Spore photoproduct lyase is a radical S-adenosyl-l-methionine (SAM) enzyme with the unusual property that addition of SAM to the [4Fe-4S]1+ enzyme absent substrate results in rapid electron transfer to SAM with accompanying homolytic S–C5′ bond cleavage. Herein, we demonstrate that this unusual reaction forms the organometallic intermediate Ω in which the unique Fe atom of the [4Fe-4S] cluster is bound to C5′ of the 5′-deoxyadenosyl radical (5′-dAdo•). During catalysis, homolytic cleavage of the Fe–C5′ bond liberates 5′-dAdo• for reaction with substrate, but here, we use Ω formation without substrate to determine the thermal stability of Ω. The reaction of Geobacillus thermodenitrificans SPL (GtSPL) with SAM forms Ω within ∼15 ms after mixing. By monitoring the decay of Ω through rapid freeze–quench trapping at progressively longer times we find an ambient temperature decay time of the Ω Fe–C5′ bond of τ ≈ 5–6 s, likely shortened by enzymatic activation as is the case with the Co–C5′ bond of B12. We have further used hand quenching at times up to 10 min, and thus with multiple SAM turnovers, to probe the fate of the 5′-dAdo• radical liberated by Ω. In the absence of substrate, Ω undergoes low-probability conversion to a stable protein radical. The WT enzyme with valine at residue 172 accumulates a Val•; mutation of Val172 to isoleucine or cysteine results in accumulation of an Ile• or Cys• radical, respectively. The structures of the radical in WT, V172I, and V172C variants have been established by detailed EPR/DFT analyses.
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTstar-Porphyrazines: synthetic, structural, and spectral investigation of complexes of the polynucleating porphyrazineoctathiolato ligandChristopher S. Velazquez, Glenn A. Fox, William E. Broderick, Kevin A. Andersen, Oren P. Anderson, Anthony G. M. Barrett, and Brian M. HoffmanCite this: J. Am. Chem. Soc. 1992, 114, 19, 7416–7424Publication Date (Print):September 1, 1992Publication History Published online1 May 2002Published inissue 1 September 1992https://doi.org/10.1021/ja00045a013Request reuse permissionsArticle Views447Altmetric-Citations96LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (2 MB) Get e-AlertscloseSupporting Info (1)»Supporting Information Supporting Information Get e-Alerts
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTThe mechanism of spin coupling in metallocenium charge-transfer salts: ferromagnetism in decamethylchromocenium tetracyanoquinodimethanideWilliam E. Broderick and Brian M. HoffmanCite this: J. Am. Chem. Soc. 1991, 113, 16, 6334–6335Publication Date (Print):July 1, 1991Publication History Published online1 May 2002Published inissue 1 July 1991https://pubs.acs.org/doi/10.1021/ja00016a095https://doi.org/10.1021/ja00016a095research-articleACS PublicationsRequest reuse permissionsArticle Views125Altmetric-Citations73LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
Maturation of [FeFe]-hydrogenase (HydA) involves synthesis of a CO, CN- , and dithiomethylamine (DTMA)-coordinated 2Fe subcluster that is inserted into HydA to make the active hydrogenase. This process requires three maturation enzymes: the radical S-adenosyl-l-methionine (SAM) enzymes HydE and HydG, and the GTPase HydF. In vitro maturation with purified maturation enzymes has been possible only when clarified cell lysate was added, with the lysate presumably providing essential components for DTMA synthesis and delivery. Here we report maturation of [FeFe]-hydrogenase using a fully defined system that includes components of the glycine cleavage system (GCS), but no cell lysate. Our results reveal for the first time an essential role for the aminomethyl-lipoyl-H-protein of the GCS in hydrogenase maturation and the synthesis of the DTMA ligand of the H-cluster. In addition, we show that ammonia is the source of the bridgehead nitrogen of DTMA.
Abstract The oxidation of the square‐pyramidal five‐coordinate Co(II) complex (I) in an aqueous solution containing 0.8 equiv. of KCN with one equiv. of H 2 O 2 leads to the isolated Co(III) complex (II).
Pyruvate formate-lyase activating enzyme (PFL-AE) is a representative member of an emerging family of enzymes that utilize iron−sulfur clusters and S-adenosylmethionine (AdoMet) to initiate radical catalysis. Although these enzymes have diverse functions, evidence is emerging that they operate by a common mechanism in which a [4Fe−4S]+ interacts with AdoMet to generate a 5'-deoxyadenosyl radical intermediate. To date, however, it has been unclear whether the iron−sulfur cluster is a simple electron-transfer center or whether it participates directly in the radical generation chemistry. Here we utilize electron paramagnetic resonance (EPR) and pulsed 35 GHz electron-nuclear double resonance (ENDOR) spectroscopy to address this question. EPR spectroscopy reveals a dramatic effect of AdoMet on the EPR spectrum of the [4Fe−4S]+ of PFL-AE, changing it from rhombic (g = 2.02, 1.94, 1.88) to nearly axial (g = 2.01, 1.88, 1.87). 2H and 13C ENDOR spectroscopy was performed on [4Fe−4S]+-PFL-AE (S = 1/2) in the presence of AdoMet labeled at the methyl position with either 2H or 13C (denoted [1+/AdoMet]). The observation of a substantial 2H coupling of ∼1 MHz (∼6−7 MHz for 1H), as well as hyperfine-split signals from the 13C, manifestly require that AdoMet lie close to the cluster. 2H and 13C ENDOR data were also obtained for the interaction of AdoMet with the diamagnetic [4Fe−4S]2+ state of PFL-AE, which is visualized through cryoreduction of the frozen [4Fe−4S]2+/AdoMet complex to form the reduced state (denoted [2+/AdoMet]red) trapped in the structure of the oxidized state. 2H and 13C ENDOR spectra for [2+/AdoMet]red are essentially identical to those obtained for the [1+/AdoMet] samples, showing that the cofactor binds in the same geometry to both the 1+ and 2+ states of PFL-AE. Analysis of 2D field-frequency 13C ENDOR data reveals an isotropic hyperfine contribution, which requires that AdoMet lie in contact with the cluster, weakly interacting with it through an incipient bond/antibond. From the anisotropic hyperfine contributions for the 2H and 13C ENDOR, we have estimated the distance from the closest methyl proton of AdoMet to the closest iron of the cluster to be ∼3.0−3.8 Å, while the distance from the methyl carbon to the nearest iron is ∼4−5 Å. We have used this information to construct a model for the interaction of AdoMet with the [4Fe−4S]2+/+ cluster of PFL-AE and have proposed a mechanism for radical generation that is consistent with these results.
ConspectusThe seeds for recognition of the vast superfamily of radical S-adenosyl-l-methionine (SAM) enzymes were sown in the 1960s, when Joachim Knappe found that the dissimilation of pyruvate was dependent on SAM and Fe(II), and Barker and co-workers made similar observations for lysine 2,3-aminomutase. These intriguing observations, coupled with the evidence that SAM and Fe were cofactors in radical catalysis by these enzyme systems, drew us in the 1990s to explore how Fe(II) and SAM initiate radical reactions. Our early work focused on the same enzyme Knappe had originally characterized: the pyruvate formate-lyase activating enzyme (PFL-AE). Our discovery of an iron–sulfur cluster in this enzyme, together with similar findings for other SAM-dependent enzymes at the time, led to the recognition of an emerging class of enzymes that use iron–sulfur clusters to cleave SAM, liberating the 5′-deoxyadenosyl radical (5′-dAdo•) that initiates radical reactions. A major bioinformatics study by Heidi Sofia and co-workers identified the enzyme superfamily denoted Radical SAM, now known to span all kingdoms of life with more than 100,000 unique sequences encoding enzymes that catalyze remarkably diverse reactions.Despite the limited sequence similarity and vastly divergent reactions catalyzed, the radical SAM enzymes appear to employ a common mechanism for initiation of radical chemistry, a mechanism we have helped to clarify over the last 25 years. A reduced [4Fe-4S]+ cluster provides the electron needed for the reductive cleavage of SAM. The resulting [4Fe-4S]2+ cluster can be rereduced either by an external reductant, with SAM acting as a cosubstrate, or by an electron provided during the reformation of SAM in cases where SAM is used as a cofactor. The amino and carboxylate groups of SAM bind to the unique iron of the catalytic [4Fe-4S] cluster, placing the sulfonium of SAM in close proximity to the cluster. Surprising recent results have shown that the initiating enzymatic cleavage of SAM generates an organometallic intermediate prior to liberation of 5′-dAdo•, which initiates radical chemistry on the substrate. This organometallic intermediate, denoted Ω, has a 5′-deoxyadenosyl moiety directly bound to the unique iron of the [4Fe-4S] cluster via the 5′-C, giving a structure that is directly analogous to the Co-(5′-C) bond of the organometallic cofactor adenosylcobalamin. Our observation that this intermediate Ω is formed throughout the superfamily suggests that this is a key intermediate in initiating radical SAM reactions, and that organometallic chemistry is much more broadly relevant in biology than previously thought.
Enzymes of the radical S -adenosyl- l -methionine (radical SAM, RS) superfamily, the largest in nature, catalyze remarkably diverse reactions initiated by H-atom abstraction. Glycyl radical enzyme activating enzymes (GRE-AEs) are a growing class of RS enzymes that generate the catalytically essential glycyl radical of GREs, which in turn catalyze essential reactions in anaerobic metabolism. Here, we probe the reaction of the GRE-AE pyruvate formate-lyase activating enzyme (PFL-AE) with the peptide substrate RVSG 734 YAV, which mimics the site of glycyl radical formation on the native substrate, pyruvate formate-lyase. Time-resolved freeze-quench electron paramagnetic resonance spectroscopy shows that at short mixing times reduced PFL-AE + SAM reacts with RVSG 734 YAV to form the central organometallic intermediate, Ω, in which the adenosyl 5′C is covalently bound to the unique iron of the [4Fe–4S] cluster. Freeze-trapping the reaction at longer times reveals the formation of the peptide G 734 • glycyl radical product. Of central importance, freeze-quenching at intermediate times reveals that the conversion of Ω to peptide glycyl radical is not concerted. Instead, homolysis of the Ω Fe–C5′ bond generates the nominally “free” 5′-dAdo• radical, which is captured here by freeze-trapping. During cryoannealing at 77 K, the 5′-dAdo• directly abstracts an H-atom from the peptide to generate the G 734 • peptide radical trapped in the PFL-AE active site. These observations reveal the 5′-dAdo• radical to be a well-defined intermediate, caught in the act of substrate H-atom abstraction, providing new insights into the mechanistic steps of radical initiation by RS enzymes.
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