that the substrate directs the orientation of the Fe(IV)=O intermediate, presenting specific frontier molecular orbitals that can activate either selective halogenation or hydroxylation.