Electronic Structure of the Ferryl Intermediate in the α‑Ketoglutarate Dependent Non-Heme Iron Halogenase SyrB2: Contributions to H Atom Abstraction Reactivity

Low temperature magnetic circular dichroism (LT MCD) spectroscopy in combination with quantum-chemical calculations are used to define the electronic structure associated with the geometric structure of the Fe^IVO intermediate in SyrB2 that was previously determined by nuclear resonance vibrational spectroscopy. These studies elucidate key frontier molecular orbitals (FMOs) and their contribution to H atom abstraction reactivity. The VT MCD spectra of the enzymatic <i>S</i> = 2 Fe^IVO intermediate with Br^– ligation contain information-rich features that largely parallel the corresponding spectra of the <i>S</i> = 2 model complex (TMG₃tren)­Fe^IVO (Srnec, M.; Wong, S. D.; England, J.; Que, L. Jr.; Solomon, E. I. <i>Proc. Natl. Acad. Sci. USA</i> <b>2012</b>, <i>109</i>, 14326–14331). However, quantitative differences are observed that correlate with π-anisotropy and oxo donor strength that perturb FMOs and affect reactivity. Due to π-anisotropy, the Fe^IVO active site exhibits enhanced reactivity in the direction of the substrate cavity that proceeds through a π-channel that is controlled by perpendicular orientation of the substrate C–H bond relative to the halide–Fe^IVO plane. Also, the increased intrinsic reactivity of the SyrB2 intermediate relative to the ferryl model complex is correlated to a higher oxyl character of the Fe^IVO at the transition states resulting from the weaker ligand field of the halogenase

Evidence That the β Subunit of <i>Chlamydia trachomatis</i> Ribonucleotide Reductase Is Active with the Manganese Ion of Its Manganese(IV)/Iron(III) Cofactor in Site 1

The reaction of a class I ribonucleotide reductase (RNR) begins when a cofactor in the β subunit oxidizes a cysteine residue ∼35 Å away in the α subunit, generating a thiyl radical. In the class Ic enzyme from <i>Chlamydia trachomatis</i> (<i>Ct</i>), the cysteine oxidant is the Mn^IV ion of a Mn^IV/Fe^III cluster, which assembles in a reaction between O₂ and the Mn^II/Fe^II complex of β. The heterodinuclear nature of the cofactor raises the question of which site, 1 or 2, contains the Mn^IV ion. Because site 1 is closer to the conserved location of the cysteine-oxidizing tyrosyl radical of class Ia and Ib RNRs, we suggested that the Mn^IV ion most likely resides in this site (i.e., ¹Mn^IV/²Fe^III), but a subsequent computational study favored its occupation of site 2 (¹Fe^III/²Mn^IV). In this work, we have sought to resolve the location of the Mn^IV ion in <i>Ct</i> RNR-β by correlating X-ray crystallographic anomalous scattering intensities with catalytic activity for samples of the protein reconstituted <i>in vitro</i> by two different procedures. In samples containing primarily Mn^IV/Fe^III clusters, Mn preferentially occupies site 1, but some anomalous scattering from site 2 is observed, implying that both ¹Mn^II/²Fe^II and ¹Fe^II/²Mn^II complexes are competent to react with O₂ to produce the corresponding oxidized states. However, with diminished Mn^II loading in the reconstitution, there is no evidence for Mn occupancy of site 2, and the greater activity of these “low-Mn” samples on a per-Mn basis implies that the ¹Mn^IV/²Fe^III-β is at least the more active of the two oxidized forms and may be the only active form

Vanadyl as a Stable Structural Mimic of Reactive Ferryl Intermediates in Mononuclear Nonheme-Iron Enzymes

The iron­(II)- and 2-(oxo)­glutarate-dependent (Fe/2OG) oxygenases catalyze an array of challenging transformations via a common iron­(IV)-oxo (ferryl) intermediate, which in most cases abstracts hydrogen (H•) from an aliphatic carbon of the substrate. Although it has been shown that the relative disposition of the Fe–O and C–H bonds can control the rate of H• abstraction and fate of the resultant substrate radical, there remains a paucity of structural information on the actual ferryl states, owing to their high reactivity. We demonstrate here that the stable vanadyl ion [(V^IV-oxo)²⁺] binds along with 2OG or its decarboxylation product, succinate, in the active site of two different Fe/2OG enzymes to faithfully mimic their transient ferryl states. Both ferryl and vanadyl complexes of the Fe/2OG halogenase, SyrB2, remain stably bound to its carrier protein substrate (l-aminoacyl-<i>S</i>-SyrB1), whereas the corresponding complexes harboring transition metals (Fe, Mn) in lower oxidation states dissociate. In the well-studied taurine:2OG dioxygenase (TauD), the disposition of the substrate C–H bond relative to the vanadyl ion defined by pulse electron paramagnetic resonance methods is consistent with the crystal structure of the reactant complex and computational models of the ferryl state. Vanadyl substitution may thus afford access to structural details of the key ferryl intermediates in this important enzyme class

Evidence for Only Oxygenative Cleavage of Aldehydes to Alk(a/e)nes and Formate by Cyanobacterial Aldehyde Decarbonylases

Cyanobacterial aldehyde decarbonylases (ADs) catalyze the conversion of C_<i>n</i> fatty aldehydes to formate (HCO₂^–) and the corresponding C_<i>n</i>‑1 alk­(a/e)­nes. Previous studies of the <i>Nostoc punctiforme</i> (<i>Np</i>) AD produced in <i>Escherichia coli</i> (<i>Ec</i>) showed that this apparently hydrolytic reaction is actually a cryptically redox oxygenation process, in which one O-atom is incorporated from O₂ into formate and a protein-based reducing system (NADPH, ferredoxin, and ferredoxin reductase; N/F/FR) provides all four electrons needed for the complete reduction of O₂. Two subsequent publications by Marsh and co-workers [Das, et al. (2011) Angew. Chem. Int. Ed. 50, 7148−7152; Eser, et al. (2011) Biochemistry 50, 10743–10750] reported that their <i>Ec</i>-expressed <i>Np</i> and <i>Prochlorococcus marinus</i> (<i>Pm</i>) AD preparations transform aldehydes to the same products more rapidly by an O₂-independent, truly hydrolytic process, which they suggested proceeded by transient substrate reduction with obligatory participation by the reducing system (they used a chemical system, NADH and phenazine methosulfate; N/PMS). To resolve this discrepancy, we re-examined our preparations of both AD orthologues by a combination of (i) activity assays in the presence and absence of O₂ and (ii) ¹⁸O₂ and H₂¹⁸O isotope-tracer experiments with <i>direct</i> mass-spectrometric detection of the HCO₂^– product. For multiple combinations of the AD orthologue (<i>Np</i> and <i>Pm</i>), reducing system (protein-based and chemical), and substrate (<i>n</i>-heptanal and <i>n</i>-octadecanal), our preparations strictly require O₂ for activity and do not support detectable hydrolytic formate production, despite having catalytic activities similar to or greater than those reported by Marsh and co-workers. Our results, especially of the ¹⁸O-tracer experiments, suggest that the activity observed by Marsh and co-workers could have arisen from contaminating O₂ in their assays. The definitive reaffirmation of the oxygenative nature of the reaction implies that the enzyme, initially designated as aldehyde decarbonylase when the C1-derived coproduct was thought to be carbon monoxide rather than formate, should be redesignated as aldehyde-deformylating oxygenase (ADO)

Circular Dichroism, Magnetic Circular Dichroism, and Variable Temperature Variable Field Magnetic Circular Dichroism Studies of Biferrous and Mixed-Valent <i>myo</i>-Inositol Oxygenase: Insights into Substrate Activation of O₂ Reactivity

<i>myo</i>-Inositol oxygenase (MIOX) catalyzes the 4e^– oxidation of <i>myo</i>-inositol (MI) to d-glucuronate using a substrate activated Fe­(II)­Fe­(III) site. The biferrous and Fe­(II)­Fe­(III) forms of MIOX were studied with circular dichroism (CD), magnetic circular dichroism (MCD), and variable temperature variable field (VTVH) MCD spectroscopies. The MCD spectrum of biferrous MIOX shows two ligand field (LF) transitions near 10000 cm^–1, split by ∼2000 cm^–1, characteristic of six coordinate (6C) Fe­(II) sites, indicating that the modest reactivity of the biferrous form toward O₂ can be attributed to the saturated coordination of both irons. Upon oxidation to the Fe­(II)­Fe­(III) state, MIOX shows two LF transitions in the ∼10000 cm^–1 region, again implying a coordinatively saturated Fe­(II) site. Upon MI binding, these split in energy to 5200 and 11200 cm^–1, showing that MI binding causes the Fe­(II) to become coordinatively unsaturated. VTVH MCD magnetization curves of unbound and MI-bound Fe­(II)­Fe­(III) forms show that upon substrate binding, the isotherms become more nested, requiring that the exchange coupling and ferrous zero-field splitting (ZFS) both decrease in magnitude. These results imply that MI binds to the ferric site, weakening the Fe­(III)−μ-OH bond and strengthening the Fe­(II)−μ-OH bond. This perturbation results in the release of a coordinated water from the Fe­(II) that enables its O₂ activation

Direct Measurement of the Radical Translocation Distance in the Class I Ribonucleotide Reductase from <i>Chlamydia trachomatis</i>

Ribonucleotide reductases (RNRs) catalyze conversion of ribonucleotides to deoxyribonucleotides in all organisms via a free-radical mechanism that is essentially conserved. In class I RNRs, the reaction is initiated and terminated by radical translocation (RT) between the α and β subunits. In the class Ic RNR from <i>Chlamydia trachomatis</i> (<i>Ct</i> RNR), the initiating event converts the active <i>S</i> = 1 Mn­(IV)/Fe­(III) cofactor to the <i>S</i> = 1/2 Mn(III)/Fe(III) “RT-product” form in the β subunit and generates a cysteinyl radical in the α active site. The radical can be trapped via the well-described decomposition reaction of the mechanism-based inactivator, 2′-azido-2′-deoxyuridine-5′-diphosphate, resulting in the generation of a long-lived, nitrogen-centered radical (N^•) in α. In this work, we have determined the distance between the Mn­(III)/Fe­(III) cofactor in β and N^• in α to be 43 ± 1 Å by using double electron–electron resonance experiments. This study provides the first structural data on the <i>Ct</i> RNR holoenzyme complex and the first direct experimental measurement of the inter-subunit RT distance in any class I RNR

Experimental Correlation of Substrate Position with Reaction Outcome in the Aliphatic Halogenase, SyrB2

The iron­(II)- and 2-(oxo)­glutarate-dependent (Fe/2OG) oxygenases catalyze an array of challenging transformations, but how individual members of the enzyme family direct different outcomes is poorly understood. The Fe/2OG halogenase, SyrB2, chlorinates C4 of its native substrate, l-threonine appended to the carrier protein, SyrB1, but hydroxylates C5 of l-norvaline and, to a lesser extent, C4 of l-aminobutyric acid when SyrB1 presents these non-native amino acids. To test the hypothesis that positioning of the targeted carbon dictates the outcome, we defined the positions of these three substrates by measuring hyperfine couplings between substrate deuterium atoms and the stable, EPR-active iron–nitrosyl adduct, a surrogate for reaction intermediates. The Fe–²H distances and N–Fe–²H angles, which vary from 4.2 Å and 85° for threonine to 3.4 Å and 65° for norvaline, rationalize the trends in reactivity. This experimental correlation of position to outcome should aid in judging from structural data on other Fe/2OG enzymes whether they suppress hydroxylation or form hydroxylated intermediates on the pathways to other outcomes

Branched Intermediate Formation Is the Slowest Step in the Protein Splicing Reaction of the Ala1 KlbA Intein from <i>Methanococcus jannaschii</i>

We report the first detailed investigation of the kinetics of protein splicing by the <i>Methanococcus jannaschii</i> KlbA (<i>Mja</i> KlbA) intein. This intein has an N-terminal Ala in place of the nucleophilic Cys or Ser residue that normally initiates splicing but nevertheless splices efficiently in vivo [Southworth, M. W., Benner, J., and Perler, F. B. (2000) <i>EMBO J.</i> <i>19</i>, 5019–5026]. To date, the spontaneous nature of the cis splicing reaction has hindered its examination in vitro. For this reason, we constructed an <i>Mja</i> KlbA intein–mini-extein precursor using intein-mediated protein ligation and engineered a disulfide redox switch that permits initiation of the splicing reaction by the addition of a reducing agent such as dithiothreitol (DTT). A fluorescent tag at the C-terminus of the C-extein permits monitoring of the progress of the reaction. Kinetic analysis of the splicing reaction of the wild-type precursor (with no substitutions in known nucleophiles or assisting groups) at various DTT concentrations shows that formation of the branched intermediate from the precursor is reversible (forward rate constant of 1.5 × 10^–3 s^–1 and reverse rate constant of 1.7 × 10^–5 s^–1 at 42 °C), whereas the productive decay of this intermediate to form the ligated exteins is faster and occurs with a rate constant of 2.2 × 10^–3 s^–1. This finding conflicts with reports about standard inteins, for which Asn cyclization has been assigned as the rate-determining step of the splicing reaction. Despite being the slowest step of the reaction, branched intermediate formation in the <i>Mja</i> KlbA intein is efficient in comparison with those of other intein systems. Interestingly, it also appears that this intermediate is protected against thiolysis by DTT, in contrast to other inteins. Evidence is presented in support of a tight coupling between the N-terminal and C-terminal cleavage steps, despite the fact that the C-terminal single-cleavage reaction occurs in variant <i>Mja</i> KlbA inteins in the absence of N-terminal cleavage. We posit that the splicing events in the <i>Mja</i> KlbA system are tightly coordinated by a network of intra- and interdomain noncovalent interactions, rendering its function particularly sensitive to minor disruptions in the intein or extein environments

A 2.8 Å Fe–Fe Separation in the Fe₂^III/IV Intermediate, X, from <i>Escherichia coli</i> Ribonucleotide Reductase

A class Ia ribonucleotide reductase (RNR) employs a μ-oxo-Fe₂^III/III/tyrosyl radical cofactor in its β subunit to oxidize a cysteine residue ∼35 Å away in its α subunit; the resultant cysteine radical initiates substrate reduction. During self-assembly of the <i>Escherichia coli</i> RNR-β cofactor, reaction of the protein’s Fe₂^II/II complex with O₂ results in accumulation of an Fe₂^III/IV cluster, termed <b>X</b>, which oxidizes the adjacent tyrosine (Y₁₂₂) to the radical (Y₁₂₂^•) as the cluster is converted to the μ-oxo-Fe₂^III/III product. As the first high-valent non-heme-iron enzyme complex to be identified and the key activating intermediate of class Ia RNRs, <b>X</b> has been the focus of intensive efforts to determine its structure. Initial characterization by extended X-ray absorption fine structure (EXAFS) spectroscopy yielded a Fe–Fe separation (<i>d</i>_Fe–Fe) of 2.5 Å, which was interpreted to imply the presence of three single-atom bridges (O^2–, HO^–, and/or μ-1,1-carboxylates). This short distance has been irreconcilable with computational and synthetic models, which all have <i>d</i>_Fe–Fe ≥ 2.7 Å. To resolve this conundrum, we revisited the EXAFS characterization of <b>X</b>. Assuming that samples containing increased concentrations of the intermediate would yield EXAFS data of improved quality, we applied our recently developed method of generating O₂ <i>in situ</i> from chlorite using the enzyme chlorite dismutase to prepare <b>X</b> at ∼2.0 mM, more than 2.5 times the concentration realized in the previous EXAFS study. The measured <i>d</i>_Fe–Fe = 2.78 Å is fully consistent with computational models containing a (μ-oxo)₂-Fe₂^III/IV core. Correction of the <i>d</i>_Fe–Fe brings the experimental data and computational models into full conformity and informs analysis of the mechanism by which <b>X</b> generates Y₁₂₂^•

Installation of the Ether Bridge of Lolines by the Iron- and 2‑Oxoglutarate-Dependent Oxygenase, LolO: Regio- and Stereochemistry of Sequential Hydroxylation and Oxacyclization Reactions

The core of the loline family of insecticidal alkaloids is the bicyclic pyrrolizidine unit with an additional strained ether bridge between carbons 2 and 7. Previously reported genetic and <i>in vivo</i> biochemical analyses showed that the presumptive iron- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase, LolO, is required for installation of the ether bridge upon the pathway intermediate, 1-<i>exo</i>-acetamidopyrrolizidine (AcAP). Here we show that LolO is, in fact, solely responsible for this biosynthetic four-electron oxidation. In sequential 2OG- and O₂-consuming steps, LolO removes hydrogens from C2 and C7 of AcAP to form both carbon–oxygen bonds in <i>N</i>-acetylnorloline (NANL), the precursor to all other lolines. When supplied with substoichiometric 2OG, LolO only hydroxylates AcAP. At higher 2OG:AcAP ratios, the enzyme further processes the alcohol to the tricyclic NANL. Characterization of the alcohol intermediate by mass spectrometry and nuclear magnetic resonance spectroscopy shows that it is 2-<i>endo</i>-hydroxy-1-<i>exo</i>-acetamidopyrrolizidine (2-<i>endo</i>-OH-AcAP). Kinetic and spectroscopic analyses of reactions with site-specifically deuteriated AcAP substrates confirm that the C2–H bond is cleaved first and that the responsible intermediate is, as expected, an Fe^IV–oxo (ferryl) complex. Analyses of the loline products from cultures fed with stereospecifically deuteriated AcAP precursors, proline and aspartic acid, establish that LolO removes the endo hydrogens from C2 and C7 and forms both new C–O bonds with retention of configuration. These findings delineate the pathway to an important class of natural insecticides and lay the foundation for mechanistic dissection of the chemically challenging oxacyclization reaction