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.

Since 1933, carbonic anhydrase research has focused on enzymes from mammals (␣ class) and plants (␤ class); however, two additional classes (␥ and ␦) were discovered recently. Cam, from the procaryote Methanosarcina thermophila, is the prototype of the ␥ class and the first carbonic anhydrase to be characterized from either an anaerobic organism or the Archaea domain. All of the enzymes characterized from the four classes have been purified aerobically and are reported to contain a catalytic zinc. Herein, we report the anaerobic reconstitution of apo- Research in the intervening years has shown that CA is one of the most widely distributed enzymes in nature (2, 3) and continues to be intensely investigated. Amino acid sequence comparisons identify four classes (␣, ␤, ␥, and ␦) of independent origins (4). Isozymes of the ␣ class are found in virtually all mammalian tissues where they function in diverse essential processes. The ␤ class is ubiquitous in plants and algae, where it is indispensable for the acquisition and concentration of CO 2 for photosynthesis. CA plays a role in the sequestration of atmospheric CO 2 in carbonates, and the global cycles of silicon and carbon are linked by CA in diatoms (5); thus, CA plays an important role in major geochemical and atmospheric processes. Members of the ␤ and ␥ classes are wide spread in physiologically diverse procaryotes from both the Bacteria and Archaea domains. Indeed, the genome of Escherichia coli contains two ␥ class homologs and two ␤ class homologs (2).Cam, from the procaryote Methanosarcina thermophila, is the prototype of the ␥ class and the first CA to be characterized from either an anaerobic organism or the Archaea domain (6). Sequence analyses approximate the evolution of the ␥ class at the estimated time of the origin of life (2). The crystal structure of Cam purified aerobically from E. coli reveals a homotrimer with a subunit fold composed of a left-handed ␤-helix motif followed by short and long ␣-helix structures (7). Each of the three active sites contain three histidines that coordinate a zinc ion. Two of the metal-binding histidines are donated by one monomer, and the third histidine from an adjacent monomer. Other residues in the active site of Cam are also donated from adjacent monomer faces and bear no resemblance to residues in the active site of the well characterized ␣ class CAs for which specific functions have been assigned.Kinetic investigations of the ␣ class CAs reveal a "zinc hydroxide" mechanism for catalysis (8) that also extends to both the ␤ and ␥ classes (9, 10). The overall enzyme-catalyzed reaction occurs in two mechanistically distinct steps,where E is enzyme, and B is buffer. The first step is the interconversion between CO 2 and bicarbonate (Equations 2 and 3) involving a nucleophilic attack of the zinc-bound hydroxyl on the CO 2 molecule. The second step is regeneration of the zinc-bound hydroxide, which involves intramolecular proton transfer from the zinc-bound water to a proton shuttle residue (Equation 4) and interm...

Binuclear non-heme iron enzymes activate O2 for diverse chemistries that include oxygenation of organic substrates and hydrogen atom abstraction. This process often involves the formation of peroxo-bridged biferric intermediates, only some of which can perform electrophilic reactions. To elucidate the geometric and electronic structural requirements to activate peroxo reactivity, the active peroxo intermediate in 4-aminobenzoate N-oxygenase (AurF) has been characterized spectroscopically and computationally. A magnetic circular dichroism study of reduced AurF shows that its electronic and geometric structures are poised to react rapidly with O2. Nuclear resonance vibrational spectroscopic definition of the peroxo intermediate formed in this reaction shows that the active intermediate has a protonated peroxo bridge. Density functional theory computations on the structure established here show that the protonation activates peroxide for electrophilic/single-electron-transfer reactivity. This activation of peroxide by protonation is likely also relevant to the reactive peroxo intermediates in other binuclear non-heme iron enzymes.

We have performed a systematic study of chemically possible peroxo-type intermediates occurring in the non-heme di-iron enzyme class Ia ribonucleotide reductase, using spectroscopically calibrated computational chemistry. Density functional computations of equilibrium structures, Fe-O and O-O stretch frequencies, Mossbauer isomer shifts, absorption spectra, J-coupling constants, electron affinities, and free energies of O(2) and proton or water binding are presented for a series of possible intermediates. The results enable structure-property correlations and a new rationale for the changes in carboxylate conformations occurring during the O(2) reaction of this class of non-heme iron enzymes. Our procedure identifies and characterizes various possible candidates for peroxo intermediates experimentally observed along the ribonucleotide reductase dioxygen activation reaction. The study explores how water or a proton can bind to the di-iron site of ribonucleotide reductase and facilitate changes that affect the electronic structure of the iron sites and activate the site for further reaction. Two potential reaction pathways are presented: one where water adds to Fe1 of the cis-mu-1,2 peroxo intermediate P causing opening of a bridging carboxylate to form intermediate P' that has an increased electron affinity and is activated for proton-coupled electron transfer to form the Fe(III)Fe(IV) intermediate X; and one that is more energetically favorable where the P to P' conversion involves addition of a proton to a terminal carboxylate ligand in the site which increases the electron affinity and triggers electron transfer to form X. Both pathways provide a mechanism for the activation of peroxy intermediates in binuclear non-heme iron enzymes for reactivity. The studies further show that water coordination can induce the conformational changes observed in crystal structures of the met state.