Co-reporter:Ryan J. Martinie, Christopher J. Pollock, Megan L. Matthews, J. Martin Bollinger Jr., Carsten Krebs, and Alexey Silakov
Inorganic Chemistry November 6, 2017 Volume 56(Issue 21) pp:13382-13382
Publication Date(Web):September 29, 2017
DOI:10.1021/acs.inorgchem.7b02113
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 [(VIV-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-S-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.
Co-reporter:Ryan J. Martinie, Christopher J. Pollock, Megan L. Matthews, J. Martin Bollinger Jr., Carsten Krebs, and Alexey Silakov
Inorganic Chemistry November 6, 2017 Volume 56(Issue 21) pp:13382-13382
Publication Date(Web):September 29, 2017
DOI:10.1021/acs.inorgchem.7b02113
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 [(VIV-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-S-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.
Co-reporter:Kiyoung Park, Ning Li, Yeonju Kwak, Martin Srnec, Caleb B. Bell, Lei V. Liu, Shaun D. Wong, Yoshitaka Yoda, Shinji Kitao, Makoto Seto, Michael Hu, Jiyong Zhao, Carsten Krebs, J. Martin Bollinger Jr., and Edward I. Solomon
Journal of the American Chemical Society May 24, 2017 Volume 139(Issue 20) pp:7062-7062
Publication Date(Web):May 1, 2017
DOI:10.1021/jacs.7b02997
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.
Co-reporter:Spencer C. Peck, Chen Wang, Laura M. K. Dassama, Bo Zhang, Yisong GuoLauren J. Rajakovich, J. Martin Bollinger Jr., Carsten Krebs, Wilfred A. van der Donk
Journal of the American Chemical Society 2017 Volume 139(Issue 5) pp:2045-2052
Publication Date(Web):January 16, 2017
DOI:10.1021/jacs.6b12147
Activation of O–H bonds by inorganic metal-oxo complexes has been documented, but no cognate enzymatic process is known. Our mechanistic analysis of 2-hydroxyethylphosphonate dioxygenase (HEPD), which cleaves the C1–C2 bond of its substrate to afford hydroxymethylphosphonate on the biosynthetic pathway to the commercial herbicide phosphinothricin, uncovered an example of such an O–H-bond-cleavage event. Stopped-flow UV–visible absorption and freeze-quench Mössbauer experiments identified a transient iron(IV)-oxo (ferryl) complex. Maximal accumulation of the intermediate required both the presence of deuterium in the substrate and, importantly, the use of 2H2O as solvent. The ferryl complex forms and decays rapidly enough to be on the catalytic pathway. To account for these unanticipated results, a new mechanism that involves activation of an O–H bond by the ferryl complex is proposed. This mechanism accommodates all available data on the HEPD reaction.
Co-reporter:Anthony J. Blaszczyk; Alexey Silakov; Bo Zhang; Stephanie J. Maiocco; Nicholas D. Lanz; Wendy L. Kelly; Sean J. Elliott; Carsten Krebs;Squire J. Booker
Journal of the American Chemical Society 2016 Volume 138(Issue 10) pp:3416-3426
Publication Date(Web):February 3, 2016
DOI:10.1021/jacs.5b12592
TsrM, an annotated radical S-adenosylmethionine (SAM) enzyme, catalyzes the methylation of carbon 2 of the indole ring of l-tryptophan. Its reaction is the first step in the biosynthesis of the unique quinaldic acid moiety of thiostrepton A, a thiopeptide antibiotic. The appended methyl group derives from SAM; however, the enzyme also requires cobalamin and iron–sulfur cluster cofactors for turnover. In this work we report the overproduction and purification of TsrM and the characterization of its metallocofactors by UV–visible, electron paramagnetic resonance, hyperfine sublevel correlation (HYSCORE), and Mössbauer spectroscopies as well as protein-film electrochemistry (PFE). The enzyme contains 1 equiv of its cobalamin cofactor in its as-isolated state and can be reconstituted with iron and sulfide to contain one [4Fe–4S] cluster with a site-differentiated Fe2+/Fe3+ pair. Our spectroscopic studies suggest that TsrM binds cobalamin in an uncharacteristic five-coordinate base-off/His-off conformation, whereby the dimethylbenzimidazole group is replaced by a non-nitrogenous ligand, which is likely a water molecule. Electrochemical analysis of the protein by PFE indicates a one-electron redox feature with a midpoint potential of −550 mV, which is assigned to a [4Fe–4S]2+/[4Fe–4S]+ redox couple. Analysis of TsrM by Mössbauer and HYSCORE spectroscopies suggests that SAM does not bind to the unique iron site of the cluster in the same manner as in other radical SAM (RS) enzymes, yet its binding still perturbs the electronic configuration of both the Fe/S cluster and the cob(II)alamin cofactors. These biophysical studies suggest that TsrM is an atypical RS enzyme, consistent with its reported inability to catalyze formation of a 5′-deoxyadenosyl 5′-radical.
Co-reporter:Esta Tamanaha; Bo Zhang; Yisong Guo; Wei-chen Chang; Eric W. Barr; Gang Xing; Jennifer St. Clair; Shengfa Ye; Frank Neese; J. Martin BollingerJr.
Journal of the American Chemical Society 2016 Volume 138(Issue 28) pp:8862-8874
Publication Date(Web):May 18, 2016
DOI:10.1021/jacs.6b04065
The enzyme isopenicillin N synthase (IPNS) installs the β-lactam and thiazolidine rings of the penicillin core into the linear tripeptide l-δ-aminoadipoyl-l-Cys-d-Val (ACV) on the pathways to a number of important antibacterial drugs. A classic set of enzymological and crystallographic studies by Baldwin and co-workers established that this overall four-electron oxidation occurs by a sequence of two oxidative cyclizations, with the β-lactam ring being installed first and the thiazolidine ring second. Each phase requires cleavage of an aliphatic C–H bond of the substrate: the pro-S-CCys,β–H bond for closure of the β-lactam ring, and the CVal,β–H bond for installation of the thiazolidine ring. IPNS uses a mononuclear non-heme-iron(II) cofactor and dioxygen as cosubstrate to cleave these C–H bonds and direct the ring closures. Despite the intense scrutiny to which the enzyme has been subjected, the identities of the oxidized iron intermediates that cleave the C–H bonds have been addressed only computationally; no experimental insight into their geometric or electronic structures has been reported. In this work, we have employed a combination of transient-state-kinetic and spectroscopic methods, together with the specifically deuterium-labeled substrates, A[d2-C]V and AC[d8-V], to identify both C–H-cleaving intermediates. The results show that they are high-spin Fe(III)-superoxo and high-spin Fe(IV)-oxo complexes, respectively, in agreement with published mechanistic proposals derived computationally from Baldwin’s founding work.
Co-reporter:Nicholas D. Lanz, Kyung-Hoon Lee, Abigail K. Horstmann, Maria-Eirini Pandelia, Robert M. Cicchillo, Carsten Krebs, and Squire J. Booker
Biochemistry 2016 Volume 55(Issue 9) pp:1372-1383
Publication Date(Web):February 3, 2016
DOI:10.1021/acs.biochem.5b01216
The prevalence of multiple and extensively drug-resistant strains of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is on the rise, necessitating the identification of new targets to combat an organism that has infected one-third of the world’s population, according to the World Health Organization. The biosynthesis of the lipoyl cofactor is one possible target, given its critical importance in cellular metabolism and the apparent lack of functional salvage pathways in Mtb that are found in humans and many other organisms. The lipoyl cofactor is synthesized de novo in two committed steps, involving the LipB-catalyzed transfer of an octanoyl chain derived from fatty acid biosynthesis to a lipoyl carrier protein and the LipA-catalyzed insertion of sulfur atoms at C6 and C8 of the octanoyl chain. A number of in vitro studies of lipoyl synthases from Escherichia coli, Sulfolobus solfataricus, and Thermosynechococcus elongatus have been conducted, but the enzyme from Mtb has not been characterized. Herein, we show that LipA from Mtb contains two [4Fe–4S] clusters and converts an octanoyl peptide substrate to the corresponding lipoyl peptide product via the same C6-monothiolated intermediate as that observed in the E. coli LipA reaction. In addition, we show that LipA from Mtb forms a complex with the H protein of the glycine cleavage system and that the strength of association is dependent on the presence of S-adenosyl-l-methionine. We also show that LipA from Mtb can complement a lipA mutant of E. coli, demonstrating the commonalities of the two enzymes. Lastly, we show that the substrate for LipA, which normally acts on a post-translationally modified protein, can be reduced to carboxybenzyl-octanoyllysine.
Co-reporter:Douglas M. Warui, Maria-Eirini Pandelia, Lauren J. Rajakovich, Carsten Krebs, J. Martin Bollinger Jr., and Squire J. Booker
Biochemistry 2015 Volume 54(Issue 4) pp:1006-1015
Publication Date(Web):December 15, 2014
DOI:10.1021/bi500847u
A two-step pathway consisting of an acyl–acyl carrier protein (ACP) reductase (AAR) and an aldehyde-deformylating oxygenase (ADO) allows various cyanobacteria to convert long-chain fatty acids into hydrocarbons. AAR catalyzes the two-electron, NADPH-dependent reduction of a fatty acid attached to ACP via a thioester linkage to the corresponding fatty aldehyde, while ADO transforms the fatty aldehyde to a Cn–1 hydrocarbon and C1-derived formate. Considering that heptadec(a/e)ne is the most prevalent hydrocarbon produced by cyanobacterial ADOs, the insolubility of its precursor, octadec(a/e)nal, poses a conundrum with respect to its acquisition by ADO. Herein, we report that AAR from the cyanobacterium Nostoc punctiforme is activated almost 20-fold by potassium and other monovalent cations of similar ionic radius, and that AAR and ADO form a tight isolable complex with a Kd of 3 ± 0.3 μM. In addition, we show that when the aldehyde substrate is supplied to ADO by AAR, efficient in vitro turnover is observed in the absence of solubilizing agents. Similarly to studies by Lin et al. with AAR from Synechococcus elongatus [Lin et al. (2013) FEBS J. 280, 4773−4781], we show that catalysis by AAR proceeds via formation of a covalent intermediate involving a cysteine residue that we have identified as Cys294. Moreover, AAR specifically transfers the pro-R hydride of NADPH to the Cys294-thioester intermediate to afford its aldehyde product. Our results suggest that the interaction between AAR and ADO facilitates either direct transfer of the aldehyde product of AAR to ADO or formation of the aldehyde product in a microenvironment allowing for its efficient uptake by ADO.
Co-reporter:Nicholas D. Lanz, Maria-Eirini Pandelia, Elizabeth S. Kakar, Kyung-Hoon Lee, Carsten Krebs, and Squire J. Booker
Biochemistry 2014 Volume 53(Issue 28) pp:4557-4572
Publication Date(Web):June 5, 2014
DOI:10.1021/bi500432r
Lipoyl synthase (LS) catalyzes the final step in lipoyl cofactor biosynthesis: the insertion of two sulfur atoms at C6 and C8 of an (N6-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 S-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 (N6-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.
Co-reporter:Wei-chen Chang;Yisong Guo;Chen Wang;Susan E. Butch;Amy C. Rosenzweig;Amie K. Boal;J. Martin Bollinger Jr.
Science 2014 Volume 343(Issue 6175) pp:1140-1144
Publication Date(Web):07 Mar 2014
DOI:10.1126/science.1248000
Carbapenems Through the Looking Glass
The carbapenem class of antibiotics is a critical weapon in the ongoing fight against drug-resistant bacteria. Microbial biosynthesis of these compounds, which contain a strained β-lactam ring motif, proceeds via a precursor that has the wrong configuration at one of the ring carbons. Chang et al. (p. 1140) combined x-ray crystallography with multiple spectroscopic probes to map out the mechanism by which the CarC enzyme inverts the precursor configuration to its mirror image.
Co-reporter:Carsten Krebs, Laura M.K. Dassama, Megan L. Matthews, Wei Jiang, John C. Price, Victoria Korboukh, Ning Li, J. Martin Bollinger Jr.
Coordination Chemistry Reviews 2013 Volume 257(Issue 1) pp:234-243
Publication Date(Web):1 January 2013
DOI:10.1016/j.ccr.2012.06.020
Co-reporter:Bigna Wörsdörfer ; Denise A. Conner ; Kenichi Yokoyama ; Jovan Livada ; Mohammad Seyedsayamdost ; Wei Jiang ; Alexey Silakov ; JoAnne Stubbe ; J. Martin Bollinger ; Jr.
Journal of the American Chemical Society 2013 Volume 135(Issue 23) pp:8585-8593
Publication Date(Web):May 16, 2013
DOI:10.1021/ja401342s
The class Ia ribonucleotide reductase (RNR) from Escherichia coli employs a free-radical mechanism, which involves bidirectional translocation of a radical equivalent or “hole” over a distance of ∼35 Å from the stable diferric/tyrosyl-radical (Y122•) cofactor in the β subunit to cysteine 439 (C439) in the active site of the α subunit. This long-range, intersubunit electron transfer occurs by a multistep “hopping” mechanism via formation of transient amino acid radicals along a specific pathway and is thought to be conformationally gated and coupled to local proton transfers. Whereas constituent amino acids of the hopping pathway have been identified, details of the proton-transfer steps and conformational gating within the β sununit have remained obscure; specific proton couples have been proposed, but no direct evidence has been provided. In the key first step, the reduction of Y122• by the first residue in the hopping pathway, a water ligand to Fe1 of the diferric cluster was suggested to donate a proton to yield the neutral Y122. Here we show that forward radical translocation is associated with perturbation of the Mössbauer spectrum of the diferric cluster, especially the quadrupole doublet associated with Fe1. Density functional theory (DFT) calculations verify the consistency of the experimentally observed perturbation with that expected for deprotonation of the Fe1-coordinated water ligand. The results thus provide the first evidence that the diiron cluster of this prototypical class Ia RNR functions not only in its well-known role as generator of the enzyme’s essential Y122•, but also directly in catalysis.
Co-reporter:Rae Ana Snyder ; Caleb B. Bell ; III; Yinghui Diao ; Carsten Krebs ; J. Martin Bollinger ; Jr.;Edward I. Solomon
Journal of the American Chemical Society 2013 Volume 135(Issue 42) pp:15851-15863
Publication Date(Web):September 25, 2013
DOI:10.1021/ja406635k
myo-Inositol oxygenase (MIOX) catalyzes the 4e– oxidation of myo-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.
Co-reporter:Laura M. K. Dassama ; Alexey Silakov ; Courtney M. Krest ; Julio C. Calixto ; Carsten Krebs ; J. Martin Bollinger ; Jr.;Michael T. Green
Journal of the American Chemical Society 2013 Volume 135(Issue 45) pp:16758-16761
Publication Date(Web):October 4, 2013
DOI:10.1021/ja407438p
A class Ia ribonucleotide reductase (RNR) employs a μ-oxo-Fe2III/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 Escherichia coli RNR-β cofactor, reaction of the protein’s Fe2II/II complex with O2 results in accumulation of an Fe2III/IV cluster, termed X, which oxidizes the adjacent tyrosine (Y122) to the radical (Y122•) as the cluster is converted to the μ-oxo-Fe2III/III product. As the first high-valent non-heme-iron enzyme complex to be identified and the key activating intermediate of class Ia RNRs, X 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 (dFe–Fe) of 2.5 Å, which was interpreted to imply the presence of three single-atom bridges (O2–, HO–, and/or μ-1,1-carboxylates). This short distance has been irreconcilable with computational and synthetic models, which all have dFe–Fe ≥ 2.7 Å. To resolve this conundrum, we revisited the EXAFS characterization of X. 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 in situ from chlorite using the enzyme chlorite dismutase to prepare X at ∼2.0 mM, more than 2.5 times the concentration realized in the previous EXAFS study. The measured dFe–Fe = 2.78 Å is fully consistent with computational models containing a (μ-oxo)2-Fe2III/IV core. Correction of the dFe–Fe brings the experimental data and computational models into full conformity and informs analysis of the mechanism by which X generates Y122•.
Co-reporter:Yeonju Kwak ; Wei Jiang ; Laura M. K. Dassama ; Kiyoung Park ; Caleb B. Bell ; III; Lei V. Liu ; Shaun D. Wong ; Makina Saito ; Yasuhiro Kobayashi ; Shinji Kitao ; Makoto Seto ; Yoshitaka Yoda ; E. Ercan Alp ; Jiyong Zhao ; J. Martin Bollinger ; Jr.; Carsten Krebs ;Edward I. Solomon
Journal of the American Chemical Society 2013 Volume 135(Issue 46) pp:17573-17584
Publication Date(Web):October 16, 2013
DOI:10.1021/ja409510d
The class Ic ribonucleotide reductase (RNR) from Chlamydia trachomatis (Ct) utilizes a Mn/Fe heterobinuclear cofactor, rather than the Fe/Fe cofactor found in the β (R2) subunit of the class Ia enzymes, to react with O2. This reaction produces a stable MnIVFeIII cofactor that initiates a radical, which transfers to the adjacent α (R1) subunit and reacts with the substrate. We have studied the MnIVFeIII cofactor using nuclear resonance vibrational spectroscopy (NRVS) and absorption (Abs)/circular dichroism (CD)/magnetic CD (MCD)/variable temperature, variable field (VTVH) MCD spectroscopies to obtain detailed insight into its geometric/electronic structure and to correlate structure with reactivity; NRVS focuses on the FeIII, whereas MCD reflects the spin-allowed transitions mostly on the MnIV. We have evaluated 18 systematically varied structures. Comparison of the simulated NRVS spectra to the experimental data shows that the cofactor has one carboxylate bridge, with MnIV at the site proximal to Phe127. Abs/CD/MCD/VTVH MCD data exhibit 12 transitions that are assigned as d–d and oxo and OH– to metal charge-transfer (CT) transitions. Assignments are based on MCD/Abs intensity ratios, transition energies, polarizations, and derivative-shaped pseudo-A term CT transitions. Correlating these results with TD-DFT calculations defines the MnIVFeIII cofactor as having a μ-oxo, μ-hydroxo core and a terminal hydroxo ligand on the MnIV. From DFT calculations, the MnIV at site 1 is necessary to tune the redox potential to a value similar to that of the tyrosine radical in class Ia RNR, and the OH– terminal ligand on this MnIV provides a high proton affinity that could gate radical translocation to the α (R1) subunit.
Co-reporter:Tyler L. Grove, Jessica H. Ahlum, Rosie M. Qin, Nicholas D. Lanz, Matthew I. Radle, Carsten Krebs, and Squire J. Booker
Biochemistry 2013 Volume 52(Issue 17) pp:
Publication Date(Web):March 11, 2013
DOI:10.1021/bi400136u
The anaerobic sulfatase-maturating enzyme from Clostridium perfringens (anSMEcpe) catalyzes the two-electron oxidation of a cysteinyl residue on a cognate protein to a formylglycyl residue (FGly) using a mechanism that involves organic radicals. The FGly residue plays a unique role as a cofactor in a class of enzymes termed arylsulfatases, which catalyze the hydrolysis of various organosulfate monoesters. anSMEcpe has been shown to be a member of the radical S-adenosylmethionine (SAM) family of enzymes, [4Fe-4S] cluster-requiring proteins that use a 5′-deoxyadenosyl 5′-radical (5′-dA•) generated from a reductive cleavage of SAM to initiate radical-based catalysis. Herein, we show that anSMEcpe contains in addition to the [4Fe-4S] cluster harbored by all radical SAM (RS) enzymes, two additional [4Fe-4S] clusters, similar to the radical SAM protein AtsB, which catalyzes the two-electron oxidation of a seryl residue to a FGly residue. We show by size-exclusion chromatography that both AtsB and anSMEcpe are monomeric proteins, and site-directed mutagenesis studies of AtsB reveal that individual Cys → Ala substitutions at seven conserved positions result in an insoluble protein, consistent with those residues acting as ligands to the two additional [4Fe-4S] clusters. Ala substitutions at an additional conserved Cys residue (C291 in AtsB and C276 in anSMEcpe) afford proteins that display intermediate behavior. These proteins exhibit reduced solubility and drastically reduced activity, behavior that is conspicuously similar to that of a critical Cys residue in BtrN, another radical SAM dehydrogenase [Grove, T. L., et al. (2010) Biochemistry49, 3783–3785]. We also show that wild-type anSMEcpe acts on peptides containing other oxidizable amino acids at the target position. Moreover, we show that the enzyme will convert threonyl peptides to the corresponding ketone product, and also allo-threonyl peptides, but with a significantly reduced efficiency, suggesting that the pro-S hydrogen atom of the normal cysteinyl substrate is stereoselectively removed during turnover. Lastly, we show that the electron generated during catalysis by AtsB and anSMEcpe can be utilized for multiple turnovers, albeit through a reduced flavodoxin-mediated pathway.
Co-reporter:Chen Wang;Wei-chen Chang;Yisong Guo;Hui Huang;Spencer C. Peck;Maria E. Pandelia;Geng-min Lin;Hung-wen Liu;J. Martin Bollinger Jr.
Science 2013 Volume 342(Issue 6161) pp:
Publication Date(Web):
DOI:10.1126/science.1240373
Just Add Peroxide
The HppE enzyme uses iron to catalyze oxidation of an alcohol to an epoxide ring in the biosynthesis of the antibiotic fosfomycin. Because this process is a two-electron oxidation, it has been unclear how the enzyme reduces its presumed oxidative partner O2 all the way to water. Where do the two extra electrons come from? Wang et al. (p. 991, published 10 October; see the Perspective by Raushel) now show that HppE is actually a peroxidase, and thus reduces H2O2, for which just two electrons are sufficient. The result expands the structural scope of iron-bearing peroxidase enzymes beyond heme motifs.
Co-reporter:Laura M. K. Dassama ; Amie K. Boal ; Carsten Krebs ; Amy C. Rosenzweig ;J. Martin Bollinger ; Jr.
Journal of the American Chemical Society 2012 Volume 134(Issue 5) pp:2520-2523
Publication Date(Web):January 12, 2012
DOI:10.1021/ja211314p
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 Chlamydia trachomatis (Ct), the cysteine oxidant is the MnIV ion of a MnIV/FeIII cluster, which assembles in a reaction between O2 and the MnII/FeII complex of β. The heterodinuclear nature of the cofactor raises the question of which site, 1 or 2, contains the MnIV 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 MnIV ion most likely resides in this site (i.e., 1MnIV/2FeIII), but a subsequent computational study favored its occupation of site 2 (1FeIII/2MnIV). In this work, we have sought to resolve the location of the MnIV ion in Ct RNR-β by correlating X-ray crystallographic anomalous scattering intensities with catalytic activity for samples of the protein reconstituted in vitro by two different procedures. In samples containing primarily MnIV/FeIII clusters, Mn preferentially occupies site 1, but some anomalous scattering from site 2 is observed, implying that both 1MnII/2FeII and 1FeII/2MnII complexes are competent to react with O2 to produce the corresponding oxidized states. However, with diminished MnII 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 1MnIV/2FeIII-β is at least the more active of the two oxidized forms and may be the only active form.
Co-reporter:Laura M. K. Dassama ; Wei Jiang ; Paul T. Varano ; Maria-Eirini Pandelia ; Denise A. Conner ; Jiajia Xie ; J. Martin Bollinger ; Jr.
Journal of the American Chemical Society 2012 Volume 134(Issue 50) pp:20498-20506
Publication Date(Web):November 16, 2012
DOI:10.1021/ja309468s
A class I ribonucleotide reductase (RNR) uses either a tyrosyl radical (Y•) or a MnIV/FeIII cluster in its β subunit to oxidize a cysteine residue ∼35 Å away in its α subunit, generating a thiyl radical that abstracts hydrogen (H•) from the substrate. With either oxidant, the inter-subunit “hole-transfer” or “radical-translocation” (RT) process is thought to occur by a “hopping” mechanism involving multiple tyrosyl (and perhaps one tryptophanyl) radical intermediates along a specific pathway. The hopping intermediates have never been directly detected in a Mn/Fe-dependent (class Ic) RNR nor in any wild-type (wt) RNR. The MnIV/FeIII cofactor of Chlamydia trachomatis RNR assembles via a MnIV/FeIV intermediate. Here we show that this cofactor-assembly intermediate can propagate a hole into the RT pathway when α is present, accumulating radicals with EPR spectra characteristic of Y•’s. The dependence of Y• accumulation on the presence of substrate suggests that RT within this “super-oxidized” enzyme form is gated by the protein, and the failure of a β variant having the subunit-interfacial pathway Y substituted by phenylalanine to support radical accumulation implies that the Y•(s) in the wt enzyme reside(s) within the RT pathway. Remarkably, two variant β proteins having pathway substitutions rendering them inactive in their MnIV/FeIII states can generate the pathway Y•’s in their MnIV/FeIV states and also effect nucleotide reduction. Thus, the use of the more oxidized cofactor permits the accumulation of hopping intermediates and the “hurdling” of engineered defects in the RT pathway.
Co-reporter:Laura M. K. Dassama, Timothy H. Yosca, Denise A. Conner, Michael H. Lee, Béatrice Blanc, Bennett R. Streit, Michael T. Green, Jennifer L. DuBois, Carsten Krebs, and J. Martin Bollinger Jr.
Biochemistry 2012 Volume 51(Issue 8) pp:
Publication Date(Web):January 20, 2012
DOI:10.1021/bi201906x
The direct interrogation of fleeting intermediates by rapid-mixing kinetic methods has significantly advanced our understanding of enzymes that utilize dioxygen. The gas’s modest aqueous solubility (<2 mM at 1 atm) presents a technical challenge to this approach, because it limits the rate of formation and extent of accumulation of intermediates. This challenge can be overcome by use of the heme enzyme chlorite dismutase (Cld) for the rapid, in situ generation of O2 at concentrations far exceeding 2 mM. This method was used to define the O2 concentration dependence of the reaction of the class Ic ribonucleotide reductase (RNR) from Chlamydia trachomatis, in which the enzyme’s MnIV/FeIII cofactor forms from a MnII/FeII complex and O2 via a MnIV/FeIV intermediate, at effective O2 concentrations as high as ∼10 mM. With a more soluble receptor, myoglobin, an O2 adduct accumulated to a concentration of >6 mM in <15 ms. Finally, the C–H-bond-cleaving FeIV–oxo complex, J, in taurine:α-ketoglutarate dioxygenase and superoxo–Fe2III/III complex, G, in myo-inositol oxygenase, and the tyrosyl-radical-generating Fe2III/IV intermediate, X, in Escherichia coli RNR, were all accumulated to yields more than twice those previously attained. This means of in situ O2 evolution permits a >5 mM “pulse” of O2 to be generated in <1 ms at the easily accessible Cld concentration of 50 μM. It should therefore significantly extend the range of kinetic and spectroscopic experiments that can routinely be undertaken in the study of these enzymes and could also facilitate resolution of mechanistic pathways in cases of either sluggish or thermodynamically unfavorable O2 addition steps.
Co-reporter:Ning Li, Wei-chen Chang, Douglas M. Warui, Squire J. Booker, Carsten Krebs, and J. Martin Bollinger Jr.
Biochemistry 2012 Volume 51(Issue 40) pp:
Publication Date(Web):September 4, 2012
DOI:10.1021/bi300912n
Cyanobacterial aldehyde decarbonylases (ADs) catalyze the conversion of Cn fatty aldehydes to formate (HCO2–) and the corresponding Cn-1 alk(a/e)nes. Previous studies of the Nostoc punctiforme (Np) AD produced in Escherichia coli (Ec) 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) Biochemistry50, 10743–10750] reported that their Ec-expressed Np and Prochlorococcus marinus (Pm) 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 direct mass-spectrometric detection of the HCO2– product. For multiple combinations of the AD orthologue (Np and Pm), reducing system (protein-based and chemical), and substrate (n-heptanal and n-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).
Co-reporter:Douglas M. Warui ; Ning Li ; Hanne Nørgaard ; Carsten Krebs ; J. Martin Bollinger ; Jr.;Squire J. Booker
Journal of the American Chemical Society 2011 Volume 133(Issue 10) pp:3316-3319
Publication Date(Web):February 22, 2011
DOI:10.1021/ja111607x
The second of two reactions in a recently discovered pathway through which saturated fatty acids are converted to alkanes (and unsaturated fatty acids to alkenes) in cyanobacteria entails scission of the C1−C2 bond of a fatty aldehyde intermediate by the enzyme aldehyde decarbonylase (AD), a ferritin-like protein with a dinuclear metal cofactor of unknown composition. We tested for and failed to detect carbon monoxide (CO), the proposed C1-derived coproduct of alkane synthesis, following the in vitro conversion of octadecanal (R−CHO, where R = n-C17H35) to heptadecane (R−H) by the Nostoc punctiforme AD isolated following its overproduction in Escherichia coli. Instead, we identified formate (HCO2−) as the stoichiometric coproduct of the reaction. Results of isotope-tracer experiments indicate that the aldehyde hydrogen is retained in the HCO2− and the hydrogen in the nascent methyl group of the alkane originates, at least in part, from solvent. With these characteristics, the reaction appears to be formally hydrolytic, but the improbability of a hydrolytic mechanism having the primary carbanion as the leaving group, the structural similarity of the ADs to other O2-activating nonheme di-iron proteins, and the dependence of in vitro AD activity on the presence of a reducing system implicate some type of redox mechanism. Two possible resolutions to this conundrum are suggested.
Co-reporter:Tyler L. Grove ; Matthew I. Radle ; Carsten Krebs ;Squire J. Booker
Journal of the American Chemical Society 2011 Volume 133(Issue 49) pp:19586-19589
Publication Date(Web):September 14, 2011
DOI:10.1021/ja207327v
The radical SAM (RS) proteins RlmN and Cfr catalyze methylation of carbons 2 and 8, respectively, of adenosine 2503 in 23S rRNA. Both reactions are similar in scope, entailing the synthesis of a methyl group partially derived from S-adenosylmethionine (SAM) onto electrophilic sp2-hybridized carbon atoms via the intermediacy of a protein S-methylcysteinyl (mCys) residue. Both proteins contain five conserved Cys residues, each required for turnover. Three cysteines lie in a canonical RS CxxxCxxC motif and coordinate a [4Fe-4S]-cluster cofactor; the remaining two are at opposite ends of the polypeptide. Here we show that each protein contains only the one “radical SAM” [4Fe-4S] cluster and the two remaining conserved cysteines do not coordinate additional iron-containing species. In addition, we show that, while wild-type RlmN bears the C355 mCys residue in its as-isolated state, RlmN that is either engineered to lack the [4Fe-4S] cluster by substitution of the coordinating cysteines or isolated from Escherichia coli cultured under iron-limiting conditions does not bear a C355 mCys residue. Reconstitution of the [4Fe-4S] cluster on wild-type apo RlmN followed by addition of SAM results in rapid production of S-adenosylhomocysteine (SAH) and the mCys residue, while treatment of apo RlmN with SAM affords no observable reaction. These results indicate that in Cfr and RlmN, SAM bound to the unique iron of the [4Fe-4S] cluster displays two reactivities. It serves to methylate C355 of RlmN (C338 of Cfr), or to generate the 5′-deoxyadenosyl 5′-radical, required for substrate-dependent methyl synthase activity.
Co-reporter:Ning Li ; Hanne Nørgaard ; Douglas M. Warui ; Squire J. Booker ; Carsten Krebs ;J. Martin Bollinger ; Jr.
Journal of the American Chemical Society 2011 Volume 133(Issue 16) pp:6158-6161
Publication Date(Web):April 4, 2011
DOI:10.1021/ja2013517
Cyanobacterial aldehyde decarbonylase (AD) catalyzes conversion of fatty aldehydes (R−CHO) to alka(e)nes (R−H) and formate. Curiously, although this reaction appears to be redox-neutral and formally hydrolytic, AD has a ferritin-like protein architecture and a carboxylate-bridged dimetal cofactor that are both structurally similar to those found in di-iron oxidases and oxygenases. In addition, the in vitro activity of the AD from Nostoc punctiforme (Np) was shown to require a reducing system similar to the systems employed by these O2-utilizing di-iron enzymes. Here, we resolve this conundrum by showing that aldehyde cleavage by the Np AD also requires dioxygen and results in incorporation of 18O from 18O2 into the formate product. AD thus oxygenates, without oxidizing, its substrate. We posit that (i) O2 adds to the reduced cofactor to generate a metal-bound peroxide nucleophile that attacks the substrate carbonyl and initiates a radical scission of the C1−C2 bond, and (ii) the reducing system delivers two electrons during aldehyde cleavage, ensuring a redox-neutral outcome, and two additional electrons to return an oxidized form of the cofactor back to the reduced, O2-reactive form.
Co-reporter:Carsten Krebs, J Martin Bollinger Jr, Squire J Booker
Current Opinion in Chemical Biology 2011 Volume 15(Issue 2) pp:291-303
Publication Date(Web):April 2011
DOI:10.1016/j.cbpa.2011.02.019
Enzymes that activate dioxygen at carboxylate-bridged non-heme diiron clusters residing within ferritin-like, four-helix-bundle protein architectures have crucial roles in, among other processes, the global carbon cycle (e.g. soluble methane monooxygenase), fatty acid biosynthesis [plant fatty acyl–acyl carrier protein (ACP) desaturases], DNA biosynthesis [the R2 or β2 subunits of class Ia ribonucleotide reductases (RNRs)], and cellular iron trafficking (ferritins). Classic studies on class Ia RNRs showed long ago how this obligatorily oxidative di-iron/O2 chemistry can be used to activate an enzyme for even a reduction reaction, and more recent investigations of class Ib and Ic RNRs, coupled with earlier studies on dimanganese catalases, have shown that members of this protein family can also incorporate either one or two Mn ions and use them in place of iron for redox catalysis. These two strategies – oxidative activation for non-oxidative reactions and use of alternative metal ions – expand the catalytic repertoire of the family, probably to include activities that remain to be discovered. Indeed, a recent study has suggested that fatty aldehyde decarbonylases (ADs) from cyanobacteria, purported to catalyze a redox-neutral cleavage of a Cn aldehyde to the Cn−1 alkane (or alkene) and CO, also belong to this enzyme family and are most similar in structure to two other members with heterodinuclear (Mn–Fe) cofactors. Here, we first briefly review both the chemical principles underlying the O2-dependent oxidative chemistry of the ‘classical’ di-iron-carboxylate proteins and the two aforementioned strategies that have expanded their functional range, and then consider what metal ion(s) and what chemical mechanism(s) might be employed by the newly discovered cyanobacterial ADs.
Co-reporter:Shengfa Ye ; John C. Price ; Eric W. Barr ; Michael T. Green ; J. Martin Bollinger ; Jr.; Carsten Krebs ;Frank Neese
Journal of the American Chemical Society 2010 Volume 132(Issue 13) pp:4739-4751
Publication Date(Web):March 10, 2010
DOI:10.1021/ja909715g
The Fe(II)- and α-ketoglutarate (αKG)-dependent enzymes are a functionally and mechanistically diverse group of mononuclear nonheme-iron enzymes that activate dioxygen to couple the decarboxylation of αKG, which yields succinate and CO2, to the oxidation of an aliphatic C−H bond of their substrates. Their mechanisms have been studied in detail by a combination of kinetic, spectroscopic, and computational methods. Two reaction intermediates have been trapped and characterized for several members of this enzyme family. The first intermediate is the C−H-cleaving Fe(IV)−oxo complex, which exhibits a large deuterium kinetic isotope effect on its decay. The second intermediate is a Fe(II):product complex. Reaction intermediates proposed to occur before the Fe(IV)−oxo intermediate do not accumulate and therefore cannot be characterized experimentally. One of these intermediates is the initial O2 adduct, which is a {FeO2}8 species in the notation introduced by Enemark and Feltham. Here, we report spectroscopic and computational studies on the stable NO-adduct of taurine:αKG dioxygenase (TauD), termed TauD−{FeNO}7, and its one-electron reduced form, TauD−{FeNO}8. The latter is isoelectronic with the proposed O2 adduct and was generated by low-temperature γ-irradiation of TauD−{FeNO}7. To our knowledge, TauD−{FeNO}8 is the first paramagnetic {FeNO}8 complex. The detailed analysis of experimental and computational results shows that TauD−{FeNO}8 has a triplet ground state. This has mechanistic implications that are discussed in this Article. Annealing of the triplet {FeNO}8 species presumably leads to an equally elusive {FeHNO}8 complex with a quintet ground state.
Co-reporter:Tyler L. Grove, Jessica H. Ahlum, Priya Sharma, Carsten Krebs and Squire J. Booker
Biochemistry 2010 Volume 49(Issue 18) pp:
Publication Date(Web):April 8, 2010
DOI:10.1021/bi9022126
BtrN catalyzes the two-electron oxidation of the C3 secondary alcohol of 2-deoxy-scyllo-inosamine to the corresponding ketone and is a member of a subclass of radical S-adenosylmethionine (SAM) enzymes called radical SAM (RS) dehydrogenases. Like all RS enzymes, BtrN contains a [4Fe-4S] cluster that delivers an electron to SAM, inducing its cleavage to the common intermediate in RS reactions, the 5′-deoxyadenosyl 5′-radical. In this work, we show that BtrN contains an additional [4Fe-4S] cluster, thought to bind in contact with the substrate to facilitate loss of the second electron in the two-electron oxidation.
Co-reporter:Wei Jiang, Jiajia Xie, Paul T. Varano, Carsten Krebs and J. Martin Bollinger Jr.
Biochemistry 2010 Volume 49(Issue 25) pp:
Publication Date(Web):May 12, 2010
DOI:10.1021/bi100037b
Catalysis by a class I ribonucleotide reductase (RNR) begins when a cysteine (C) residue in the α2 subunit is oxidized to a thiyl radical (C•) by a cofactor ∼35 Å away in the β2 subunit. In a class Ia or Ib RNR, a stable tyrosyl radical (Y•) is the C oxidant, whereas a MnIV/FeIII cluster serves this function in the class Ic enzyme from Chlamydia trachomatis (Ct). It is thought that, in either case, a chain of Y residues spanning the two subunits mediates C oxidation by forming transient “pathway” Y•s in a multistep electron transfer (ET) process that is “gated” by the protein so that it occurs only in the ready holoenzyme complex. The drug hydroxyurea (HU) inactivates both Ia/b and Ic β2 subunits by reducing their C oxidants. Reduction of the stable cofactor Y• (Y122•) in Escherichia coli class Ia β2 is faster in the presence of α2 and a substrate (CDP), leading to speculation that HU might intercept a transient ET pathway Y• under these turnover conditions. Here we show that this mechanism is one of two that are operant in HU inactivation of the Ct enzyme. HU reacts with the MnIV/FeIII cofactor to give two distinct products: the previously described homogeneous MnIII/FeIII-β2 complex, which forms only under turnover conditions (in the presence of α2 and the substrate), and a distinct, diamagnetic Mn/Fe cluster, which forms ∼900-fold less rapidly as a second phase in the reaction under turnover conditions and as the sole outcome in the reaction of MnIV/FeIII-β2 only. Formation of MnIII/FeIII-β2 also requires (i) either Y338, the subunit-interfacial ET pathway residue of β2, or Y222, the surface residue that relays the “extra electron” to the MnIV/FeIV intermediate during activation of β2 but is not part of the catalytic ET pathway, and (ii) W51, the cofactor-proximal residue required for efficient ET between either Y222 or Y338 and the cofactor. The combined requirements for the catalytic subunit, the substrate, and, most importantly, a functional surface-to-cofactor electron relay system imply that HU effects the MnIV/FeIII → MnIII/FeIII reduction by intercepting a Y• that forms when the ready holoenzyme complex is assembled, the ET gate is opened, and the MnIV oxidizes either Y222 or Y338.
Co-reporter:J. Martin Bollinger, Jr.;Victoria Korneeva Korboukh;Ning Li
PNAS 2010 Volume 107 (Issue 36 ) pp:15722-15727
Publication Date(Web):2010-09-07
DOI:10.1073/pnas.1002785107
The nonheme di-iron oxygenase, AurF, converts p-aminobenzoate (Ar-NH2, where Ar = 4-carboxyphenyl) to p-nitrobenzoate (Ar-NO2) in the biosynthesis of the antibiotic, aureothin, by Streptomyces thioluteus. It has been reported that this net six-electron oxidation proceeds in three consecutive, two-electron steps, through p-hydroxylaminobenzoate (Ar-NHOH) and p-nitrosobenzoate (Ar-NO) intermediates, with each step requiring one equivalent of O2 and two exogenous reducing equivalents. We recently demonstrated that a peroxodiiron(III/III) complex (peroxo--AurF) formed by addition of O2 to the diiron(II/II) enzyme (-AurF) effects the initial oxidation of Ar-NH2, generating a μ-(oxo)diiron(III/III) form of the enzyme (μ-oxo--AurF) and (presumably) Ar-NHOH. Here we show that peroxo--AurF also oxidizes Ar-NHOH. Unexpectedly, this reaction proceeds through to the Ar-NO2 final product, a four-electron oxidation, and produces -AurF, with which O2 can combine to regenerate peroxo--AurF. Thus, conversion of Ar-NHOH to Ar-NO2 requires only a single equivalent of O2 and (starting from -AurF or peroxo--AurF) is fully catalytic in the absence of exogenous reducing equivalents, by contrast to the published stoichiometry. This
novel type of four-electron N-oxidation is likely also to occur in the reaction sequences of nitro-installing di-iron amine oxygenases in the biosyntheses
of other natural products.
Co-reporter:Victoria Korneeva Korboukh ; Ning Li ; Eric W. Barr ; J. Martin Bollinger ; Jr.
Journal of the American Chemical Society 2009 Volume 131(Issue 38) pp:13608-13609
Publication Date(Web):September 4, 2009
DOI:10.1021/ja9064969
The amine oxygenase AurF from Streptomyces thioluteus catalyzes the six-electron oxidation of p-aminobenzoate (pABA) to p-nitrobenzoate (pNBA). In this work, we have studied the reaction of its reduced Fe2(II/II) cofactor with O2, which results in generation of a peroxo-Fe2(III/III) intermediate. In the absence of substrate, this intermediate is unusually stable (t1/2 = 7 min at 20 °C), allowing for its accumulation to almost stoichiometric amounts. Its decay is accelerated ∼105-fold by the substrate, pABA, implying that it is the complex that effects the two-electron oxidation of the amine to the hydroxylamine. The nearly quantitative conversion of pABA to pNBA by solutions containing an excess of the intermediate suggests that it may also be competent for the two subsequent two-electron oxidations leading to the product.
Co-reporter:J. Martin Bollinger, Yinghui Diao, Megan L. Matthews, Gang Xing and Carsten Krebs
Dalton Transactions 2009 (Issue 6) pp:905-914
Publication Date(Web):26 Nov 2008
DOI:10.1039/B811885J
The enzyme myo-inositol oxygenase (MIOX) catalyzes conversion of myo-inositol (cyclohexan-1,2,3,5/4,6-hexa-ol or MI) to D-glucuronate (DG), initiating the only known pathway in humans for catabolism of the carbon skeleton of cell-signaling inositol (poly)phosphates and phosphoinositides. Recent kinetic, spectroscopic and crystallographic studies have shown that the enzyme activates its substrates, MI and O2, at a carboxylate-bridged nonheme diiron(II/III) cluster, making it the first of many known nonheme diiron oxygenases to employ the mixed-valent form of its cofactor. Evidence suggests that: (1) the Fe(III) site coordinates MI via its C1 and C6 hydroxyl groups; (2) the Fe(II) site reversibly coordinates O2 to produce a superoxo-diiron(III/III) intermediate; and (3) the pendant oxygen atom of the superoxide ligand abstracts hydrogen from C1 to initiate the unique C–C-bond-cleaving, four-electron oxidation reaction. This review recounts the studies leading to the recognition of the novel cofactor requirement and catalytic mechanism of MIOX and forecasts how remaining gaps in our understanding might be filled by additional experiments.
Co-reporter:Megan L. Matthews, Courtney M. Krest, Eric W. Barr, Frédéric H. Vaillancourt, Christopher T. Walsh, Michael T. Green, Carsten Krebs and J. Martin Bollinger Jr.
Biochemistry 2009 Volume 48(Issue 20) pp:
Publication Date(Web):February 26, 2009
DOI:10.1021/bi900109z
Aliphatic halogenases activate O2, cleave α-ketoglutarate (αKG) to CO2 and succinate, and form haloferryl [X−Fe(IV)═O; X = Cl or Br] complexes that cleave aliphatic C−H bonds to install halogens during the biosynthesis of natural products by non-ribosomal peptide synthetases (NRPSs). For the related αKG-dependent dioxygenases, it has been shown that reaction of the Fe(II) cofactor with O2 to form the C−H bond-cleaving ferryl complex is “triggered” by binding of the target substrate. In this study, we have tested for and defined structural determinants of substrate triggering (ST) in the halogenase, SyrB2, from the syringomycin E biosynthetic NRPS of Pseudomonas syringae B301D. As for other halogenases, the substrate of SyrB2 is complex, consisting of l-Thr tethered via a thioester linkage to a covalently bound phosphopantetheine (PPant) cofactor of a carrier protein, SyrB1. Without an appended amino acid, SyrB1 does not trigger formation of the chloroferryl intermediate state in SyrB2, even in the presence of free l-Thr or its analogues, but SyrB1 charged either by l-Thr (l-Thr-S-SyrB1) or by any of several non-native amino acids does trigger the reaction by as much as 8000-fold (for the native substrate). Triggering efficacy is sensitive to the structures of both the amino acid and the carrier protein, being diminished by 5−24-fold when the native l-Thr is replaced with another amino acid and by ∼40-fold when SyrB1 is replaced with the heterologous carrier protein, CytC2. The directing effect of the carrier protein and consequent tolerance for profound modifications to the target amino acid allow the chloroferryl state to be formed in the presence of substrates that perturb the ratio of its two putative coordination isomers, lack the target C−H bond (l-Ala-S-SyrB1), or contain a C−H bond of enhanced strength (l-cyclopropylglycyl-S-SyrB1). For the latter two cases, the SyrB2 chloroferryl state so formed exhibits unprecedented stability (t1/2 = 30−110 min at 0 °C), can be trapped at high concentration and purity by manual freezing without a cryosolvent, and represents an ideal target for structural characterization. As initial steps toward this goal, extended X-ray absorption fine structure (EXAFS) spectroscopy has been used to determine the Fe−O and Fe−Cl distances and density functional theory (DFT) calculations have been used to confirm that the measured distances are consistent with the anticipated structure of the intermediate.
Co-reporter:Kyung-Hoon Lee, Lana Saleh, Brian P. Anton, Catherine L. Madinger, Jack S. Benner, David F. Iwig, Richard J. Roberts, Carsten Krebs and Squire J. Booker
Biochemistry 2009 Volume 48(Issue 42) pp:
Publication Date(Web):September 8, 2009
DOI:10.1021/bi900939w
RimO, encoded by the yliG gene in Escherichia coli, has been recently identified in vivo as the enzyme responsible for the attachment of a methylthio group on the β-carbon of Asp88 of the small ribosomal protein S12 [Anton, B. P., Saleh, L., Benner, J. S., Raleigh, E. A., Kasif, S., and Roberts, R. J. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 1826−1831]. To date, it is the only enzyme known to catalyze methylthiolation of a protein substrate; the four other naturally occurring methylthio modifications have been observed on tRNA. All members of the methylthiotransferase (MTTase) family, to which RimO belongs, have been shown to contain the canonical CxxxCxxC motif in their primary structures that is typical of the radical S-adenosylmethionine (SAM) family of proteins. MiaB, the only characterized MTTase, and the enzyme experimentally shown to be responsible for methylthiolation of N6-isopentenyladenosine of tRNA in E. coli and Thermotoga maritima, has been demonstrated to harbor two distinct [4Fe-4S] clusters. Herein, we report in vitro biochemical and spectroscopic characterization of RimO. We show by analytical and spectroscopic methods that RimO, overproduced in E. coli in the presence of iron−sulfur cluster biosynthesis proteins from Azotobacter vinelandii, contains one [4Fe-4S]2+ cluster. Reconstitution of this form of RimO (RimOrcn) with 57Fe and sodium sulfide results in a protein that contains two [4Fe-4S]2+ clusters, similar to MiaB. We also show by mass spectrometry that RimOrcn catalyzes the attachment of a methylthio group to a peptide substrate analogue that mimics the loop structure bearing aspartyl 88 of the S12 ribosomal protein from E. coli. Kinetic analysis of this reaction shows that the activity of RimOrcn in the presence of the substrate analogue does not support a complete turnover. We discuss the possible requirement for an assembled ribosome for fully active RimO in vitro. Our findings are consistent with those of other enzymes that catalyze sulfur insertion, such as biotin synthase, lipoyl synthase, and MiaB.
Co-reporter:Megan L. Matthews;Christopher S. Neumann;Linde A. Miles;Tyler L. Grove;Squire J. Booker;Christopher T. Walsh;J. Martin Bollinger, Jr.
PNAS 2009 Volume 106 (Issue 42 ) pp:17723-17728
Publication Date(Web):2009-10-20
DOI:10.1073/pnas.0909649106
The α-ketoglutarate-dependent hydroxylases and halogenases employ similar reaction mechanisms involving hydrogen-abstracting
Fe(IV)-oxo (ferryl) intermediates. In the halogenases, the carboxylate residue from the His2(Asp/Glu)1“facial triad” of iron ligands found in the hydroxylases is replaced by alanine, and a halide ion (X−) coordinates at the vacated site. Halogenation is thought to result from “rebound” of the halogen radical from the X-Fe(III)-OH
intermediate produced by hydrogen (H•) abstraction to the substrate radical. The alternative decay pathway for the X-Fe(III)-OH intermediate, rebound of the hydroxyl
radical to the substrate radical (as occurs in the hydroxylases), reportedly does not compete. Here we show for the halogenase
SyrB2 that positioning of the alkyl group of the substrate away from the oxo/hydroxo ligand and closer to the halogen ligand
sacrifices H•-abstraction proficiency for halogen-rebound selectivity. Upon replacement of l-Thr, the C4 amino acid tethered to the SyrB1 carrier protein in the native substrate, by the C5 amino acid l-norvaline, decay of the chloroferryl intermediate becomes 130× faster and the reaction outcome switches to primarily hydroxylation
of C5, consistent with projection of the methyl group closer to the oxo/hydroxo by the longer side chain. Competing H• abstraction from C4 results primarily in chlorination, as occurs at this site in the native substrate. Consequently, deuteration
of C5, which slows attack at this site, switches both the regioselectivity from C5 to C4 and the chemoselectivity from hydroxylation
to chlorination. Thus, substrate-intermediate disposition and the carboxylate → halide ligand swap combine to specify the
halogenation outcome.
Co-reporter:Carsten Krebs;J. Martin Bollinger Jr.
Photosynthesis Research 2009 Volume 102( Issue 2-3) pp:
Publication Date(Web):2009 December
DOI:10.1007/s11120-009-9406-6
57Fe-Mössbauer spectroscopy is a method that probes transitions between the nuclear ground state (I = 1/2) and the first nuclear excited state (I = 3/2). This technique provides detailed information about the chemical environment and electronic structure of iron. Therefore, it has played an important role in studies of the numerous iron-containing proteins and enzymes. In conjunction with the freeze-quench method, 57Fe-Mössbauer spectroscopy allows for monitoring changes of the iron site(s) during a biochemical reaction. This approach is particularly powerful for detection and characterization of reactive intermediates. Comparison of experimentally determined Mössbauer parameters to those predicted by density functional theory for hypothetical model structures can then provide detailed insight into the structures of reactive intermediates. We have recently used this methodology to study the reactions of various mononuclear non-heme-iron enzymes by trapping and characterizing several Fe(IV)-oxo reaction intermediates. In this article, we summarize these findings and demonstrate the potential of the method.
Co-reporter:Allison H. Saunders, Amy E. Griffiths, Kyung-Hoon Lee, Robert M. Cicchillo, Loretta Tu, Jeffrey A. Stromberg, Carsten Krebs and Squire J. Booker
Biochemistry 2008 Volume 47(Issue 41) pp:
Publication Date(Web):September 20, 2008
DOI:10.1021/bi801268f
Quinolinate synthase (NadA) catalyzes a unique condensation reaction between iminoaspartate and dihydroxyacetone phosphate, affording quinolinic acid, a central intermediate in the biosynthesis of nicotinamide adenine dinucleotide (NAD). Iminoaspartate is generated via the action of l-aspartate oxidase (NadB), which catalyzes the first step in the biosynthesis of NAD in most prokaryotes. NadA from Escherichia coli was hypothesized to contain an iron−sulfur cluster as early as 1991, because of its observed labile activity, especially in the presence of hyperbaric oxygen, and because its primary structure contained a CXXCXXC motif, which is commonly found in the [4Fe-4S] ferredoxin class of iron−sulfur (Fe/S) proteins. Indeed, using analytical methods in concert with Mössbauer and electron paramagnetic resonance spectroscopies, the protein was later shown to harbor a [4Fe-4S] cluster. Recently, the X-ray structure of NadA from Pyrococcus horikoshii was solved to 2.0 Å resolution [Sakuraba, H., Tsuge, H.,Yoneda, K., Katunuma, N., and Ohshima, T. (2005) J. Biol. Chem. 280, 26645−26648]. This protein does not contain a CXXCXXC motif, and no Fe/S cluster was observed in the structure or even mentioned in the report. Moreover, rates of quinolinic acid production were reported to be 2.2 μmol min−1 mg−1, significantly greater than that of E. coli NadA containing an Fe/S cluster (0.10 μmol min−1 mg−1), suggesting that the [4Fe-4S] cluster of E. coli NadA may not be necessary for catalysis. In the study described herein, nadA genes from both Mycobacterium tuberculosis and Pyrococcus horikoshii were cloned, and their protein products shown to contain [4Fe-4S] clusters that are absolutely required for activity despite the absence of a CXXCXXC motif in their primary structures. Moreover, E. coli NadA, which contains nine cysteine residues, is shown to require only three for turnover (C113, C200, and C297), of which only C297 resides in the CXXCXXC motif. These results are consistent with a bioinformatics analysis of NadA sequences, which indicates that three cysteines are strictly conserved across all species. This study concludes that all currently annotated quinolinate synthases harbor a [4Fe-4S] cluster, that the crystal structure reported by Sakuraba et al. does not accurately represent the active site of the protein, and that the “activity” reported does not correspond to quinolinate formation.
Co-reporter:Wei Jiang, Jiajia Xie, Hanne Nørgaard, J. Martin Bollinger Jr. and Carsten Krebs
Biochemistry 2008 Volume 47(Issue 15) pp:
Publication Date(Web):March 22, 2008
DOI:10.1021/bi702085z
We recently showed that the class Ic ribonucleotide reductase (RNR) from the human pathogen Chlamydia trachomatis (Ct) uses a MnIV/FeIII cofactor in its R2 subunit to initiate catalysis [Jiang, W., Yun, D., Saleh, L., Barr, E. W., Xing, G., Hoffart, L. M., Maslak, M.-A., Krebs, C., and Bollinger, J. M., Jr. (2007) Science 316, 1188–1191]. The MnIV site of the novel cofactor functionally replaces the tyrosyl radical used by conventional class I RNRs to initiate substrate radical production. As a first step in evaluating the hypothesis that the use of the alternative cofactor could make the RNR more robust to reactive oxygen and nitrogen species [RO(N)S] produced by the host’s immune system [Högbom, M., Stenmark, P., Voevodskaya, N., McClarty, G., Gräslund, A., and Nordlund, P. (2004) Science 305, 245–248], we have examined the reactivities of three stable redox states of the Mn/Fe cluster (MnII/FeII, MnIII/FeIII, and MnIV/FeIII) toward hydrogen peroxide. Not only is the activity of the MnIV/FeIII−R2 intermediate stable to prolonged (>1 h) incubations with as much as 5 mM H2O2, but both the fully reduced (MnII/FeII) and one-electron-reduced (MnIII/FeIII) forms of the protein are also efficiently activated by H2O2. The MnIII/FeIII−R2 species reacts with a second-order rate constant of 8 ± 1 M−1 s−1 to yield the MnIV/FeIV−R2 intermediate previously observed in the reaction of MnII/FeII−R2 with O2 [Jiang, W., Hoffart, L. M., Krebs, C., and Bollinger, J. M., Jr. (2007) Biochemistry 46, 8709–8716]. As previously observed, the intermediate decays by reduction of the Fe site to the active MnIV/FeIII−R2 complex. The reaction of the MnII/FeII−R2 species with H2O2 proceeds in three resolved steps: sequential oxidation to MnIII/FeIII−R2 (k = 1.7 ± 0.3 mM−1 s−1) and MnIV/FeIV−R2, followed by decay of the intermediate to the active MnIV/FeIII−R2 product. The efficient reaction of both reduced forms with H2O2 contrasts with previous observations on the conventional class I RNR from Escherichia coli, which is efficiently converted from the fully reduced (Fe2II/II) to the “met” (Fe2III/III) form [Gerez, C., and Fontecave, M. (1992) Biochemistry 31, 780–786] but is then only very inefficiently converted from the met to the active (Fe2III/III−Y•) form [Sahlin, M., Sjöberg, B.-M., Backes, G., Loehr, T., and Sanders-Loehr, J. (1990) Biochem. Biophys. Res. Commun. 167, 813–818].
Co-reporter:Tyler L. Grove, Kyung-Hoon Lee, Jennifer St. Clair, Carsten Krebs and Squire J. Booker
Biochemistry 2008 Volume 47(Issue 28) pp:
Publication Date(Web):June 18, 2008
DOI:10.1021/bi8004297
Sulfatases catalyze the cleavage of a variety of cellular sulfate esters via a novel mechanism that requires the action of a protein-derived formylglycine cofactor. Formation of the cofactor is catalyzed by an accessory protein and involves the two-electron oxidation of a specific cysteinyl or seryl residue on the relevant sulfatase. Although some sulfatases undergo maturation via mechanisms in which oxygen serves as an electron acceptor, AtsB, the maturase from Klebsiella pneumoniae, catalyzes the oxidation of Ser72 on AtsA, its cognate sulfatase, via an oxygen-independent mechanism. Moreover, it does not make use of pyridine or flavin nucleotide cofactors as direct electron acceptors. In fact, AtsB has been shown to be a member of the radical S-adenosylmethionine superfamily of proteins, suggesting that it catalyzes this oxidation via an intermediate 5′-deoxyadenosyl 5′-radical that is generated by a reductive cleavage of S-adenosyl-l-methionine. In contrast to AtsA, very little in vitro characterization of AtsB has been conducted. Herein we show that coexpression of the K. pneumoniae atsB gene with a plasmid that encodes genes that are known to be involved in iron−sulfur cluster biosynthesis yields soluble protein that can be characterized in vitro. The as-isolated protein contained 8.7 ± 0.4 irons and 12.2 ± 2.6 sulfides per polypeptide, which existed almost entirely in the [4Fe-4S]2+ configuration, as determined by Mössbauer spectroscopy, suggesting that it contained at least two of these clusters per polypeptide. Reconstitution of the as-isolated protein with additional iron and sulfide indicated the presence of 12.3 ± 0.2 irons and 9.9 ± 0.4 sulfides per polypeptide. Subsequent characterization of the reconstituted protein by Mössbauer spectroscopy indicated the presence of only [4Fe-4S] clusters, suggesting that reconstituted AtsB contains three per polypeptide. Consistent with this stoichiometry, an as-isolated AtsB triple variant containing Cys → Ala substitutions at each of the cysteines in its CX3CX2C radical SAM motif contained 7.3 ± 0.1 irons and 7.2 ± 0.2 sulfides per polypeptide while the reconstituted triple variant contained 7.7 ± 0.1 irons and 8.4 ± 0.4 sulfides per polypeptide, indicating that it was unable to incorporate an additional cluster. UV−visible and Mössbauer spectra of both samples indicated the presence of only [4Fe-4S] clusters. AtsB was capable of catalyzing multiple turnovers and exhibited a Vmax/[ET] of ∼0.36 min−1 for an 18-amino acid peptide substrate using dithionite to supply the requisite electron and a value of ∼0.039 min−1 for the same substrate using the physiologically relevant flavodoxin reducing system. Simultaneous quantification of formylglycine and 5′-deoxyadenosine as a function of time indicates an approximate 1:1 stoichiometry. Use of a peptide substrate in which the target serine is changed to cysteine also gives rise to turnover, supporting approximately 4-fold the activity of that observed with the natural substrate.
Co-reporter:Wei Jiang, Lana Saleh, Eric W. Barr, Jiajia Xie, Monique Maslak Gardner, Carsten Krebs and J. Martin Bollinger Jr.
Biochemistry 2008 Volume 47(Issue 33) pp:
Publication Date(Web):July 26, 2008
DOI:10.1021/bi800881m
A conventional class I (subclass a or b) ribonucleotide reductase (RNR) employs a tyrosyl radical (Y•) in its R2 subunit for reversible generation of a 3′-hydrogen-abstracting cysteine radical in its R1 subunit by proton-coupled electron transfer (PCET) through a network of aromatic amino acids spanning the two subunits. The class Ic RNR from the human pathogen Chlamydia trachomatis (Ct) uses a MnIV/FeIII cofactor (specifically, the MnIV ion) in place of the Y• for radical initiation. Ct R2 is activated when its MnII/FeII form reacts with O2 to generate a MnIV/FeIV intermediate, which decays by reduction of the FeIV site to the active MnIV/FeIII state. Here we show that the reduction step in this sequence is mediated by residue Y222. Substitution of Y222 with F retards the intrinsic decay of the MnIV/FeIV intermediate by ∼10-fold and diminishes the ability of ascorbate to accelerate the decay by ∼65-fold but has no detectable effect on the catalytic activity of the MnIV/FeIII−R2 product. By contrast, substitution of Y338, the cognate of the subunit interfacial R2 residue in the R1 ⇔ R2 PCET pathway of the conventional class I RNRs [Y356 in Escherichia coli (Ec) R2], has almost no effect on decay of the MnIV/FeIV intermediate but abolishes catalytic activity. Substitution of W51, the Ct R2 cognate of the cofactor-proximal R1 ⇔ R2 PCET pathway residue in the conventional class I RNRs (W48 in Ec R2), both retards reduction of the MnIV/FeIV intermediate and abolishes catalytic activity. These observations imply that Ct R2 has evolved branched pathways for electron relay to the cofactor during activation and catalysis. Other R2s predicted also to employ the Mn/Fe cofactor have Y or W (also competent for electron relay) aligning with Y222 of Ct R2. By contrast, many R2s known or expected to use the conventional Y•-based system have redox-inactive L or F residues at this position. Thus, the presence of branched activation- and catalysis-specific electron relay pathways may be functionally important uniquely in the Mn/Fe-dependent class Ic R2s.
Co-reporter:Wei Jiang, Danny Yun, Lana Saleh, J. Martin Bollinger Jr. and Carsten Krebs
Biochemistry 2008 Volume 47(Issue 52) pp:13736-13744
Publication Date(Web):December 5, 2008
DOI:10.1021/bi8017625
The β2 subunit of a class Ia or Ib ribonucleotide reductase (RNR) is activated when its carboxylate-bridged Fe2II/II cluster reacts with O2 to oxidize a nearby tyrosine (Y) residue to a stable radical (Y•). During turnover, the Y• in β2 is thought to reversibly oxidize a cysteine (C) in the α2 subunit to a thiyl radical (C•) by a long-distance (∼35 Å) proton-coupled electron-transfer (PCET) step. The C• in α2 then initiates reduction of the 2′ position of the ribonucleoside 5′-diphosphate substrate by abstracting the hydrogen atom from C3′. The class I RNR from Chlamydia trachomatis (Ct) is the prototype of a newly recognized subclass (Ic), which is characterized by the presence of a phenylalanine (F) residue at the site of β2 where the essential radical-harboring Y is normally found. We recently demonstrated that Ct RNR employs a heterobinuclear MnIV/FeIII cluster for radical initiation. In essence, the MnIV ion of the cluster functionally replaces the Y• of the conventional class I RNR. The Ct β2 protein also autoactivates by reaction of its reduced (MnII/FeII) metal cluster with O2. In this reaction, an unprecedented MnIV/FeIV intermediate accumulates almost stoichiometrically and decays by one-electron reduction of the FeIV site. This reduction is mediated by the near-surface residue, Y222, a residue with no functional counterpart in the well-studied conventional class I RNRs. In this review, we recount the discovery of the novel Mn/Fe redox cofactor in Ct RNR and summarize our current understanding of how it assembles and initiates nucleotide reduction.
Co-reporter:Carsten Krebs, Danica Galonić Fujimori, Christopher T. Walsh and J. Martin Bollinger Jr.
Accounts of Chemical Research 2007 Volume 40(Issue 7) pp:484
Publication Date(Web):June 2, 2007
DOI:10.1021/ar700066p
High-valent non-heme iron–oxo intermediates have been proposed for decades as the key intermediates in numerous biological oxidation reactions. In the past three years, the first direct characterization of such intermediates has been provided by studies of several αKG-dependent oxygenases that catalyze either hydroxylation or halogenation of their substrates. In each case, the Fe(IV)–oxo intermediate is implicated in cleavage of the aliphatic C–H bond to initiate hydroxylation or halogenation. The observation of non-heme Fe(IV)–oxo intermediates and Fe(II)-containing product(s) complexes with almost identical spectroscopic parameters in the reactions of two distantly related αKG-dependent hydroxylases suggests that members of this subfamily follow a conserved mechanism for substrate hydroxylation. In contrast, for the αKG-dependent non-heme iron halogenase, CytC3, two distinct Fe(IV) complexes form and decay together, suggesting that they are in rapid equilibrium. The existence of two distinct conformers of the Fe site may be the key factor accounting for the divergence of the halogenase reaction from the more usual hydroxylation pathway after C–H bond cleavage. Distinct transformations catalyzed by other mononuclear non-heme enzymes are likely also to involve initial C–H bond cleavage by Fe(IV)–oxo complexes, followed by diverging reactivities of the resulting Fe(III)–hydroxo/substrate radical intermediates.
Co-reporter:Wei Jiang;Danny Yun;Lana Saleh;Eric W. Barr;Gang Xing;Lee M. Hoffart;Monique-Anne Maslak;J. Martin Bollinger Jr.
Science 2007 Volume 316(Issue 5828) pp:
Publication Date(Web):
DOI:10.1126/science.1141179
Abstract
In a conventional class I ribonucleotide reductase (RNR), a diiron(II/II) cofactor in the R2 subunit reacts with oxygen to produce a diiron(III/IV) intermediate, which generates a stable tyrosyl radical (Y⚫). The Y⚫ reversibly oxidizes a cysteine residue in the R1 subunit to a cysteinyl radical (C⚫), which abstracts the 3′-hydrogen of the substrate to initiate its reduction. The RNR from Chlamydia trachomatis lacks the Y⚫, and it had been proposed that the diiron(III/IV) complex in R2 directly generates the C⚫ in R1. By enzyme activity measurements and spectroscopic methods, we show that this RNR actually uses a previously unknown stable manganese(IV)/iron(III) cofactor for radical initiation.
Co-reporter:J. Martin Bollinger Jr.;John C. Price;Lee M. Hoffart;Eric W. Barr
European Journal of Inorganic Chemistry 2005 Volume 2005(Issue 21) pp:
Publication Date(Web):16 SEP 2005
DOI:10.1002/ejic.200500476
The iron(II)- and α-ketoglutarate-dependent dioxygenases comprise enzymes that catalyze a variety of important reactions in biology, including steps in the biosynthesis of collagen and antibiotics, the degradation of xenobiotics, the repair of alkylated DNA, and the sensing of oxygen and response to hypoxia. In these reactions, the reductive activation of oxygen is coupled to hydroxylation of the substrate and decarboxylation of the co-substrate, α-ketoglutarate. It is believed that a single, conserved mechanistic pathway for formation of a high-valent iron intermediate that attacks the substrate is operant in all members of this family. Application of a combination of rapid kinetic and spectroscopic techniques to the reaction of taurine/α-ketoglutarate dioxygenase (TauD), one member of this large enzyme family, has led to the detection of two reaction intermediates. The first intermediate, which is termed J, is a high-spin FeIV-oxo complex. Decay of J exhibits a large, normal C1 deuterium kinetic isotope effect, demonstrating that it is the species activating the C–H bond for hydroxylation. The second intermediate is an FeII-containing product(s) complex. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)
Co-reporter:Maria-Eirini Pandelia, Nicholas D. Lanz, Squire J. Booker, Carsten Krebs
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research (June 2015) Volume 1853(Issue 6) pp:
Publication Date(Web):1 June 2015
DOI:10.1016/j.bbamcr.2014.12.005
•Mössbauer spectroscopy is pivotal for characterizing Fe/S clusters.•Nuclearity and redox states of Fe/S cofactors can be uniquely addressed.•Geometric and electronic properties of Fe metallocofactors can be established.•Unparallel insight into novel ‘non-traditional’ polynuclear Fe/S cluster forms.•Mössbauer studies on Fe/S centers can be carried out both in-vivo and in-vitro.Iron-sulfur (Fe/S) clusters are structurally and functionally diverse cofactors that are found in all domains of life. 57Fe Mössbauer spectroscopy is a technique that provides information about the chemical nature of all chemically distinct Fe species contained in a sample, such as Fe oxidation and spin state, nuclearity of a cluster with more than one metal ion, electron spin ground state of the cluster, and delocalization properties in mixed-valent clusters. Moreover, the technique allows for quantitation of all Fe species, when it is used in conjunction with electron paramagnetic resonance (EPR) spectroscopy and analytical methods. 57Fe-Mössbauer spectroscopy played a pivotal role in unraveling the electronic structures of the “well-established” [2Fe-2S]2+/+, [3Fe-4S]1+/0, and [4Fe-4S]3+/2+/1+/0 clusters and -more-recently- was used to characterize novel Fe/S clustsers, including the [4Fe-3S] cluster of the O2-tolerant hydrogenase from Aquifex aeolicus and the 3Fe-cluster intermediate observed during the reaction of lipoyl synthase, a member of the radical SAM enzyme superfamily.
Co-reporter:J. Martin Bollinger, Yinghui Diao, Megan L. Matthews, Gang Xing and Carsten Krebs
Dalton Transactions 2009(Issue 6) pp:NaN914-914
Publication Date(Web):2008/11/26
DOI:10.1039/B811885J
The enzyme myo-inositol oxygenase (MIOX) catalyzes conversion of myo-inositol (cyclohexan-1,2,3,5/4,6-hexa-ol or MI) to D-glucuronate (DG), initiating the only known pathway in humans for catabolism of the carbon skeleton of cell-signaling inositol (poly)phosphates and phosphoinositides. Recent kinetic, spectroscopic and crystallographic studies have shown that the enzyme activates its substrates, MI and O2, at a carboxylate-bridged nonheme diiron(II/III) cluster, making it the first of many known nonheme diiron oxygenases to employ the mixed-valent form of its cofactor. Evidence suggests that: (1) the Fe(III) site coordinates MI via its C1 and C6 hydroxyl groups; (2) the Fe(II) site reversibly coordinates O2 to produce a superoxo-diiron(III/III) intermediate; and (3) the pendant oxygen atom of the superoxide ligand abstracts hydrogen from C1 to initiate the unique C–C-bond-cleaving, four-electron oxidation reaction. This review recounts the studies leading to the recognition of the novel cofactor requirement and catalytic mechanism of MIOX and forecasts how remaining gaps in our understanding might be filled by additional experiments.
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