Spectroscopic, Steady-State Kinetic, and Mechanistic Characterization of the Radical SAM Enzyme QueE, Which Catalyzes a Complex Cyclization Reaction in the Biosynthesis of 7‑Deazapurines

7-Carboxy-7-deazaguanine (CDG) synthase (QueE) catalyzes the complex heterocyclic radical-mediated conversion of 6-carboxy-5,6,7,8-tetrahydropterin (CPH₄) to CDG in the third step of the biosynthetic pathway to all 7-deazapurines. Here we present a detailed characterization of QueE from <i>Bacillus subtilis</i> to delineate the mechanism of conversion of CPH₄ to CDG. QueE is a member of the radical <i>S</i>-adenosyl-l-methionine (SAM) superfamily, all of which use a bound [4Fe-4S]⁺ cluster to catalyze the reductive cleavage of the SAM cofactor to generate methionine and a 5′-deoxyadenosyl radical (5′-dAdo^•), which initiates enzymatic transformations requiring hydrogen atom abstraction. The ultraviolet–visible, electron paramagnetic resonance, and Mössbauer spectroscopic features of the homodimeric QueE point to the presence of a single [4Fe-4S] cluster per monomer. Steady-state kinetic experiments indicate a <i>K</i>_m of 20 ± 7 μM for CPH₄ and a <i>k</i>_cat of 5.4 ± 1.2 min^–1 for the overall transformation. The kinetically determined <i>K</i>_app for SAM is 45 ± 1 μM. QueE is also magnesium-dependent and exhibits a <i>K</i>_app for the divalent metal ion of 0.21 ± 0.03 mM. The SAM cofactor supports multiple turnovers, indicating that it is regenerated at the end of each catalytic cycle. The mechanism of rearrangement of QueE was probed with CPH₄ isotopologs containing deuterium at C-6 or the two prochiral positions at C-7. These studies implicate 5′-dAdo^• as the initiator of the ring contraction reaction catalyzed by QueE by abstraction of the H atom from C-6 of CPH₄

Importance of the Maintenance Pathway in the Regulation of the Activity of <i>Escherichia coli</i> Ribonucleotide Reductase

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. The <i>Escherichia coli</i> class Ia RNR is composed of α and β subunits that form an α₂β₂ active complex. β contains the diferric tyrosyl radical (Y^•) cofactor that is essential for the reduction process that occurs on α. [Y^•] in vitro is proportional to RNR activity, and its regulation in vivo potentially represents a mechanism for controlling RNR activity. To examine this thesis, N- and C-terminal StrepII-tagged β under the control of an l-arabinose promoter were constructed. Using these constructs and with [l-arabinose] varying from 0 to 0.5 mM in the growth medium, [β] could be varied from 4 to 3300 µM. [Y^•] in vivo and on affinity-purified Strep-β in vitro was determined by EPR spectroscopy and Western analysis. In both cases, there was 0.1–0.3 Y^• radical per β. To determine if the substoichiometric Y^• level was associated with apo β or diferric β, titrations of crude cell extracts from these growths were carried out with reduced YfaE, a 2Fe2S ferredoxin involved in cofactor maintenance and assembly. Each titration, followed by addition of O₂ to assemble the cofactor and EPR analysis to quantitate Y^•, revealed that β is completely loaded with a diferric cluster even when its concentration in vivo is 244 µM. These titrations, furthermore, resulted in 1 Y^• radical per β, the highest levels reported. Whole cell Mössbauer analysis on cells induced with 0.5 mM arabinose supports high iron loading in β. These results suggest that modulation of the level of Y^• in vivo in <i>E. coli</i> is a mechanism of regulating RNR activity

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

Iron–Sulfur Cluster Engineering Provides Insight into the Evolution of Substrate Specificity among Sulfonucleotide Reductases

Assimilatory sulfate reduction supplies prototrophic organisms with reduced sulfur that is required for the biosynthesis of all sulfur-containing metabolites, including cysteine and methionine. The reduction of sulfate requires its activation <i>via</i> an ATP-dependent activation to form adenosine-5′-phosphosulfate (APS). Depending on the species, APS can be reduced directly to sulfite by APS reductase (APR) or undergo a second phosphorylation to yield 3′-phosphoadenosine-5′-phosphosulfate (PAPS), the substrate for PAPS reductase (PAPR). These essential enzymes have no human homologue, rendering them attractive targets for the development of novel antibacterial drugs. APR and PAPR share sequence and structure homology as well as a common catalytic mechanism, but the enzymes are distinguished by two features, namely, the amino acid sequence of the phosphate-binding loop (P-loop) and an iron–sulfur cofactor in APRs. On the basis of the crystal structures of APR and PAPR, two P-loop residues are proposed to determine substrate specificity; however, this hypothesis has not been tested. In contrast to this prevailing view, we report here that the P-loop motif has a modest effect on substrate discrimination. Instead, by means of metalloprotein engineering, spectroscopic, and kinetic analyses, we demonstrate that the iron–sulfur cluster cofactor enhances APS reduction by nearly 1000-fold, thereby playing a pivotal role in substrate specificity and catalysis. These findings offer new insights into the evolution of this enzyme family and extend the known functions of protein-bound iron–sulfur clusters

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

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

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)

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

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

Evidence for a Catalytically and Kinetically Competent Enzyme–Substrate Cross-Linked Intermediate in Catalysis by Lipoyl Synthase

Lipoyl synthase (LS) catalyzes the final step in lipoyl cofactor biosynthesis: the insertion of two sulfur atoms at C6 and C8 of an (<i>N</i>⁶-octanoyl)-lysyl residue on a lipoyl carrier protein (LCP). LS is a member of the radical SAM superfamily, enzymes that use a [4Fe–4S] cluster to effect the reductive cleavage of <i>S</i>-adenosyl-l-methionine (SAM) to l-methionine and a 5′-deoxyadenosyl 5′-radical (5′-dA^•). In the LS reaction, two equivalents of 5′-dA^• are generated sequentially to abstract hydrogen atoms from C6 and C8 of the appended octanoyl group, initiating sulfur insertion at these positions. The second [4Fe–4S] cluster on LS, termed the auxiliary cluster, is proposed to be the source of the inserted sulfur atoms. Herein, we provide evidence for the formation of a covalent cross-link between LS and an LCP or synthetic peptide substrate in reactions in which insertion of the second sulfur atom is slowed significantly by deuterium substitution at C8 or by inclusion of limiting concentrations of SAM. The observation that the proteins elute simultaneously by anion-exchange chromatography but are separated by aerobic SDS-PAGE is consistent with their linkage through the auxiliary cluster that is sacrificed during turnover. Generation of the cross-linked species with a small, unlabeled (<i>N</i>⁶-octanoyl)-lysyl-containing peptide substrate allowed demonstration of both its chemical and kinetic competence, providing strong evidence that it is an intermediate in the LS reaction. Mössbauer spectroscopy of the cross-linked intermediate reveals that one of the [4Fe–4S] clusters, presumably the auxiliary cluster, is partially disassembled to a 3Fe-cluster with spectroscopic properties similar to those of reduced [3Fe–4S]⁰ clusters